JPWO2019178296A5 - - Google Patents

Description

The present application claims priority to US Provisional Patent Application No. 62/642538 filed on March 13, 2018, which is incorporated herein by reference in its entirety for all purposes.

Aging is characterized by gradual loss of function occurring at the molecular, cellular, tissue and biological levels. At chromatin levels, aging is associated with progressive epigenetic error accumulation that ultimately results in abnormal gene regulation, stem cell depletion, aging and deregulated cell / tissue homeostasis. Nuclear reprogramming techniques for pluripotency due to overexpression of a small number of transcription factors restore both age and identity of either cell to that of germ cells by driving epigenetic reprogramming. Can be made to. Undesirable eradication of cell uniqueness poses problems for the development of rejuvenation therapies due to the resulting disruption of structure, function and cell type distribution in tissues and organs.

(Gist of the invention)
Given the above, there is a need for improved methods of rejuvenating cells that avoid dedifferentiation and loss of cell uniqueness. This disclosure addresses these needs and, in addition, provides additional benefits.

The present disclosure relates to cell rejuvenation, tissue engineering and regenerative medicine in general. In particular, the present disclosure rejuvenates and rejuvenates aged cells and tissues by transient exposure to non-integrated mRNA encoding a reprogramming factor that rejuvenates cells while retaining them in a differentiated state. Concerning compositions and methods for recovery.

The present disclosure relates to cell-based therapies utilizing rejuvenated cells. In particular, the present disclosure rejuvenates and rejuvenates aged cells and tissues by transient exposure to non-integrated mRNA encoding a reprogramming factor that rejuvenates cells while retaining them in a differentiated state. Regarding how to recover.

In one embodiment, a method of rejuvenating a cell, in which one or more non-integrated messenger RNAs encoding one or more cell reprogramming factors are transfected into the cell for up to 5 consecutive days. Provided herein is a method comprising the step of producing rejuvenated cells.

In some embodiments, methods for treating an age-related disease or condition, cartilage degenerative disorder, neurodegenerative disorder and / or musculoskeletal dysfunction of a subject are provided herein. The method comprises administering a therapeutically effective amount of cells containing one or more non-integrated messenger RNAs encoding one or more cell reprogramming factors.

In some embodiments, methods are provided herein for treating an age-related disease or condition of a subject, a cartilage degenerative disorder, and / or a subject having musculoskeletal dysfunction. The method comprises administering a therapeutically effective amount of one or more non-integrated messenger RNAs encoding one or more cell reprogramming factors.

In some embodiments, a method of rejuvenating an engineered tissue with Exvivo is provided herein. The method transfects tissue with one or more non-integrated messenger RNAs encoding one or more cellular reprogramming factors for up to 5 consecutive days, thereby rejuvenating the engineered tissue. Includes steps to produce.

In some embodiments, a pharmaceutical composition comprising rejuvenated cells obtained by transfecting cells with one or more non-integrated messenger RNAs encoding one or more cell reprogramming factors for up to 5 consecutive days. The material is provided herein.

Thus, in one aspect, the present disclosure is a method of rejuvenating a cell a) in the step of transfecting a cell with one or more non-integrated messenger RNAs encoding one or more cell reprogramming factors. The transfecting step is performed once a day for at least 2 days and 4 days or less, and b) translating one or more non-integrated messenger RNAs into one or more cells. A step of producing a cell reprogramming factor and resulting in transient reprogramming of the cell, comprising a step-complicated method of rejuvenating the cell without dedifferentiating into a stem cell. The method can be performed in vitro, ex vivo or in vivo in cells.

In certain embodiments, transfection with one or more non-integrated messenger RNAs encoding one or more cell reprogramming factors is performed once daily for 2 days, 3 days or 4 days.

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

The method can be performed on any type of cell. In some embodiments, the cell is a mammalian cell (eg, human, non-human primate, rodent, cat, dog, cow, horse, pig, goat, etc.). For example, the method can be performed on fibroblasts, endothelial cells, chondrocytes or skeletal muscle stem cells. In another embodiment, the cells are derived from an elderly subject.

In certain embodiments, transient reprogramming involves increased expression of HP1γ, H3K9me3, lamina-supporting proteins LAP2α and SIRT1, decreased expression of GMSCF, IL18 and TNFα, decreased nuclear folds, and decreased vesicle formation. , Increased cellular autophagosome formation, increased chymotrypsin-like proteasome activity, increased mitochondrial membrane potential, or decreased reactive oxygen species (ROS).

In certain embodiments, the cells are in a tissue or organ. Transient reprogramming according to the methods described herein can restore the function of cells in a tissue or organ, increase the ability of cells to differentiate in a tissue or organ, tissue or It can reduce the number of aged cells in an organ, enhance the ability of cells in a tissue or organ to replicate, or extend the lifespan of cells in a tissue or organ.

In another aspect, the disclosure is a method for treating a subject's age-related disease or condition a) one or more non-integrated messenger RNAs encoding one or more cellular reprogramming factors. , A step of transfecting a cell of interest, wherein the transfecting step is performed once a day for at least 2 days and 4 days or less, and b) one or more cells in the subject. A step of expressing a cell reprogramming factor and resulting in transient reprogramming of the cell, comprising a step comprising rejuvenating the cell without dedifferentiating into a stem cell. Cells can be transfected in vivo or in vivo.

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

In certain embodiments, the age-related disease or condition is degenerative disease, neurodegenerative disease, cardiovascular disease, peripheral vascular disease, skin disease, eye disease, autoimmune disease, endocrine disorder, metabolic disorder, musculoskeletal disorder. Disorders, digestive system disorders or respiratory disorders.

In another embodiment, the disclosure is a method for treating a disease or disorder associated with chondrocyte degeneration of interest, a) one or more non-integrations encoding one or more cell reprogramming factors. A step of transfecting a messenger RNA into a chondrocyte of interest, wherein the transfecting step is performed once a day for at least 2 days and 4 days or less, and b) 1 in chondrocytes. A step of expressing a cell reprogramming factor of a species or more and resulting in transient reprogramming of chondrocytes, which comprises a step of rejuvenating chondrocytes without dedifferentiating into stem cells. The rejuvenated chondrocytes can be transplanted, for example, into the arthritic joint of interest.

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

In certain embodiments, the disease or disorder associated with cartilage degeneration is arthritis (eg, osteoarthritis or rheumatoid arthritis).

In certain embodiments, treatment reduces inflammation in the subject.

In certain embodiments, the treatment / treatment reduces the expression of RANKL, iNOS, IL6, IL8, BDNF, IFNα, IFNγ and LIF by chondrocytes and increases the expression of COL2A1.

In another aspect, the disclosure is a method for treating a disease or disorder associated with muscle degeneration in a subject, a) one or more non-integrated messengers encoding one or more cell reprogramming factors. A step of transfecting RNA into a skeletal muscle stem cell of interest, wherein the transfecting step is performed once a day for at least 2 days and 4 days or less, and b) in skeletal muscle stem cells. A step that expresses one or more cell reprogramming factors and results in transient reprogramming of skeletal muscle stem cells, in which the skeletal muscle stem cells rejuvenate without losing their ability to differentiate into muscle cells. Including methods to include.

The method can be performed in vivo, in vitro or in vivo. In one embodiment, skeletal muscle stem cells are isolated from a muscle tissue sample obtained from a subject, transfected with Exvivo, and then transplanted into muscle in need of repair or regeneration in the subject.

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

In certain embodiments, treatment / treatment restores the ability of skeletal muscle stem cells to differentiate. In certain embodiments, treatment / treatment results in muscle fiber regeneration.

The method of the present disclosure may be performed on 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 the elderly.

The above and other embodiments of the subject disclosure are readily envisioned by one of ordinary skill in the art in light of the disclosure herein.

We show that transient reprogramming restores aged physiology in fibroblasts towards a more youthful state. It is a diagram showing a representative plot demonstrating the variance of effect (ROS) by duration of treatment: 2 days vs. 4 days of transient reprogramming, both with 2 days of subsequent relaxation. All boxplot and bar plots are generated after combining data across all individual cells, biological and technical replications for clarity. The significance level shown allows flexibility in one pairwise comparison with respect to patient-to-patient variability. We show that transient reprogramming restores aged physiology in fibroblasts towards a more youthful state. FIG. 6 shows quantification of heterochromatin markers H3K9me3 and HP1γ at the single cell level using immunocytochemistry. We show that transient reprogramming restores aged physiology in fibroblasts towards a more youthful state. It is a diagram showing the quantification of the presence of the nuclear lamina-supporting polypeptide LAP2α in a single cell and the percentage of abnormal nuclei (folded or vesicled) in each population using immunocytochemistry. We show that transient reprogramming restores aged physiology in fibroblasts towards a more youthful state. The results of live cell imaging with a florescent-tagged substrate that is cleaved during autophagosome formation in a single cell and chymotrypsin-like 20S proteolytic activity in the total population are shown. We show that transient reprogramming restores aged physiology in fibroblasts towards a more youthful state. It is a figure which shows the individual cell mitochondria membrane potential and ROS level quantified by the mitochondria-specific dye. We show that transient reprogramming restores aged physiology in fibroblasts towards a more youthful state. It is a figure which shows the single cell quantification of the immunostaining with respect to SIRT1. We show that transient reprogramming restores aged physiology in fibroblasts towards a more youthful state. It is a figure which shows the result of telomere quantitative fluorescence in situ hybridization (QFISH) in a single cell. We show that transient reprogramming restores aged physiology in fibroblasts towards a more youthful state. It is a figure which shows the result of SAβGal staining with respect to the aged population. We show that transient reprogramming restores aged physiology in fibroblasts towards a more youthful state. Analyte for Multiplex Cytometry Figure showing quantification of inflammatory cytokine profiling using a panel of antibody-conjugated beads. We show that transient reprogramming restores aged physiology in fibroblasts towards a more youthful state. It is a figure which shows the typical plot which shows the maintenance of a youthful shift in relaxation for a longer period of 4 and 6 days after 4 days of transient reprogramming. Cells in each cohort were then subjected to 80 base pair paired end read data RNA sequencing to obtain transcriptome profiles for each group (young, aging and treatment-R4X2). We show that transient reprogramming restores aged physiology in fibroblasts towards a more youthful state. It is a figure which shows the principal component analysis in the subspace defined by the aging signature. We show that transient reprogramming restores aged physiology in fibroblasts towards a more youthful state. It is a figure which shows the comparison of the logarithmic multiple change between young age and aging (x-axis) and treatment and aging (y-axis). All dark gray dots are genes of the aging signature, while light gray dots are genes that are significantly different between treatment and aging and also overlap with the treatment signature. The majority of the genes are located along the y = x-ray, indicating that the magnitude of treatment changes closely matched the magnitude of the difference between young and aging. Significance is calculated by Student's t-test, pairwise during treatment and aging, and groupwise when compared to younger patients. P-values: ** 0.05, *** <0.01, *** <0.001, asterisk color matches the population being compared. We show that transient reprogramming restores aged physiology in endothelial cells towards a more youthful state. It is a diagram showing a representative plot demonstrating the variance in effect by duration of treatment (ROS): 2 days vs. 4 days of transient reprogramming, both with 2 days of subsequent relaxation. All boxplot and bar plots are generated after combining data across all individual cells, biological and technical replications for clarity. The significance level shown allows flexibility in one pairwise comparison with respect to patient-to-patient variability. We show that transient reprogramming restores aged physiology in endothelial cells towards a more youthful state. FIG. 6 shows quantification of heterochromatin markers H3K9me3 and HP1γ at the single cell level using immunocytochemistry. We show that transient reprogramming restores aged physiology in endothelial cells towards a more youthful state. It is a diagram showing the quantification of the presence of the nuclear lamina-supporting polypeptide LAP2α in a single cell and the percentage of abnormal nuclei (folded or vesicled) in each population using immunocytochemistry. We show that transient reprogramming restores aged physiology in endothelial cells towards a more youthful state. FIG. 6 shows the results of live cell imaging with a florescent-tagged substrate that is cleaved during autophagosome formation in a single cell and chymotrypsin-like 20S proteolytic activity in the total population. We show that transient reprogramming restores aged physiology in endothelial cells towards a more youthful state. It is a figure which shows the individual cell mitochondria membrane potential and ROS level quantified by the mitochondria-specific dye. We show that transient reprogramming restores aged physiology in endothelial cells towards a more youthful state. The single cell quantification of immunostaining for SIRT1 is shown. We show that transient reprogramming restores aged physiology in endothelial cells towards a more youthful state. It is a figure which shows the result of telomere quantitative fluorescence in situ hybridization (QFISH) in a single cell. We show that transient reprogramming restores aged physiology in endothelial cells towards a more youthful state. It is a figure which shows the result of SAβGal staining with respect to the aged population. We show that transient reprogramming restores aged physiology in endothelial cells towards a more youthful state. It is a figure which shows the typical plot which shows the maintenance of a youthful shift in relaxation for a longer period of 4 and 6 days after 4 days of transient reprogramming. Cells in each cohort were then subjected to 80 base pair paired end read data RNA sequencing to obtain transcriptome profiles for each group (young, aging and treatment-R4X2). We show that transient reprogramming restores aged physiology in endothelial cells towards a more youthful state. It is a figure which shows the principal component analysis in the subspace defined by the aging signature. We show that transient reprogramming restores aged physiology in endothelial cells towards a more youthful state. It is a figure which shows the comparison of the logarithmic multiple change between young age and aging (x-axis) and treatment and aging (y-axis). All dark gray dots are genes of the aging signature, while light gray dots are genes that are significantly different between treatment and aging and also overlap with the treatment signature. The majority of the genes are located along the y = x-ray, indicating that the magnitude of treatment changes closely matched the magnitude of the difference between young and aging. Significance is calculated by Student's t-test, pairwise during treatment and aging, and groupwise when compared to younger patients. P-values: ** 0.05, *** <0.01, *** <0.001, asterisk color matches the population being compared FIGS. 3A-3I show that transient reprogramming alleviates the osteoarthritis phenotype in affected chondrocytes: all box whiskers and bar plots are biological for clarity. And combined over technical duplication. The significance levels shown allow flexibility in one pairwise comparison with respect to patient-to-patient variability. Treatment refers to optimized 3-day reprogramming and 2-day relaxation. It is a figure which shows the population result of the cell viability staining. Transient reprogramming has been shown to reduce the osteoarthritis phenotype in affected chondrocytes: all box whiskers and rod plots are combined across biological and technical replication for clarity. .. The significance levels shown allow flexibility in one pairwise comparison with respect to patient-to-patient variability. Treatment refers to optimized 3-day reprogramming and 2-day relaxation. It is a figure which shows the qRT-PCR evaluation of the RNA level of the anabolic factor COL2A1. Transient reprogramming has been shown to reduce the osteoarthritis phenotype in affected chondrocytes: all box whiskers and rod plots are combined across biological and technical replication for clarity. .. The significance levels shown allow flexibility in one pairwise comparison with respect to patient-to-patient variability. Treatment refers to optimized 3-day reprogramming and 2-day relaxation. It is a figure which shows the quantification of the ATP concentration in each cohort. Transient reprogramming has been shown to reduce the osteoarthritis phenotype in affected chondrocytes: all box whiskers and rod plots are combined across biological and technical replication for clarity. .. The significance levels shown allow flexibility in one pairwise comparison with respect to patient-to-patient variability. Treatment refers to optimized 3-day reprogramming and 2-day relaxation. It is a figure which shows the qRT-PCR evaluation of the RNA level of the antioxidant SOD2. It should be noted that the younger level is below OA because the increase in SOD2 is only beneficial in the presence of ROS, i.e., in the OA state (FIG. 3E). Transient reprogramming has been shown to reduce the osteoarthritis phenotype in affected chondrocytes: all box whiskers and rod plots are combined across biological and technical replication for clarity. .. The significance levels shown allow flexibility in one pairwise comparison with respect to patient-to-patient variability. Treatment refers to optimized 3-day reprogramming and 2-day relaxation. The increase in SOD2, which is a diagram showing the qRT-PCR evaluation of RNA levels of the antioxidant SOD2, is beneficial only in the presence of ROS, i.e., in the OA state (FIG. 3E), so younger levels have OA. Please note that it is below. Transient reprogramming has been shown to reduce the osteoarthritis phenotype in affected chondrocytes: all box whiskers and rod plots are combined across biological and technical replication for clarity. .. The significance levels shown allow flexibility in one pairwise comparison with respect to patient-to-patient variability. Treatment refers to optimized 3-day reprogramming and 2-day relaxation. It is a figure which shows the qRT-PCR evaluation of the RNA level of the catabolism MMP13 (FIG. 3F). Transient reprogramming has been shown to reduce the osteoarthritis phenotype in affected chondrocytes: all box whiskers and rod plots are combined across biological and technical replication for clarity. .. The significance levels shown allow flexibility in one pairwise comparison with respect to patient-to-patient variability. Treatment refers to optimized 3-day reprogramming and 2-day relaxation. It is a figure which shows the qRT-PCR evaluation of the RNA level of a catabolist MMP3 (FIG. 3G). Transient reprogramming has been shown to reduce the osteoarthritis phenotype in affected chondrocytes: all box whiskers and rod plots are combined across biological and technical replication for clarity. .. The significance levels shown allow flexibility in one pairwise comparison with respect to patient-to-patient variability. Treatment refers to optimized 3-day reprogramming and 2-day relaxation. Analyte for Multiplex Cytometry Analysis A panel of antibody-conjugated beads is used to show RT-PCR evaluation of RNA levels of pro-inflammatory factor RANKL (FIG. 3H) profiling. Significance is pairwise during treatment and aging, groupwise when compared to younger patients, calculated by Student's t-test, P-values: ** 0.05, ** <0.01, *** <0.001. Transient reprogramming has been shown to reduce the osteoarthritis phenotype in affected chondrocytes: all box whiskers and rod plots are combined across biological and technical replication for clarity. .. The significance levels shown allow flexibility in one pairwise comparison with respect to patient-to-patient variability. Treatment refers to optimized 3-day reprogramming and 2-day relaxation. Analyte for Multiplex Cytometry Analysis A panel of antibody-conjugated beads is used to show RT-PCR evaluation of RNA levels of pro-inflammatory factor iNOS (FIG. 3I) profiling. Significance is pairwise during treatment and aging, groupwise when compared to younger patients, calculated by Student's t-test, P-values: ** 0.05, ** <0.01, *** <0.001. We show that transient reprogramming restores age-related muscle stem cell differentiation potential. It is a figure which shows the measurement of MuSC activation from a rest state. Freshly isolated aged MuSCs were incubated with EdU and fixed after 2 days of treatment and 1 or 2 days of relaxation. All boxplot and bar plots are combined over biological and technical replication for easy viewing. The significance levels shown allow flexibility in one pairwise comparison with respect to patient-to-patient variability. We show that transient reprogramming restores age-related muscle stem cell differentiation potential. FIG. 6 shows quantified results of bioluminescence measured from mice 11 days after transplantation in TA muscle of treated / untreated + luciferase mice MuSC at different time points after transplantation and injury. We show that transient reprogramming restores age-related muscle stem cell differentiation potential. FIG. 4B shows the quantification of immunofluorescent staining of GFP expression in the cross section of TA muscle in mice, imaged and quantified in FIG. 4B. We show that transient reprogramming restores age-related muscle stem cell differentiation potential. It is a figure which shows the quantification of the area of the cross section of the donor-derived GFP + fiber in the TA muscle which was the recipient of transplanted MuSC. We show that transient reprogramming restores age-related muscle stem cell differentiation potential. It is a figure which shows the result of the bioluminescence imaging of the TA muscle which was injured again 60 days after transplantation (second injury). A second injury was performed to examine whether the bioluminescence signal was increased as a result of activation and augmentation of luciferase + / GFP + MuSC that was initially transplanted and engrafted beneath the basal layer. We show that transient reprogramming restores age-related muscle stem cell differentiation potential. It is a figure which shows the quantified result of bioluminescence measured from the mouse 11 days after transplantation in the TA muscle of treated luciferase + human MuSC. We show that transient reprogramming restores age-related muscle stem cell differentiation potential. FIG. 5 shows the difference in the ratio of bioluminescence between treated and untreated MuSCs obtained from healthy donors of different age groups. Significance is calculated by Student's t-test, pairwise during treatment and aging, and groupwise when compared to younger patients. P-values: ** 0.05, *** <0.01, *** <0.001, asterisk color matches the population being compared. We show that transient reprogramming restores aged physiology in human fibroblasts and endothelial cells towards a more youthful state. Fibroblasts and endothelial cells were obtained from otherwise healthy young and aged individuals. It is a figure which shows the distribution of epigenetic and nuclear markers about H3K9me3. We show that transient reprogramming restores aged physiology in human fibroblasts and endothelial cells towards a more youthful state. Fibroblasts and endothelial cells were obtained from otherwise healthy young and aged individuals. It is a figure which shows the distribution of epigenetic and nuclear markers about HP1γ. We show that transient reprogramming restores aged physiology in human fibroblasts and endothelial cells towards a more youthful state. Fibroblasts and endothelial cells were obtained from otherwise healthy young and aged individuals. It is a figure which shows the distribution of epigenetic and nuclear markers about LAP2α. We show that transient reprogramming restores aged physiology in human fibroblasts and endothelial cells towards a more youthful state. Fibroblasts and endothelial cells were obtained from otherwise healthy young and aged individuals. It is a figure which shows the distribution of nutrition and energy regulation with respect to SIRT1. We show that transient reprogramming restores aged physiology in human fibroblasts and endothelial cells towards a more youthful state. Fibroblasts and endothelial cells were obtained from otherwise healthy young and aged individuals. It is a figure which shows the distribution of nutrition and energy regulation with respect to Mito membrane potential. We show that transient reprogramming restores aged physiology in human fibroblasts and endothelial cells towards a more youthful state. Fibroblasts and endothelial cells were obtained from otherwise healthy young and aged individuals. It is a figure which shows the distribution of nutrition and energy regulation about Mito ROS. We show that transient reprogramming restores aged physiology in human fibroblasts and endothelial cells towards a more youthful state. Fibroblasts and endothelial cells were obtained from otherwise healthy young and aged individuals. It is a figure which shows the distribution in an autophagosome and the clearance and aging of a large amount of waste. We show that transient reprogramming restores aged physiology in human fibroblasts and endothelial cells towards a more youthful state. Fibroblasts and endothelial cells were obtained from otherwise healthy young and aged individuals. It is a figure which shows the proteasome activity of a young, aging and treated cells. We show that transient reprogramming restores aged physiology in human fibroblasts and endothelial cells towards a more youthful state. Fibroblasts and endothelial cells were obtained from otherwise healthy young and aged individuals. It is a figure which shows the aging activity of a young age, the aging and the treated cell. We show that transient reprogramming restores aged physiology in human fibroblasts and endothelial cells towards a more youthful state. Fibroblasts and endothelial cells were obtained from otherwise healthy young and aged individuals. FIG. 6 shows cytokines secreted in young, aging and treated cells. Transcriptome and methylome analysis of aged fibroblasts and endothelial cells is shown. It is a figure which shows the juvenile vs. aging transcriptome profile of fibroblast. The data show that 961 genes (5.85%) in fibroblasts (678 upregulated, 289 downregulated) have a significance criterion p <0.05 and a log-multiple change cutoff +/-. A value of 0.5 indicates that there was a difference between young and aged cells. Transcriptome and methylome analysis of aged fibroblasts and endothelial cells is shown. It is a figure which shows the PCA analysis of a fibroblast. Transcriptome and methylome analysis of aged fibroblasts and endothelial cells is shown. It is a figure which shows the expression analysis of a fibroblast. Transcriptome and methylome analysis of aged fibroblasts and endothelial cells is shown. It is a figure which shows the young vs. aging profile of the endothelial cell. The data show that 748 genes (4.80%) in endothelial cells (389 upregulated, 377 downregulated) had a significance criterion p <0.05 and a logarithmic multiple change cutoff +/- 0. At .5, it is shown that there is a difference between young and aged cells. Transcriptome and methylome analysis of aged fibroblasts and endothelial cells is shown. It is a figure which shows the PCA analysis of the endothelial cell. Transcriptome and methylome analysis of aged fibroblasts and endothelial cells is shown. It is a figure which shows the expression analysis of the endothelial cell. Transcriptome and methylome analysis of aged fibroblasts and endothelial cells is shown. It is a graph of the methylation age of fibroblasts evaluated by the Horvath clock before and after treatment, and the data is a diagram showing the general tendency of decline. Transcriptome and methylome analysis of aged fibroblasts and endothelial cells is shown. It is a graph of the methylation age of endothelial cells as assessed by the Holvas clock before and after treatment, and the data are diagrams showing a general trend of decline. Transcriptome and methylome analysis of aged fibroblasts and endothelial cells is shown. FIG. 6I is an unsupervised cluster showing unsupervised clustering in methylation patterns separated by treatment status, gender, patient and cell type. Clustering demonstrates the collective retention of cell uniqueness, at least when comparing fibroblasts and endothelial cells. We show transient reprogramming in osteoarthritis chondrocytes and mesenchymal stem cells. Data showing that transient reprogramming reduces the inflammatory phenotype in affected chondrocytes. Chondrocytes were obtained from a cartilage biopsy from an aged patient diagnosed with late-stage osteoarthritis (OA). Aged OA cells and transiently reprogrammed OA cells were evaluated for an OA-specific phenotype. All boxplot and bar plots are combined over biological and technical replication for easy viewing. The overall significance ranking is set by the second strictest p-value. Significance was calculated by Student's t-test. P-value: ** 0.05, ** <0.01, *** <0.001. Error bars indicate RMSE (Root Mean Square Error). FIG. 5 shows an increase in ATP levels due to treatment in chondrocytes by measurement of ATP concentration using fluorophore-based glycerol. We show transient reprogramming in osteoarthritis chondrocytes and mesenchymal stem cells. Data showing that transient reprogramming reduces the inflammatory phenotype in affected chondrocytes. Chondrocytes were obtained from a cartilage biopsy from an aged patient diagnosed with late-stage osteoarthritis (OA). Aged OA cells and transiently reprogrammed OA cells were evaluated for an OA-specific phenotype. All boxplot and bar plots are combined over biological and technical replication for easy viewing. The overall significance ranking is set by the second strictest p-value. Significance was calculated by Student's t-test. P-value: ** 0.05, ** <0.01, *** <0.001. Error bars indicate RMSE (Root Mean Square Error). FIG. 5 shows that ROS activity by tracking live single cell images of cells incorporating superoxide-induced fluorescent dyes shows a reduced signal after treatment. We show transient reprogramming in osteoarthritis chondrocytes and mesenchymal stem cells. Data showing that transient reprogramming reduces the inflammatory phenotype in affected chondrocytes. Chondrocytes were obtained from a cartilage biopsy from an aged patient diagnosed with late-stage osteoarthritis (OA). Aged OA cells and transiently reprogrammed OA cells were evaluated for an OA-specific phenotype. All boxplot and bar plots are combined over biological and technical replication for easy viewing. The overall significance ranking is set by the second strictest p-value. Significance was calculated by Student's t-test. P-value: ** 0.05, ** <0.01, *** <0.001. Error bars indicate RMSE (Root Mean Square Error). It is a figure which shows the result of the qRT-PCR evaluation of the RNA level of the antioxidant SOD2 which was increased by the treatment. We show transient reprogramming in osteoarthritis chondrocytes and mesenchymal stem cells. Data showing that transient reprogramming reduces the inflammatory phenotype in affected chondrocytes. Chondrocytes were obtained from a cartilage biopsy from an aged patient diagnosed with late-stage osteoarthritis (OA). Aged OA cells and transiently reprogrammed OA cells were evaluated for an OA-specific phenotype. All boxplot and bar plots are combined over biological and technical replication for easy viewing. The overall significance ranking is set by the second strictest p-value. Significance was calculated by Student's t-test. P-value: ** 0.05, ** <0.01, *** <0.001. Error bars indicate RMSE (Root Mean Square Error). It is a figure which shows the cell proliferation in a young age, aging and aging / treated chondrocytes. Aging and treated cells shift towards levels near young cells. We show transient reprogramming in osteoarthritis chondrocytes and mesenchymal stem cells. Data showing that transient reprogramming reduces the inflammatory phenotype in affected chondrocytes. Chondrocytes were obtained from a cartilage biopsy from an aged patient diagnosed with late-stage osteoarthritis (OA). Aged OA cells and transiently reprogrammed OA cells were evaluated for an OA-specific phenotype. All boxplot and bar plots are combined over biological and technical replication for easy viewing. The overall significance ranking is set by the second strictest p-value. Significance was calculated by Student's t-test. P-value: ** 0.05, ** <0.01, *** <0.001. Error bars indicate RMSE (Root Mean Square Error). FIG. 6 shows data from qRT-PCR that reflect elevated RNA levels of extracellular matrix protein component COL2A1 in young, aging and aging / treated chondrocytes. Aging and treated cells shift towards levels near young cells. We show transient reprogramming in osteoarthritis chondrocytes and mesenchymal stem cells. Data showing that transient reprogramming reduces the inflammatory phenotype in affected chondrocytes. Chondrocytes were obtained from a cartilage biopsy from an aged patient diagnosed with late-stage osteoarthritis (OA). Aged OA cells and transiently reprogrammed OA cells were evaluated for an OA-specific phenotype. All boxplot and bar plots are combined over biological and technical replication for easy viewing. The overall significance ranking is set by the second strictest p-value. Significance was calculated by Student's t-test. P-value: ** 0.05, ** <0.01, *** <0.001. Error bars indicate RMSE (Root Mean Square Error). Data showing that the qRT-PCR levels of the cartilage formation identity and functional transcription factor SOX9 are retained after treatment. We show transient reprogramming in osteoarthritis chondrocytes and mesenchymal stem cells. Data showing that transient reprogramming reduces the inflammatory phenotype in affected chondrocytes. Chondrocytes were obtained from a cartilage biopsy from an aged patient diagnosed with late-stage osteoarthritis (OA). Aged OA cells and transiently reprogrammed OA cells were evaluated for an OA-specific phenotype. All boxplot and bar plots are combined over biological and technical replication for easy viewing. The overall significance ranking is set by the second strictest p-value. Significance was calculated by Student's t-test. P-value: ** 0.05, ** <0.01, *** <0.001. Error bars indicate RMSE (Root Mean Square Error). It is a figure summarizing the RT-PCR evaluation showing that the treatment reduces the intracellular RNA level of the NF-κB ligand RANKL. We show transient reprogramming in osteoarthritis chondrocytes and mesenchymal stem cells. Data showing that transient reprogramming reduces the inflammatory phenotype in affected chondrocytes. Chondrocytes were obtained from a cartilage biopsy from an aged patient diagnosed with late-stage osteoarthritis (OA). Aged OA cells and transiently reprogrammed OA cells were evaluated for an OA-specific phenotype. All boxplot and bar plots are combined over biological and technical replication for easy viewing. The overall significance ranking is set by the second strictest p-value. Significance was calculated by Student's t-test. P-value: ** 0.05, ** <0.01, *** <0.001. Error bars indicate RMSE (Root Mean Square Error). Figure summarizing RT-PCR assessments showing that treatment reduces the level of iNOS to produce nitric oxide in response and propagates inflammatory stimuli by a closer shift to that of young chondrocytes. Is. We show transient reprogramming in osteoarthritis chondrocytes and mesenchymal stem cells. Data showing that transient reprogramming reduces the inflammatory phenotype in affected chondrocytes. Chondrocytes were obtained from a cartilage biopsy from an aged patient diagnosed with late-stage osteoarthritis (OA). Aged OA cells and transiently reprogrammed OA cells were evaluated for an OA-specific phenotype. All boxplot and bar plots are combined over biological and technical replication for easy viewing. The overall significance ranking is set by the second strictest p-value. Significance was calculated by Student's t-test. P-value: ** 0.05, ** <0.01, *** <0.001. Error bars indicate RMSE (Root Mean Square Error). Data reflecting that the cytokine profiling of chondrocyte secretion shows increased pro-inflammatory cytokines that are reduced by treatment. We show transient reprogramming in osteoarthritis chondrocytes and mesenchymal stem cells. FIG. 5 is a graph showing patient-by-patient distribution shifts towards p16 levels reduced by treatment in mesenchymal stem cells. We show transient reprogramming in osteoarthritis chondrocytes and mesenchymal stem cells. It is a graph which shows the distribution shift for each patient toward p21 of the level decreased by the treatment in mesenchymal stem cells. We show transient reprogramming in osteoarthritis chondrocytes and mesenchymal stem cells. It is a figure which shows the multiple change corresponding to the increase of cell proliferation in aging and treatment mesenchymal stem cells. We show transient reprogramming in osteoarthritis chondrocytes and mesenchymal stem cells. It is a figure which shows the percentage of the aged aging and treated mesenchymal stem cells corresponding to the decrease of cell aging. Shows the effect of transient reprogramming of manipulated skin tissue. The skin aging parameters of fibroblasts and keratinocytes are shown. Histological scores incorporating metrics for morphology, structure and organization show improvement with mRNA treatment, but not with retinoic acid for commonly sold skin treatments. Shows the effect of transient reprogramming of manipulated skin tissue. The skin aging parameters of fibroblasts and keratinocytes are shown. Reduction of aging parameters by mRNA treatment is shown in FIG. 8B (SaβGal) and FIG. 8C left panel (p16), and inflammatory parameters are shown in FIG. 8C center panel (IL-8) and FIG. 8C right panel (MMP-1). ), With further comparison with the effect of retinoic acid. Shows the effect of transient reprogramming of manipulated skin tissue. The skin aging parameters of fibroblasts and keratinocytes are shown. Reduction of aging parameters by mRNA treatment is shown in FIG. 8B (SaβGal) and FIG. 8C left panel (p16), and inflammatory parameters are shown in FIG. 8C center panel (IL-8) and FIG. 8C right panel (MMP-1). ), With further comparison with the effect of retinoic acid. Shows the effect of transient reprogramming of manipulated skin tissue. Shows muscle regeneration in satellite cells. It is a figure which shows the quantified result of bioluminescence measured from the mouse 11 days after transplantation in the TA muscle of treated luciferase ⁺ human MuSC. Shows the effect of transient reprogramming of manipulated skin tissue. Shows muscle regeneration in satellite cells. It is a figure which shows the bioluminescence of a cohort 10-30 days old, 30-55 days old and 60-80 days old. Differences in the ratio of bioluminescence between treated and untreated MuSCs obtained from healthy donors of different age groups. Significance is calculated by Student's t-test, pairwise during treatment and aging, and groupwise when compared to younger patients (age group, 10-30: n = 5; 30-55: n). = 7; 60-80: n = 5). P-values: ** 0.05, *** <0.01, *** <0.001, asterisk color matches the population being compared. Shows the effect of transient reprogramming of manipulated skin tissue. Shows muscle regeneration in satellite cells. It is a figure which shows the tetanic force measurement of the tetanic force of the aging muscle which was injured and transplanted with the aging MuSC. TA muscles were dissected and electrophysiological examinations were performed with Exvivo to measure tetanus. Baselines of non-transplanted muscle force production were measured in young (4 months, dashed blue) and aged (27 months, dashed red) mice. Treated aged MuSCs were transplanted into TA muscles of aged mice and force production was measured 30 days later (n = 5). Shows the effect of transient reprogramming of manipulated skin tissue. Shows muscle regeneration in satellite cells. FIG. 6 shows quantified results of treatment, aging and bioluminescence of young cells at different time points after transplantation and injury (n = 10). Shows the effect of transient reprogramming of manipulated skin tissue. Shows muscle regeneration in satellite cells. It is a figure which shows the quantification of the immunofluorescent staining in the TA muscle cross section of the mouse which was transplanted with the aging / treated and the aging / untreated cells (n = 5). Shows the effect of transient reprogramming of manipulated skin tissue. Shows muscle regeneration in satellite cells. FIG. 6 is a graph showing the quantification of the cross-sectional area of donor-derived GFP + fibers in TA muscle that was the recipient of the transplanted MuSC (n = 5). Shows the effect of transient reprogramming of manipulated skin tissue. Shows muscle regeneration in satellite cells. It is a figure which shows the result of the bioluminescence imaging of the TA muscle which was injured again 60 days after MuSC transplantation (second injury) (n = 6). A second injury was performed to examine whether the bioluminescence signal was increased as a result of activation and augmentation of luciferase ⁺ / GFP ⁺ MuSC that was initially transplanted and engrafted beneath the basal layer. We show the transfection of corneal epithelial cells by transiently reprogrammed cells. It is a figure which shows the decrease of aging measured by the expression of p16 in the aging-paired cell. We show the transfection of corneal epithelial cells by transiently reprogrammed cells. It is a figure which shows the decrease of aging measured by the expression of p21 in the aging-paired cell. We show the transfection of corneal epithelial cells by transiently reprogrammed cells. It is a figure which shows the decrease of the inflammatory factor IL8 in the aging-paired cell. We show the transfection of corneal epithelial cells by transiently reprogrammed cells. It is a figure which shows the increase of mitochondrial neoplasia measured by PGC1α expression. FIG. 6 is a chart showing P-values of changes in cell-specific markers between treated and aged cells using RNAseq analysis. Of the 8 fibroblasts and 50 endothelial cell markers, none showed significant changes by treatment in each cell type, suggesting retention of cell uniqueness. FIG. 6 is a chart showing P-values of changes in cell-specific markers between treated and aged cells using RNAseq analysis. Of the 8 fibroblasts and 50 endothelial cell markers, none showed significant changes by treatment in each cell type, suggesting retention of cell uniqueness. FIG. 6 is a chart showing P-values of changes in cell-specific markers between treated and aged cells using RNAseq analysis. Of the 8 fibroblasts and 50 endothelial cell markers, none showed significant changes by treatment in each cell type, suggesting retention of cell uniqueness. It is a figure which shows the characteristic of aging analyzed using the panel of 11 kinds of established assays.

Implementation of the techniques described herein is within the skill of one of ordinary skill in the art, medical, cell biology, pharmacology, chemistry, biochemistry, molecular biology and recombinant DNA techniques and, unless otherwise indicated. Use conventional methods of immunology. Such techniques are well described in the literature. For example, G. Vunjak-Novakovic and R.V. I. Freshney Culture of Cells for Blackwell Engineering (Wiley-Lis, 1st Edition, 2006); Artritis Research: Materials and Protocols, Volumes 1 and 2: (Massimo Sabatini) Methods in Molecular Medicine, M. Sabatini P. Pastoureau and F. De Cenincck ed., Humana Press; 2004; Handbook of Experimental Library, I-IV ); A. L. See Lehninger, Biochemistry (Worth Publicsers, Inc., current edition); and Sambrook et al., Molecular Cloning: A Laboratory Manual (3rd edition, 2001).

All publications, patents and patent applications cited herein, whether above or below, are hereby incorporated herein by reference in their entirety.

I. Definitions In the description of this disclosure, the following terms are used and are intended to be defined as shown below.

As used herein and in the appended claims, the singular forms "one (a)", "one (an)" and "the (the)" unless the content explicitly indicates otherwise. It should be noted that "" includes multiple referents. Thus, for example, a reference to "a cell" includes a mixture of two or more cells, and others.

Throughout the specification, for example, "one embodiment", "one embodiment", "another embodiment", "specific embodiment", "related embodiment", "certain embodiment", "additional embodiment". The specific features, structures or features described in connection with such embodiments are included in at least one embodiment of the present disclosure. Means. Therefore, throughout the specification, the appearance of the above-mentioned words and phrases in various places does not necessarily refer to the same embodiment. In addition, specific features, structures or features may be combined in any suitable manner in one or more embodiments.

As used herein, the term "about" means a range of values that include a specified value, which one of ordinary skill in the art would consider reasonably similar to the specified value. In embodiments, the term "about" means within standard deviation using measurements generally accepted in the art. In embodiments, about means the range stretched to +/- 10% of the specified value. In embodiments, about means a specified value.

Throughout this specification, the words "comprise", "comprises" and "comprising" are described as steps or elements or elements, unless the context otherwise requires. It is understood to imply inclusion of a step or group of elements, but not any other step or element or exclusion of a group of steps or elements. By "consisting of" is meant to include, and be limited to, anything following the phrase "consisting of". Thus, the phrase "consisting of" indicates that the contained element is required or required and that no other element may be present. "Becomes essential" to any other element that includes any of the elements listed after this phrase and that does not interfere with or contribute to the activity or action specified in this disclosure with respect to the listed elements. Means to be limited. Thus, the phrase "becomes essential from" means that the listed element is required or required, but the other elements are not optional and affect the activity or action of the listed element. Indicates that it may or may not exist depending on whether it is given or not.

As used herein, the term "biocompatibility" generally refers to a material and any metabolite or degradation product thereof that is generally non-toxic to the recipient and does not cause any significant adverse effects on the subject.

As used herein, the term "cell" refers to a living cell of natural origin or modified, intact. Cells that are mixed with other cells in culture or in tissues (partially or intact) or organisms can be isolated from other cells. The methods described herein can be performed, for example, on a sample containing a single cell, a population of cells, or a tissue or organ containing cells.

As used herein, the term "non-integration" with reference to messenger RNA (mRNA) refers to an mRNA molecule that is neither intrachromosomally or extrachromosomally integrated into the host genome nor into a vector.

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". Numerous transfection techniques are commonly known in the art. For example, Graham et al. (1973) Virology, 52: 456, Sambrook et al. (2001) Molecular Cloning, a laboratory manual, 3rd edition, Cold Spring Harbor Biology, New York, See 2nd Edition, McGraw-Hill and Chu et al. (1981) Gene 13: 197. Such techniques can be used to introduce one or more exogenous DNA or RNA molecules into cells. The term refers to both stable and transient uptake of DNA or RNA molecules. For example, transfection can be used for transient uptake of mRNA encoding a cell reprogramming factor into cells that require rejuvenation.

As used herein, the term "transient reprogramming" is sufficient to rejuvenate cells (ie, eliminate all or part of the characteristics of aging), but cause dedifferentiation into stem cells. Refers to the exposure of cells to cellular reprogramming factors during periods of not long enough. Such transient reprogramming results in rejuvenated cells that retain their identity (ie, differentiated cell types).

As used herein, the term "rejuvenated cell (s)" is used so that the cell has a transcriptome profile of a younger cell while still retaining one or more cell-specific markers. Refers to aged cells that have been treated or transiently reprogrammed by cell reprogramming factors above the species.

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

As used herein, the term "stem cell" refers to a cell that retains its ability to regenerate itself by mitosis and is capable of differentiating into a diverse range of specialized cell types. Mammalian stem cells can be divided into three broad categories: embryonic stem cells derived from blastocysts, adult stem cells found in adult tissues, and cord blood stem cells found in the umbilical cord. In developing embryos, stem cells can differentiate into all specialized embryonic tissues. In adult organisms, stem cells and progenitor cells act as the body's repair system by replenishing specialized cells. Totipotent stem cells are produced from the fusion of egg and sperm cells. The cells produced by the first few divisions of the fertilized egg are also totipotent. These cells can differentiate into embryonic and extraembryonic cell types. Pluripotent stem cells are progeny of totipotent cells and can differentiate into cells derived from any of the three germ layers. Multipotent stem cells can only produce cells of a closely related family of cells (eg, hematopoietic stem cells differentiate into erythrocyte cells, leukocyte 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 cells such as, for example, skin or blood cell cells.

As used herein, the term "transcriptome profile" refers to a set of total RNA molecules in a cell or cell population. It may also be used to refer to total RNA or simply mRNA, depending on the particular experiment. It differs from exome in that it contains only RNA molecules found in a given cell population and usually contains the amount or concentration of each RNA molecule in addition to molecular identity. Methods for obtaining transcriptome profiles include DNA microarrays such as RNA-Seq and next generation sequencing techniques. Transcription can be tested at the individual cell level by unicellular transcriptomics. There are two general methods for inferring transcriptome arrays. One approach maps sequence read data to the reference genome of either the organism itself (its transcriptome is being tested) or a closely related species. The other approach, the de novo transcriptome assembly, uses software that infers transcripts directly from short sequence read data.

As used herein, the term "root mean square error" or "RMSE" refers to the standard deviation of the residuals (prediction error). Residual is a measure of the distance of a data point from the regression line. RMSE is a measure of the spread of these residuals. In other words, this shows how much data is concentrated around the best fit line.

As used herein, the term "cell viability" refers to a measure of the number of living or dead cells based on a total cell sample. The high cell viability as defined herein is a cell population in which more than 85% of all cells are viable, preferably more than 90-95%, more preferably more than 99%. Refers to a population characterized by high cell viability containing possible cells.

As used herein, the term "autophagosome" refers to a spherical structure with a bilayer membrane. This is a key structure in macroautophagy, which is also the intracellular degradation system of cytoplasmic contents (eg, abnormal intracellular proteins, excess or damaged organelles) and invading microorganisms. After formation, autophagosomes deliver cytoplasmic components to lysosomes. The outer membrane of autophagosomes fuses with lysosomes to form autophagosomes. Lysosome hydrolases degrade the contents delivered to the autophagosomes and their endometrium.

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

As used herein, the term "mitochondrial membrane potential" refers to a potential and proton gradient that results from the redox conversion associated with the activity of the Krebs cycle and serves as an intermediate form of energy storage for the production of ATP. It is produced by a proton pump and is an essential process of energy storage in oxidative phosphorylation. It plays a key role in mitochondrial homeostasis by the selective elimination of dysfunctional mitochondria.

As used herein, the term "pharmaceutically acceptable excipient or carrier" may optionally be included in the compositions of the present disclosure and does not cause a significant toxicological effect on the patient. Refers to a form.

As used herein, the term "reactive oxygen species" or "ROS" is a chemically reactive species containing oxygen. Examples include peroxides, superoxide, hydroxyl radicals, singlet oxygen and alpha-oxygen. In the biological context, ROS are formed as a natural by-product of the normal metabolism of oxygen and have important roles in cellular signaling and homeostasis.

As used herein, the term "cell senescence-related secretory phenomenon" or "SASP" refers to a number of diverse cytokines, chemokines, growth factors and proteases that are characteristic features of aged cells. Aged cells are still metabolically active and show stable upregulation of a wide range of genes, including genes encoding secretory proteins such as inflammatory cytokines, chemokines, extracellular matrix remodeling factors and growth factors. It is a non-dividing cell. These secretory proteins function physiologically in the tissue microenvironment, which allows secretory proteins to propagate stress responses and communicate with neighboring cells. This phenotype, named Cellular Senescence-Associated Secretory Phenomenon (SASP), reveals the paraclinic function of aged cells and removes aged cells from non-senescent cell cycle arrest cells such as quiescent cells and terminally differentiated cells. It is an important distinguishing feature. "SASP cytokine" specifically refers to a cytokine produced by aged cells to create a cellular senescence-related secretory phenomenon. Cytokines include, but are not limited to, IL18, IL1A, GROA, IL22 and IL9.

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

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-organization epigenetic clock, the Horvath epigenetic clock or "Horvath clock", was developed by Steve Horvath (Horvath 2013).

As used herein, the term "cell reprogramming factor" refers to a set of transcription factors capable of converting adult or differentiated cells into pluripotent stem cells. In embodiments herein, this factor comprises OCT4, SOX2, KLF4, c-MYC, LIN28 and NANOG.

Correspondence of "pharmaceutically acceptable salts" as either salts prepared with inorganic acids such as amino acid salts, chlorides, sulfates, phosphates, diphosphates, bromides and nitrates, or precursors. Salts prepared from inorganic acid forms such as hydrochloride, or malate, maleate, fumarate, tartrate, succinate, ethyl succinate, citrate, acetate, lactate, Salts prepared with organic acids such as methanesulfonate, benzoate, ascorbate, para-toluenesulfonate, pamoate, salicylate and stearate, as well as estrates and gluceptates. And lactobionates, but are not limited to these. Similarly, pharmaceutically acceptable cation-containing salts include, but are not limited to, sodium, potassium, calcium, aluminum, lithium and ammonium (including substituted ammonium).

As used herein, the term "transplant" refers to the transfer of cells, tissues or organs from another source to a subject. The term is not limited to a particular mobile mechanism. The cells can be transplanted by any suitable method, such as injection or surgical implantation.

As used herein, the term "arthritis" includes, but is not limited to, osteoarthritis, rheumatoid arthritis, lupus-related arthritis, juvenile idiopathic arthritis, reactive arthritis, enteritis arthritis and psoriatic arthritis.

As used herein, the term "age-related disease or condition" refers to neurodegenerative diseases (eg, Alzheimer's disease, Parkinson's disease, Huntington's disease, muscular atrophic lateral sclerosis, dementia and stroke), cardiovascular and peripheral blood vessels. Diseases (eg, porphyritic arteriosclerosis, peripheral arterial disease (PAD), hematoma, calcification, thrombosis, embolism and aneurysms), eye diseases (eg, age-related yellow spot degeneration, glaucoma, cataracts, dry eyes, diabetes) Retinopathy, loss of vision), skin disorders (skin atrophy and thinning, elastic fibrosis and wrinkles, sebaceous gland hyperplasia or hypoplasia, senile kuroko and other pigmentation abnormalities, gray hair , Hair loss or thinning of hair, and chronic skin ulcers), autoimmune diseases (eg, rheumatic polymyopathy (PMR), giant cell arteritis (GCA), rheumatoid arthritis (RA), crystalline arthritis and Spine arthritis (SPA)), endocrine and metabolic dysfunction (eg, adult pituitary dysfunction, thyroid dysfunction, aspirational thyroid poisoning, osteoporosis, true diabetes, adrenal dysfunction, various forms of gonad dysfunction Diseases and endocrine malignant lesions), musculoskeletal disorders (eg, arthritis, osteoporosis, myeloma, gout, Paget's disease, fractures, myelopathy syndrome, joint toughness, diffuse idiopathic osteoproliferative disease, hematogenous myelitis, muscle atrophy, Peripheral neuropathy, multiple sclerosis, muscular atrophic lateral sclerosis (ALS), Duchenne muscular dystrophy, primary lateral sclerosis and severe myasthenia), digestive disorders (eg, liver cirrhosis, hepatic fibrosis) , Barrett's esophagus), respiratory disease (eg, pulmonary fibrosis, chronic obstructive pulmonary disease, asthma, chronic bronchitis, pulmonary embolism (PE), lung cancer and infectious diseases), and any other with age. Refers to any age-related condition, disease or disorder, including but not limited to diseases and disorders.

As used herein, the term "disorder or disorder associated with cartilage degeneration" is any disease or disorder involving cartilage and / or joint degeneration. The term "diseases or disorders associated with cartilage degeneration" includes, as such, conditions, disorders, syndromes, diseases and injuries affecting spinal discs or joints (eg, joint joints) in animals, including humans. , Arthritis, chondropasia, spondyloarthropathies, tonic spondylitis, erythematosus, relapsing polychondritis and Schegren's syndrome, but not limited to these.

As used herein, the term "muscle degenerative disease or disorder" is any disease or disorder in which muscle degeneration is involved. The terms are muscle atrophy, disused muscle, muscle rupture, burns, surgery, peripheral neuropathy, multiple sclerosis, amyotrophic lateral sclerosis (ALS), Duchenne muscular dystrophy, primary lateral sclerosis. , Severe muscular asthenia, cancer, AIDS, congestive heart failure, chronic obstructive pulmonary disease (COPD), liver disease, renal failure, feeding disorders, malnutrition, starvation, infectious diseases, or treatment with glucocorticoids. Includes, but is not limited to, conditions, disorders, syndromes, diseases and injuries that affect muscular tissue.

A "therapeutically effective dose or amount" is intended to provide a positive therapeutic response to a subject in need of tissue repair or regeneration, such as an amount that restores function at the treatment site and / or results in the formation of new tissue. It is intended for the amount of rejuvenated cells or non-integrated messenger RNA. Rejuvenated cells can be produced by in vitro, ex vivo or in vivo transfection with one or more non-integrated messenger RNAs encoding one or more cell reprogramming factors, as described herein. .. Thus, for example, a "positive therapeutic response" may include restored tissue functionality, decreased pain, improved stamina, increased intensity, increased mobility and / or improved cognitive function. , Improvement in treatment-related age-related diseases or conditions, and / or improvement in one or more symptoms of treatment-related age-related diseases or conditions. The exact amount (of cells or mRNA) required will vary between subjects depending on the species, age and general condition of the subject, the severity of the condition being treated, the mechanism of administration and others. Appropriate "effective" amounts in any individual case may be determined by one of ordinary skill in the art using routine experimental methods, based on the information provided herein.

For example, when administered as described herein, a therapeutically effective dose or amount of rejuvenated chondrocytes, such as an amount that results in the production of new cartilage at the treatment site (eg, a damaged joint). Intended to be an amount that provides a positive therapeutic response to subjects with cartilage damage or loss. For example, a therapeutically effective dose or amount may be used to treat degenerative diseases such as cartilage damage or loss due to traumatic injury, or other diseases associated with arthritis or cartilage degeneration. Preferably, the therapeutically effective amount restores function and / or reduces pain and inflammation associated with cartilage damage or loss.

In another example, the therapeutically effective dose or amount of rejuvenated skeletal muscle stem cells is as described herein, such as the amount that results in the production of new muscle fibers at the treatment site (eg, damaged muscle). When administered, it is intended to provide an amount that provides a positive therapeutic response to subjects with muscle damage or loss. For example, a therapeutically effective dose or amount may be used to treat a disease or disorder associated with muscle injury or loss due to traumatic injury, or muscle degeneration. Preferably, the therapeutically effective amount improves muscle strength and muscle function.

As used herein, the terms "subject", "individual" and "patient" are used interchangeably herein, including humans and other non-human primates such as chimpanzees and other apes and monkey species. Primates; livestock such as cows, sheep, pigs, goats and horses; domestic mammals such as dogs and cats; rodents such as mice, rats, rabbits, hamsters and guinea pigs; chickens, chimpanzees and other poultry birds, ducks, Refers to any vertebrate subject, including, but not limited to, breeding, wild and hunting (game) birds such as geese and others. In some cases, the methods of the present disclosure find use in laboratory animals, veterinary applications, and the development of animal models for diseases. This term does not indicate a particular age. Therefore, it is intended to cover both adult and neonatal individuals.

II. METHODS: Before describing this disclosure in detail, it should be understood that the present disclosure is not limited to such particular formulation or process parameters, as the particular formulation or process parameters may of course vary. It should also be understood that the terminology used herein is intended to describe only certain embodiments of the present disclosure and is not intended to be limiting.

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

The present disclosure relates to aged cells and tissues, for example, by transient overexpression of mRNA that affects mitochondrial function, proteolytic activity, heterochromatin levels, histone methylation, nuclear lamina polypeptides, cytokine secretion or aging. It's about how to rejuvenate and restore functionality. In particular, we present fibroblasts, endothelial cells, chondrocytes and skeletal muscle while the mRNAs encoding OCT4, SOX2, KLF4, c-MYC, LIN28 and NANOG retain the cells in a differentiated cellular state. It has been shown that it can be used to rejuvenate various cell types, including stem cells.

For 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.

a. Cell Rejuvenation In one embodiment, a method of rejuvenating a cell, the cell is transfected with one or more non-integrated messenger RNAs encoding one or more cell reprogramming factors for up to 5 consecutive days. However, this provides a method herein comprising the step of producing rejuvenated cells.

In embodiments, the rejuvenated cells have a phenotype or activity profile similar to that of young cells. The phenotype or activity profile is one or more of the 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. include.

In embodiments, the rejuvenated cells have a transcriptome profile similar to the transcriptome profile of young cells. In embodiments, the transcriptome profile of rejuvenated cells is the gene for one or more genes selected from RPL37, RHOA, SRSF3, EPHB4, ARHGAP18, RPL31, FKBP2, MAP1LC3B2, Elf1, Phf8, Pol2s2, Taf1 and Sin3a. Includes increased expression. In embodiments, the transcriptome profile of rejuvenated cells comprises increased gene expression of RPL37. In embodiments, the transcriptome profile of rejuvenated cells comprises increased expression of the RHOA gene. In embodiments, the transcriptome profile of the rejuvenated cells comprises increased gene expression of SRSF3. In embodiments, the transcriptome profile of the rejuvenated cells comprises increased expression of the EPHB4 gene. In embodiments, the transcriptome profile of rejuvenated cells comprises increased gene expression of ARHGAP18. In embodiments, the transcriptome profile of rejuvenated cells comprises increased gene expression of RPL31. In embodiments, the transcriptome profile of rejuvenated cells comprises increased FKBP2 gene expression. In embodiments, the transcriptome profile of the rejuvenated cells comprises increased gene expression of MAP1LC3B2. In embodiments, the transcriptome profile of the rejuvenated cells comprises increased expression of the Elf1 gene. In embodiments, the transcriptome profile of the rejuvenated cells comprises increased expression of the Phf8 gene. In embodiments, the transcriptome profile of the rejuvenated cells comprises increased expression of the Pol2s2 gene. In embodiments, the transcriptome profile of the rejuvenated cells comprises increased expression of the Taf1 gene. In embodiments, the transcriptome profile of rejuvenated cells comprises increased Sin3a gene expression. In embodiments, the transcriptome profile of rejuvenated cells comprises increased gene expression of RPL37, RHOA, SRSF3, EPHB4, ARHGAP18, RPL31, FKBP2, MAP1LC3B2, Elf1, Phf8, Pol2s2, Taf1 and Sin3a.

In embodiments, rejuvenated cells exhibit increased gene expression of one or more nuclei and / or epigenetic markers as compared to reference values. In embodiments, one or more nuclear and / or epigenetic markers are selected from HP1 gamma, H3K9me3, lamina support protein LAP2alpha and SIRT1 protein. In embodiments, rejuvenated cells exhibit increased gene expression of HP1 gamma. In embodiments, rejuvenated cells exhibit increased gene expression of H3K9me3. In embodiments, rejuvenated cells exhibit increased gene expression of the lamina-supporting protein LAP2alpha. In embodiments, rejuvenated cells exhibit increased gene expression of SIRT1 protein. In embodiments, rejuvenated cells exhibit increased gene expression of HP1 gamma, H3K9me3, lamina-supporting protein LAP2alpha and SIRT1 protein.

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

In embodiments, rejuvenated cells exhibit improved mitochondrial health and function as compared to reference values. In embodiments, improved mitochondrial health and function are measured as increased mitochondrial membrane potential, decreased reactive oxygen species (ROS), or a combination thereof. In embodiments, improved mitochondrial health and function are measured as increased mitochondrial membrane potential. In embodiments, improved mitochondrial health and function is measured as reduced reactive oxygen species (ROS). In embodiments, improved mitochondrial health and function are measured as increased mitochondrial membrane potential and decreased reactive oxygen species (ROS).

In embodiments, rejuvenated cells exhibit reduced expression of one or more SASP cytokines as compared to reference values. In embodiments, one or more SASP cytokines include IL18, IL1A, GROA, IL22 and IL9. In embodiments, rejuvenated cells exhibit reduced expression of IL18. In embodiments, rejuvenated cells exhibit reduced expression of IL1A. In embodiments, rejuvenated cells exhibit reduced expression of GROA. In embodiments, rejuvenated cells exhibit reduced expression of IL22. In embodiments, rejuvenated cells exhibit reduced expression of IL9. In embodiments, rejuvenated cells exhibit reduced expression of IL18, IL1A, GROA, IL22 and IL9.

In embodiments, rejuvenated cells exhibit a reversal of the methylated landscape. In embodiments, the inversion of the methylated landscape is measured by estimation with a Horvath clock.

In embodiments, reference values are obtained from aged cells.

In embodiments, cells are rejuvenated by transient reprogramming with mRNA encoding one or more cell reprogramming factors. Transient reprogramming is achieved by transfecting cells once daily with non-integrated mRNA for at least 2 and 5 days or less. "Non-integration" means that the mRNA molecule is such that reprogramming is transient and does not disrupt the identity of the rejuvenated cell (ie, the cell retains its ability to differentiate into its adult cell type). Means that they have not been integrated into or out of the chromosome into the host genome and have not been integrated into the vector. In embodiments, transient reprogramming of cells eliminates various features of aging while avoiding complete dedifferentiation of cells into stem cells.

In embodiments, the steps of transfecting cells with messenger RNA include lipofectamine and LT-1-mediated transfection, dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, electroporation, encapsulation of mRNA in liposomes, and direct. It can be achieved by a transfection method selected from microinjection. In embodiments, the step of transfecting cells with messenger RNA can be accomplished by lipofectamine and LT-1-mediated transfection. In embodiments, the step of transfecting cells with messenger RNA can be accomplished by dextran-mediated transfection. In embodiments, the step of transfecting cells with messenger RNA can be accomplished by calcium phosphate precipitation. In embodiments, the step of transfecting cells with messenger RNA can be accomplished by polybrene-mediated transfection. In embodiments, the step of transfecting cells with messenger RNA can be accomplished by electroporation. In embodiments, the step of transfecting a cell with messenger RNA can be accomplished by encapsulation of the mRNA in liposomes. In embodiments, the step of transfecting cells with messenger RNA can be accomplished by direct microinjection.

Aging reversal or rejuvenation of cells is achieved by transient overexpression of one or more mRNAs encoding cell reprogramming factors. Such cellular reprogramming factors are transcription factors, epigenetic remodeling factors or small molecules that affect mitochondrial function, proteolytic activity, heterochromatin levels, histone methylation, nuclear lamina polypeptides, cytokine secretion or aging. Can contain molecules. In embodiments, the cell reprogramming factor comprises one or more of OCT4, SOX2, KLF4, c-MYC, LIN28 and NANOG. In another embodiment, cell reprogramming factors include OCT4, SOX2, KLF4, c-MYC, LIN28 and NANOG. In certain embodiments, the cell reprogramming factor consists of OCT4, SOX2, KLF4, c-MYC, LIN28 and NANOG.

In embodiments, the methods provided herein can be applied to any type of cell that requires rejuvenation. Cells that are mixed with other cells in culture or in tissues (partially or intact) or living organisms can be isolated from other cells. The methods described herein can be performed, for example, on a sample containing a single cell, a population of cells, or a tissue or organ containing cells. The cells selected for rejuvenation depend on the desired therapeutic effect on the treatment of age-related diseases or conditions.

In embodiments, the cells are mammalian cells. In an embodiment, the cell is a human cell. In embodiments, the cells are derived from an elderly subject.

In embodiments, the methods provided herein are nervous system, muscular system, respiratory system, cardiovascular system, skeletal system, reproductive system, integumentary system, lymphatic system, excretory system, endocrine system (eg, endocrine and endocrine system). It can be done in exocrine) or in cells, tissues or organs of the digestive system. These include epithelial cells (eg, squamous epithelial, cubic, columnar and pseudo-layered epithelial cells), endothelial cells (eg, vein, arterial and lymphatic endothelial cells), and connective tissue, muscle and nervous system cells. Any type of cell, including, but not limited to, may potentially rejuvenate as described herein. Such cells include epidermal cells, fibroblasts, cartilage cells, skeletal muscle cells, satellite cells, myocardial cells, smooth muscle cells, keratinocytes, basal cells, enamel blast cells, exocrine secretory cells, myoepithelial cells, osteoblasts. , Osteolithic cells, neurons (eg, sensory neurons, motor neurons and intervening neurons), glial cells (eg, oligodendrocytes, astrosites, coat cells, microglia, Schwan cells and satellite cells), pillar cells, fat cells, Perisite, stellate cells, lung cells, blood and immune system cells (eg, erythrocytes, monospheres, dendritic cells, macrophages, neutrophils, eosinophils, mast cells, T cells, B cells, natural killer cells) , Hormone-secreting cells, germ cells, stromal cells, crystalline cells, photoreceptor cells, taste receptor cells and olfactory cells; as well as kidney, liver, pancreas, stomach, spleen, bile sac, intestine, bladder, lung, prostate, breast , Urogenital tract, pituitary cells, oral cavity, esophagus, skin, hair, nails, thyroid gland, parathyroid gland, adrenal gland, eyes, nose or brain-derived cells and / or tissues.

In some embodiments, the cells are 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 embodiments, the cells are endothelial cells. In an embodiment, the cell is a chondrocyte. In embodiments, the cells are skeletal muscle stem cells. In embodiments, the cells are keratinocytes. In embodiments, the cells are mesenchymal stem cells. In an embodiment, the cell is a corneal epithelial cell.

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

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

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

In embodiments, rejuvenated skeletal muscle stem cells have higher proliferative capacity, enhanced ability to differentiate into myoblasts and myoblasts, lower, recovery of activation kinetics from rest, muscle microniche. Shows the ability to rejuvenate, restore youthful power in muscle, or a combination of these.

In embodiments, rejuvenated keratinocytes have higher proliferative capacity, reduced inflammatory phenotype, lower RNAKL and INOS2 expression, decreased expression of cytokines MIP1A, IL6, IFNa, MCP3, increased ATP, increased levels of SOD2. And COL2A1 expression.

In embodiments, rejuvenated mesenchymal stem cells exhibit decreased aging parameters, increased cell proliferation and / or decreased ROS levels. In embodiments, rejuvenated mesenchymal stem cells exhibit reduced aging parameters. In embodiments, aging parameters include p16 expression, p21 expression and positive SAβGal staining. In embodiments, rejuvenated mesenchymal stem cells show increased cell proliferation. In embodiments, rejuvenated mesenchymal stem cells show reduced ROS levels. In embodiments, rejuvenated mesenchymal stem cells exhibit decreased aging parameters, increased cell proliferation and decreased ROS levels.

In embodiments, rejuvenated corneal epithelial cells exhibit reduced aging parameters. In embodiments, the aging parameter comprises one or more of p21 expression, p16 expression, mitochondrial neoplastic PGC1α, and inflammatory factor IL8 expression. In embodiments, the aging parameter comprises p21. In embodiments, the aging parameter comprises expression of p16. In embodiments, the aging parameter comprises mitochondrial neoplasia PGC1α. In embodiments, the aging parameter comprises expression of the inflammatory factor IL8. In embodiments, the aging parameter comprises one or more of p21 expression, p16 expression, mitochondrial neoplastic PGC1α, and inflammatory factor IL8 expression.

The methods of the present disclosure can be used to rejuvenate cells in culture (eg, exvivo or in vitro) to improve function and differentiation potential for use in cell therapy. The cells used in the treatment of the patient may be autologous or allogeneic. Preferably, the cells are from the patient or the matched donor. For example, in Exvivo therapy, cells are obtained directly from the patient to be treated, transfected with mRNA encoding the cellular reprogramming factors described herein and transplanted back into the patient. Such cells can be obtained, for example, from a biopsy or surgical procedure performed on the patient. Instead, cells in need of rejuvenation may be directly transfected in vivo with mRNA encoding a cell reprogramming factor.

Transfection results in transient uptake of mRNA encoding a cell reprogramming factor into cells in need of rejuvenation (ie, for transient reprogramming), any of those known in the art. It can be done using a suitable method. In embodiments, methods for ex vivo, in vitro or in vivo delivery of mRNA to cells of interest include lipofectamine and LT-1-mediated transfection, dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, electroporation, liposomes. It can include encapsulation of mRNA in cells, direct microinjection of mRNA into cells, or a method selected from combinations thereof.

b. Composition In some embodiments, the cell comprises rejuvenated cells obtained by the step of transfecting one or more non-integrated messenger RNAs encoding one or more cell reprogramming factors for up to 5 consecutive days. Pharmaceutical compositions are provided herein.

In embodiments, the rejuvenated cells are autologous. In embodiments, the rejuvenated cells are allogeneic.

In embodiments, 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.

In embodiments, rejuvenated cells exhibit one or more of the following: increased expression of HP1 gamma, H3K9me3, LAP2alpha, SIRT1, increased mitochondrial membrane potential and decreased reactive oxygen species, and decreased SASP cytokines: Expression. In embodiments, the SASP cytokine comprises one or more of IL18, IL1A, GROA, IL22 and IL9.

In certain embodiments, compositions comprising rejuvenated cells for use in cell therapy include nutrients, cytokines, growth factors, extracellular matrix (ECM) components, antibiotics to improve cell function or viability. It can further include one or more additional factors such as substances, antioxidants or immunosuppressants. The composition may further comprise a pharmaceutically acceptable carrier.

Examples of growth factors include fibroblast growth factor (FGF), insulin-like growth factor (IGF), transforming growth factor beta (TGF-β), epiregulin, epithelial growth factor (“EGF”), endothelial cell growth factor (“EGF”). "ECGF"), nerve growth factor ("NGF"), leukemia suppressor ("LIF"), bone-forming protein-4 ("BMP-4"), hepatocellular growth factor ("HGF"), vascular endothelial growth factor -A ("VEGF-A") and cholecystinine octapeptides include, but are not limited to.

Examples of ECM components include proteoglycans (eg chondroitin sulfate, heparan sulfate and keratan sulfate), non-proteoglycan polysaccharides (eg hyaluronic acid), fibers (eg collagen and elastin) and other ECM components (eg fibronectin and laminin). However, it is not limited to these.

Examples of immunosuppressive agents include steroidal (eg, prednisone) or non-steroidal (eg, sirolimus (Rapamune, Wyeth-Ayerst Canda), tacrolimus (Prograf, Fujisawa Canda) and anti-IL2R daclizumab (Zenapax, Roche Cana)). Other immunosuppressive agents include, but are not limited to, 15-deoxyspagarin, cyclosporin, methotrexate, rapamycin, rapamune (sirolimus / rapamycin), FK506 or lithophylline (LSF).

It may contain one or more pharmaceutically acceptable excipients. Examples include, but are not limited to, carbohydrates, inorganic salts, antibacterial agents, antioxidants, surfactants, buffers, acids, bases, and combinations thereof.

For example, an antibacterial agent for preventing or blocking 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, thimerosal, and these. The combination of. Antibiotic agents also include antibiotics that can also be used to prevent bacterial infections. Examples of antibiotics include amoxicillin, penicillin, sulfa agents, cephalosporin, erythromycin, streptomycin, gentamicin, tetracycline, clarithromycin, ciprofloxacin, azithromycin and others. Also included are antifungal agents such as miconazole and terconazole.

Various antioxidants may be included, such as reduced glutathione (GSH) or precursors thereof, glutathione or glutathione analogs, glutathione monoesters, and molecules with thiol groups such as N-acetylcysteine. Other suitable antioxidants include superoxide dismutase, catalase, vitamin E, trolox, lipoic acid, lazaroid, butylatedhydroxyanisole (BHA), vitamin K and others.

Suitable excipients for injectable compositions include water, alcohols, polyols, glycerin, vegetable oils, phospholipids and surfactants. Derivatized sugars such as sugars, alditols and aldonic acids, esterified sugars and / or carbohydrates such as sugar polymers can be present as excipients. Specific carbohydrate excipients are, for example: monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, sorbitol and others; disaccharides such as lactose, sucrose, trehalose, cellobiose and others; raffinose, melesitose, etc. Polysaccharides such as maltodextrin, dextran, starch and others; and argitol such as mannitol, xylitol, martitol, lactitol, xylitol, sorbitol (glucitol), pyranosylsorbitol, myoinositol and others. Excipients 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.

Acids or bases can also be present as excipients. Non-limiting examples of acids that can be used include hydrochloric acid, acetic acid, phosphoric acid, citric acid, malic acid, lactic acid, formic acid, trichloroacetic acid, nitrate, perchloric acid, phosphoric acid, sulfuric acid, fumaric acid, and these. Acids selected from the group consisting of combinations of. Examples of suitable bases are sodium hydroxide, sodium acetate, ammonium hydroxide, potassium hydroxide, ammonium acetate, potassium acetate, sodium phosphate, potassium phosphate, sodium citrate, sodium formate, sodium sulfate, potassium sulfate, fumal. Includes, without limitation, potassium acid (potassium ferrate), and bases selected from the group consisting of combinations thereof.

Typically, the optimum amount of any individual excipient is prepared by routine experimental methods, i.e., to prepare a composition containing varying amounts of excipients (small to large), stability and stability. It is determined by testing other parameters and then determining the extent to which optimal results are achieved without significant adverse effects. However, in general, the excipient (s) are about 1% to about 99% by weight, preferably about 5% to about 98% by weight, more preferably about 15 to about 95% by weight. It is present in the composition in the amount of the excipient, and concentrations less than 30% by weight are most preferred. These aforementioned pharmaceutical excipients, along with other excipients, "Remington: The Science & Practice of Pharmacy" 19th Edition, Williams & Williams (1995), "Physician's Desk Reference" 52nd Edition, Montvale. NJ (1998) and Kibe, A. et al. H. , Handbook of Physical Excipients, 3rd Edition, American Pharmacistical Association, Washington, D. et al. C. , 2000.

c. Administration The methods of the present disclosure can be used to treat subjects with respect to age-related diseases or conditions. For example, cell therapies involving transient reprogramming of cells by transfection with non-integrated mRNAs encoding reprogramming factors (eg, in vitro, exvivo or in vivo) include neurodegenerative diseases (eg, Alzheimer's disease, Parkinson). Diseases, Huntington's disease, atrophic lateral sclerosis, dementia and stroke), cardiovascular and peripheral vascular diseases (eg, porphyritic arteriosclerosis, peripheral arterial disease (PAD), hematoma, calcification, thrombosis, embolism And aneurysms), eye diseases (eg, age-related yellow spot degeneration, glaucoma, cataracts, dry eyes, diabetic retinopathy, loss of vision), skin diseases (skin atrophy and thinning, elastic fibrosis) ) And skin wrinkles, sebaceous gland hyperplasia or hypoplasia, senile kuroko and other pigmentation abnormalities, gray hair, hair loss or thinning, and chronic skin ulcers), autoimmune disorders (eg, rheumatic polymuscular muscles) Pain (PMR), giant cell arteritis (GCA), rheumatoid arthritis (RA), crystalline arthritis and spondyloarthropathies (SPA)), endocrine and metabolic dysfunction (eg, adult pituitary dysfunction, thyroid function) Deterioration, aspirational thyroid poisoning, osteoporosis, true diabetes, adrenal insufficiency, various forms of gonad dysfunction and endocrine malignant lesions), musculoskeletal disorders (eg, arthritis, osteoporosis, myeloma, gout, Paget's disease, fractures) , Myelopathy syndrome, joint tonicity, diffuse idiopathic osteoproliferative disease, hematogenous myelitis, muscle atrophy, peripheral neuropathy, multiple sclerosis, myortic lateral sclerosis (ALS), Duchenne-type muscular dystrophy, primary Lateral sclerosis and severe myasthenia), gastrointestinal disorders (eg liver cirrhosis, liver fibrosis, Barrett esophagus), respiratory disorders (eg pulmonary fibrosis, chronic obstructive pulmonary disease, asthma, chronic bronchitis) , Pulmonary embolism (PE), lung cancer and infectious diseases), and any other age-related diseases and disorders, including, but not limited to, treatment of subjects relating to various age-related diseases and conditions. Can be used.

In some embodiments, methods for treating an age-related disease or condition, cartilage degenerative disorder, neurodegenerative disorder and / or musculoskeletal dysfunction of a subject are provided herein. The method comprises administering a therapeutically effective amount of cells containing one or more non-integrated messenger RNAs encoding one or more cell reprogramming factors.

At least one therapeutically effective treatment cycle by transfection with one or more non-integrated messenger RNAs encoding one or more cell reprogramming factors may be administered to a subject for the treatment of age-related diseases or conditions. ..

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

In embodiments, the subject has a cartilage degeneration disorder. In embodiments, the disorder is selected from arthritis, achondroplasia, spondyloarthropathies, ankylosing spondylitis, erythematosus, relapsing polychondritis and Sjogren's syndrome. In embodiments, the disorder is arthritis. In embodiments, the disorder is achondroplasia. In embodiments, the disorder is spondyloarthropathies. 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.

In embodiments, treatment reduces 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 is measured by one or more of increased ATP levels, decreased ROS and increased SOD2, increased COL2A1, and overall proliferation by chondrocytes.

In embodiments, treatment of a subject with cells rejuvenated by Exvivo or in vitro transfection in cell culture, a composition for transplanting rejuvenated cells, typically injects into an area requiring tissue regeneration or repair. Alternatively, it is administered by surgical implantation, but this is not always the case.

In embodiments, therapeutically effective amounts of rejuvenated cells are selected from fibroblasts, endothelial cells, chondrocytes, skeletal muscle stem cells, keratinocytes, mesenchymal stem cells and corneal epithelial cells. In embodiments, the therapeutically effective amount of rejuvenated cells is fibroblasts. In embodiments, the therapeutically effective amount of rejuvenated cells is endothelial cells. In embodiments, the therapeutically effective amount of rejuvenated cells is chondrocytes. In embodiments, the therapeutically effective amount of rejuvenated cells is skeletal muscle stem cells. In embodiments, the therapeutically effective amount of rejuvenated cells is keratinocytes. In embodiments, the therapeutically effective amount of rejuvenated cells is mesenchymal stem cells. In embodiments, the therapeutically effective amount of rejuvenated cells are corneal epithelial cells.

In embodiments, the rejuvenated corneal epithelium exhibits reduced aging parameters. In embodiments, the aging parameter comprises one or more of the expression of p21 and p16, the mitochondrial neoplasia PGC1α, and the inflammatory factor IL8.

In one embodiment, the chondrocytes in the area of cartilage damage or loss encode one or more cell reprogramming factors sufficient to result in rejuvenation of the chondrocytes and generation of new cartilage at the treatment site. An effective amount of the above non-integrated messenger RNA is transfected in vivo. Instead, rejuvenated chondrocytes produced by Exvivo or in vitro transfection can be administered topically to areas of cartilage damage or loss, such as the subject's damaged joint or other suitable treatment site. A therapeutically effective dose or amount of rejuvenated chondrocytes is an amount that provides a positive therapeutic response to a subject with cartilage damage or loss, such as an amount that results in the production of new cartilage at the treatment site (eg, damaged joint). Intended. For example, a therapeutically effective dose or amount may be used to treat degenerative diseases such as cartilage damage or loss due to traumatic injury, or other diseases associated with arthritis or cartilage degeneration. Preferably, the therapeutically effective amount restores function and / or reduces pain and inflammation associated with cartilage damage or loss.

In another embodiment, the skeletal muscle stem cells are sufficient to bring about the rejuvenation (ie, regaining potency) of the skeletal muscle stem cells and the production of new muscle fibers at the treatment site (eg, damaged muscle). An effective amount of one or more non-integrated messenger RNAs encoding a cell reprogramming factor of one or more species is transfected in vivo. Instead, rejuvenated skeletal muscle stem cells produced by Exvivo or in vitro transfection can be administered topically to damaged muscle in need of repair or regeneration. For example, a therapeutically effective dose or amount may be used to treat a disease or disorder involving muscle damage or loss, muscle atrophy, or muscle degeneration due to traumatic injury. A therapeutically effective dose or amount of rejuvenated skeletal muscle stem cells provides a positive therapeutic response to a subject with muscle damage or loss, such as an amount that results in the production of new muscle fibers at the treatment site (eg, damaged muscle). Intended quantity. Preferably, the therapeutically effective amount improves muscle strength and function, reduces pain, improves stamina, and / or increases mobility.

In some embodiments, as described herein, methods for treating a subject's age-related disease or condition, cartilage degenerative disorders, and / or a subject with musculoskeletal dysfunction are described herein. Provided to. The method comprises administering a therapeutically effective amount of one or more non-integrated messenger RNAs encoding one or more cell reprogramming factors, as described herein.

In embodiments, cells in a subject are rejuvenated by in vivo transfection with an effective amount of one or more non-integrated messenger RNAs encoding one or more cell reprogramming factors, as described herein. Can be made.

In some embodiments, a method of rejuvenating an engineered tissue with Exvivo is provided herein. The method transfects tissue with one or more non-integrated messenger RNAs encoding one or more cellular reprogramming factors for up to 5 consecutive days, thereby rejuvenating the engineered tissue. Includes steps to produce.

In embodiments, the engineered tissue exhibits aging parameters, decreased pro-inflammatory factors, improved histological scores, or a combination thereof. In embodiments, the manipulated tissue exhibits one or more reductions in aging parameters. In embodiments, the aging parameter is selected from p16 expression, positive SAβGal staining, and pro-inflammatory factors IL8 and MMP1 expression. In embodiments, the engineered tissue exhibits reduced p16 expression. In embodiments, the engineered tissue exhibits reduced positive SAβGal staining. In embodiments, the engineered tissue exhibits reduced expression of pro-inflammatory factors IL8 and MMP1. In embodiments, the engineered tissue exhibits an improvement in histological score. In embodiments, histological scores include morphology, organization and / or quality.

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

d. Kit The present disclosure also comprises one or more containers holding a composition comprising one or more non-integrated messenger RNAs encoding one or more cell reprogramming factors for transient reprogramming of cells. Provide a kit containing. The kit can further include 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 and / or other composition encoding the cell reprogramming factor may be in liquid form or lyophilized. Such kits can also contain components that preserve or maintain mRNA, which protects it from degradation. Such components may be RNAse-free or can be protected from RNAses. Suitable containers for the composition include, for example, bottles, vials, syringes and test tubes. The container may be made of various materials, including glass or plastic. The container can have a sterile access port (eg, the container may be an intravenous solution bag or vial with a stopper that can be penetrated with a hypodermic needle).

The kit may further include a second container containing a pharmaceutically acceptable buffer such as phosphate buffered saline, Ringer's solution or dextrose solution. It 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.

The kit may also include a package insert containing written instructions for how to treat age-related diseases or conditions. The package insert may be an unapproved draft package insert, or it may be a package insert approved by the Food and Drug administration (FDA) or other regulatory bodies.

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 OCT4, SOX2, KLF4, c-MYC, LIN28 and NANOG cell reprogramming factors.

III. Below are examples of specific embodiments for carrying out the present disclosure. The examples are provided for illustration purposes only and are by no means intended to limit the scope of this disclosure.

Attempts have been made to ensure accuracy with respect to the numbers used (eg, quantity, temperature, etc.), but of course some experimental error and deviation should be tolerated.

The examples and embodiments described herein are for purposes of illustration only, and various modifications or modifications in consideration thereof are suggested to those skilled in the art, and the main purpose of the present application and the accompanying claims and the present invention. It is understood that it should be included in the category. All publications, patents and patent applications cited herein are hereby incorporated by reference in their entirety for any purpose.

[Example 1]: Transient and non-integrated cell reprogramming promotes multifaceted reversal of aging The experiments described herein are stopped before cell uniqueness is irreversibly lost. Depicts the degree of aging reversal effect that can be achieved by a transient reprogramming protocol. Recent evidence has also shown that partially transgenic reprogramming can ameliorate age-related features and prolong lifespan in progeria mice. However, it is unclear how this form of "epigenetic rejuvenation" is widely applied to natural aging and, importantly, how it can be safely bridged to human cells. .. The data herein indicate that transient reprogramming based on mRNA technology reverses the physiologic aging characteristics and reduces the age-related disease phenotype in somatic and stem cells obtained from human clinical samples. , Indicates that the regenerative response, which has been reduced with age, is restored. The non-incorporated methods of transient cell reprogramming described herein are for the treatment of exvivo cell rejuvenation aimed at regenerative medicine, as well as the physiological decay of natural aging and the pathology of age-related diseases. It paves the way for new, more bridging strategies for in vivo tissue rejuvenation therapy that delays or reverses development.

To test whether any substantial and measurable reprogramming of cell age can be achieved prior to the irreversible limit, and whether this can result in amelioration of either cellular or physiological function. Evaluate the effect of transient reprogramming on the aging physiological function of fibroblasts and endothelial cells, two distinct cell types derived from otherwise healthy human subjects, and harvested from young donors. Compared with the same cell type. Fibroblasts were derived from chopped arm and abdominal skin biopsies (young control 25-35 years, n = 3, and aging group 60-70 years, n = 3), while endothelial cells. Extracted from collagenase digestion of iliac veins and arteries (young control 15-25 years, n = 3, and aging group 45-50 years, n = 3).

We used a non-embedded reprogramming protocol. The protocol was optimized based on a cocktail of mRNAs expressing OCT4, SOX2, KLF4, c-MYC, LIN28 and NANOG (OSKMLN). Exploring multiple reprogramming durations revealed that both cell types showed rapid changes in many aging parameters as early as R2X2 (two reprogramming transfections and two days of relaxation to return to the ground state). Although presented, the most prominent effect was seen in R4X2 (FIGS. 1A and 2A). The protocol consistently produces induced pluripotent stem cell (iPSC) colonies after 12 to 15 daily transfections, regardless of donor age; Applicants are endogenous pluripotency-related lncRNAs. Based on the observation that the first detectable expression of is occurring on day 5, we conclude that the PNR on our platform occurs on about 5 days of reprogramming. Therefore, a transient reprogramming protocol was adopted in which OSKMLN was transfected daily for 4 consecutive days, and gene expression analysis was performed 2 days after discontinuation.

Paired end bulk RNA sequencing was performed on both cell types for the same three cohorts: young (Y), untreated / aging (UA) and treated / aging (TA). First, we compared juvenile and untreated / aged cell quantile-normalized transcriptomes (“Y vs. UA”) by cell type. Data include 961 genes (5.85%) in fibroblasts (678 upregulated, 289 downregulated) and 748 genes (4.80%) (389 up) in endothelial cells. Modulation (377 downregulation) was shown to be different between young and aged cells with a significance criterion p <0.05 and a logarithmic multiple change cutoff +/- 0.5 (FIG. 6). ). These gene sets were enriched in many of the known aging pathways identified in the characteristic gene set collections of the molecular signature database. When the direction of expression above or below the average of each gene is mapped, there is a clear similarity between treatment and young cells for both fibroblasts and endothelial cells, as opposed to aged cells. Was observed. Principal component analysis (PCA) in this gene set space determined that young and aging populations were separable along the first principal component (PC1), which is a fibroblast. 64.8% of the dispersion in cells and 60.9% of the dispersion in endothelial cells were explained. Intriguingly, the treated cells also clustered along PC1 closer to the younger population (FIGS. 1K and 2J).

When comparing untreated and treated-aged populations (“UA vs. TA”) using the same significance criteria defined above, 1042 genes in fibroblasts (734 upregulated, It was found that 308 species were downwardly regulated) and 992 species in endothelial cells (461 species were upwardly regulated and 531 species were downwardly regulated) were differentially expressed. Interestingly, even within these gene sets, Applicants have found enrichment of the aging pathway in the molecular signature database. Profiles of each cell type When comparing young vs. untreated / aging (“Y vs. UA”) and untreated / aging vs. aging (“UA vs. TA”), 24.7 of fibroblasts % Duplication (odds ratio 4.53, p <0.05) and 16.7% duplication of endothelial cells (odds ratio 3.84, p <0.05) were observed, and the direction of changes in gene expression was Matched that of younger ones (ie, higher in younger age, higher in treatment / aging); less than 0.5% migrated reversely in any cell type.

These transcriptome profiles were then used to verify retention of cell uniqueness after transient reprogramming. To this end, using established cell-specific markers, Applicants validated that no significant changes were made with treatment (FIG. 10). In addition, Applicants were unable to detect the expression of any pluripotency-related marker (other than the transfected OSKMLN mRNA) (FIG. 10). In short, analysis of transcriptome signatures revealed that transient reprogramming induces a more youthful gene expression profile while preserving cell uniqueness.

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 states, including longevity. Exogenous expression of canonical reprogramming factors (OSKM) is known to restore the epigenetic age of primary cells to their prenatal state. Two epigenetic clocks applied to human fibroblasts and endothelial cells were used to examine whether transient expression of OSKMLN could reverse the epigenetic clock: Horvas' original pan-tissue. Epigenetic clocks (based on 353 cytosine-phosphate-guanine pairs) and skin & blood clocks (based on 391 CpG).

According to the pan-tissue epigenetic clock, transient OSKMLN significantly restored the DNA methylation age (bilateral mixed effect model P value = 0.023) (mean age difference = -3.40 years, standard error 1). .17). The rejuvenating effect is on endothelial cells (mean age difference = -4.94 years, SE = 1.63, FIG. 6H) and fibroblasts (mean age difference = -1.84, SE = 1.46, FIG. 6G). It was more prominent than in. Using a skin and blood epigenetic clock, qualitatively similar but less significant results could be obtained (overall rejuvenation effect-1.35 years, SE = 0.67, unilateral mixture). Effect model P value = 0.042, average rejuvenation in endothelial cells and fibroblasts is -1.62 years and -1.07, respectively).

Inspired by these results, we analyzed the effects of transient reprogramming on various features of cell-physiological aging. Quantitative changes and distribution of single cells throughout the cell population were performed using a panel of 11 established assays that span the characteristics of aging (Figure 11) and most of the analysis was performed using single cell high-throughput imaging. Captured the shift. All analyzes were performed separately for each individual cell line (19 fibroblast lines in total: 3 young, 8 aging and 8 treatments / aging; 17 endothelial cells in total). Strains: 3 types of young age, 7 types of aging and 7 types of treatment / aging). Statistical analysis was performed on each of the paired sample sets; data were subsequently pooled by age category for ease of display (see Materials and Methods for a detailed description of the statistical methods used). .. Control experiments were performed by adopting the same transfection scheme using mRNA encoding GFP.

To extend our knowledge of epigenetics, we conducted experiments to quantitatively measure epigenetic suppression marks H3K9me3, heterochromatin-related protein HP1γ and nuclear lamina-supporting protein LAP2α by immunofluorescence (IF) (Fig. 1B, FIG. 1B). 1C, 2B, 2C, 5A-5C). Aged fibroblasts and endothelial cells showed reduced 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. Both pathways involved in cellular proteolytic activity were then tested by measuring autophagosome formation and chymotrypsin-like proteasome activity, which decreases with age. Treatment increases both pathways to levels similar to or even higher than that of young cells, suggesting that early steps in reprogramming promote active clearance of degraded biomolecules. (FIGS. 1D, 2D, 5G-5H).

In terms of energy metabolism, aged cells exhibit reduced mitochondrial activity, reactive oxygen species (ROS) accumulation, and deregulated nutrient sensing. Therefore, by measuring mitochondrial membrane potential, mitochondrial ROS, and sirtuin 1 protein (SIRT1) levels in cells, the effect of treatment on aged cells was examined. Transient reprogramming increased mitochondrial membrane potential in both cell types (FIG. 1E left panel, FIG. 2E left panel, FIG. 5E), which is a juvenile cell (FIGS. 1F, 2F and 5D). ), ROS were decreased (FIG. 1E right panel, FIG. 2E right panel, FIG. 5F) and SIRT1 protein levels were increased in fibroblasts. Aging-related beta-galactosidase staining showed a significant reduction in the number of aged cells in aged endothelial cells (FIGS. 1H, 2H and 5I). This decrease was also associated with a decrease in pro-inflammatory cellular senescence-related secretory phenomenon (SASP) cytokines in endothelial cells (FIG. 5J). Finally, in both cell types, telomere length measured by quantitative fluorescent in situ hybridization did not show significant elongation by treatment (FIGS. 1G and 2G), and the cells were reactivated with telomerase activity. It suggests that it did not dedifferentiate into a stem cell-like state.

Next, when the persistence of these effects was evaluated, it was found that most of them were significantly retained 4 and 6 days after the interruption of reprogramming. By repeating the same set of experiments on fibroblasts and endothelial cells transfected for only two consecutive days, we investigated how rapidly these physiological rejuvenation changes manifested. Notably, the data showed that most of the rejuvenation effects were already visible after two days of treatment, although most were more modest.

Taken together, this data demonstrates that transient expression of OSKMLN can induce a rapid persistent reversal of cell age in human cells at the transcriptome, epigenetic and cellular levels. Importantly, these data are from the process of "cell rejuvenation", named epigenetic reprogramming or "ERA" herein, very early and rapidly in the iPSC reprogramming process. Demonstrate involvement in. These epigenetic and transcriptional changes occur before any epigenetic reprogramming of cell uniqueness takes place.

Experiments were conducted to investigate whether ERA can also reverse the inflammatory phenotype associated with aging with these signs of the beneficial effects of ERA on cell aging. After obtaining preliminary evidence of this reversal in endothelial cells (Fig. 5J), osteoarthritis is a disease that is strongly associated with aging and is characterized by a prominent inflammatory spectrum affecting chondrocytes in the joints. The analysis was extended to. Chondrocytes were isolated from the cartilage of patients aged 60 to 70 years who underwent total artificial joint replacement surgery for their advanced stage OA, and the treatment results were compared with the cartilage cells isolated from young individuals. Transient reprogramming was performed for 2 or 3 days and analysis was performed 2 days after reprogramming interruption, but a more consistent effect across patients was achieved by longer treatment. Treatment showed a significant reduction in intracellular mRNA levels of pro-inflammatory cytokines (FIG. 7I), RANKL and iNOS2, as well as levels of inflammatory factors secreted by the cells (FIGS. 3H-3I and 7I). In addition, ERA has been shown to promote cell proliferation (FIGS. 3A and 7D), increase ATP production (FIGS. 3C and 7A), and be downregulated in reduced mitochondrial RNA and OA. Oxidative stress was reduced (FIGS. 3D, 3E, 7B and 7D), as evidenced by the elevated RNA levels of the antioxidant SOD2. ERA does not affect the expression level of SOX9 (a transcription factor central to chondrocyte specificity and function) and significantly increases the expression level of COL2A1 (major collagen in articular cartilage) (FIGS. 3B, 7E). And qRT-PCR in FIG. 7F), suggesting retention of chondrogenic cell uniqueness. Taken together, these results indicate that transient expression of OSKMLN can promote partial reversal of gene expression and cell physiology in aged OA chondrocytes towards a healthier state.

Loss of stem cells in function and regenerative capacity represents another important feature of aging. Experiments were conducted to evaluate the effect of transient reprogramming on age-related changes in somatic stem cells that impair regeneration. First, the effect of transient reprogramming was examined in mouse-derived skeletal muscle stem cells (MuSC). MuSC was treated for 2 days using an artificial niche while remaining stationary. Initial experiments were performed using young (3 months) and aged (20-24 months) mouse MuSCs isolated by FACS. Treatment of aging MuSC reduced both the time of initial division close to the faster activation kinetics of quiescent young MuSC, and mitochondrial mass. In addition, the treatment partially rescued the reduced ability of a single MuSC to form a colony. Upon further culture of such cells, the data showed that the treatment did not alter the expression of the myogenic marker MyoD, but instead improved its ability to differentiate into myotubes. It suggests that transient reprogramming does not destroy myogenic fate, but can enhance myogenic potential.

Next, the MuSC function and differentiation potential to regenerate new tissue in vivo were examined. To do this, young, aged or transiently reprogrammed aged MuSCs are transduced with a lentivirus expressing luciferase and green fluorescent protein (GFP), followed by injury to immunocompromised mice. The cells were transplanted into the tibialis anterior (TA) muscle. Long-axis bioluminescence imaging (BLI) showed the highest signal in early treatment-aged MuSC-implanted muscles (4th day, FIG. 4B), but by 11th day after transplantation, it was young. It was shown to be comparable to the muscles with age MuSC; conversely, the untreated and age MuSC muscles showed a lower signal at all time points after transplantation (FIG. 4B). Immunofluorescence analysis further revealed (GFP ⁺ ) muscle fibers from a higher number of donors in TAs transplanted with treated MuSCs compared to untreated and aged MuSCs (FIG. 4C). In addition, treated / aged cell-derived GFP ⁺ muscle fibers showed an increased cross-sectional area compared to their untreated counterpart, and in fact were even larger than the young control (FIG. 4D). ). Taken together, these results suggest an improved tissue regeneration capacity of transiently reprogrammed age-related MuSCs. Three months later, all mice were autopsied and no neoplastic lesions or teratomas were found.

To test for potential long-term benefits of treatment, a second injury was induced 60 days after cell transplantation, and again, the data show that the TA muscles transplanted with transiently reprogrammed age-related MuSC. It was shown that a higher BLI signal was produced (Fig. 4E).

Sarcopenia is an aging condition characterized by loss of muscle mass and force production. Similarly, in mice, muscle function exhibits progressive degeneration with age. Young (4 months) or aging (27 months) to test whether transient reprogramming of aging MuSC improves cell-based procedures in the recovery of muscular function in older mice. ) Electrophysiological tests were performed to measure the production of tetanus in TA muscles isolated from immunocompromised mice. The data show that TA muscles from aged mice have lower tetanus compared to young mice, suggesting age-related loss of force production (FIG. 8F). Next, MuSC was isolated from aged mice (20-24 months). After treatment with age-related MuSC, cells were transplanted into TA muscle with cardiotoxic injury in aged (27 months) immunocompromised mice. Thirty (30) days were allowed to give the transplanted muscle sufficient time to regenerate. An electrophysiological test was performed to measure the production of tetanus. Muscles transplanted with untreated and aged MuSC showed comparable strength to non-transplanted muscles derived from age-controlled mice (Fig. 4h). Conversely, treated / aged MuSC-treated muscles showed tetanus power comparable to untransplanted muscles from young control mice. These results support that transient reprogramming in combination with MuSC-based therapies can restore the physiological function of aged muscles to that of youthful muscles.

Finally, these results were bridged to human MuSC. Surgical samples from patients of different age ranges (10-80 years) were transduced with GFP and luciferase-expressing lentiviral vectors into them and the test was repeated. Similar to mice, transplanted transiently reprogrammed aged human MuSCs yield increased BLI signals compared to untreated MuSCs from the same individual, to those observed in young MuSCs. It was comparable (Fig. 8D). Interestingly, the BLI signal ratio between contralateral muscles with treated and untreated MuSCs was in the older age group (60-80 years) and in the younger age group (10-30 or 30-55 years). ), Which suggests that ERA restores lost function in aged cells to younger levels (FIG. 8E). Taken together, these results show that transient reprogramming partially restores the differentiation potential of aging MuSCs to a degree similar to that of young MuSCs, thus regenerative medicine. It suggests that it has potential as a cell therapy in.

Fibroblasts and keratinocytes from patients over 65 years of age were combined to reconstruct three-dimensional (3D) in vitro manipulated skin and transfected by adding a cocktail of reprogramming factors to culture medium. Histological analysis was performed to assess quality and numerical scores were assigned (Fig. 8A). Rejuvenation was observed with reprogramming factors, as measured by the increased numerical scores compared to the control untreated and retinoic acid treated samples.

Retinal epithelial cells were cultured in Exvivo and transiently reprogrammed with OSKMN for 2 or 3 days. The results showed a significant reduction in expression of p16 (FIG. 9A), p21 (FIG. 9B), IL8 (FIG. 9C) and PGC1a (FIG. 9D).

Nuclear reprogramming into induced pluripotent stem cells (iPSCs) is a polyphasic process involving induction, maturation and stabilization. After the completion of such dynamic and complex "epigenetic reprogramming", iPSCs are not only pluripotent but also youthful. The data herein are non-incorporated, mRNA-based platforms of transient cell reprogramming that very rapidly reverse the characteristics of aging in the evoked phase in which cell-specific epigenetic erasure has not yet occurred. Demonstrated that it can be done. The data show that the process of rejuvenation occurs in aged human cells by restoring lost functionality in affected and aged stem cells while preserving cell uniqueness.

[Example 2]: Method
mRNA Transfection : Using the manufacturer's protocol, mRNA-In (mTI Global Stem) for fibroblasts and chondrocytes that reduce cytotoxicity, as well as endothelial cells and MuSCs that are more difficult to transfect. Cells were transfected using any of the lipofectamine MessengerMax (Thermo Fisher). Culture medium was exchanged for fibroblasts and endothelial cells 4 hours after transfection but not for chondrocytes or MuSC as overnight incubation is required for the production of significant uptake of mRNA. rice field. Both GFP mRNA and immunostaining of individual factors in the OSKMNL cocktail confirmed the efficiency of delivery. MRNA synthesis and transfection optimization were performed at the facility at ESI BIO, where he advises, along with Jens Durlution-Durruthy, who is also a member of the Sebastiano Lab.

Fibroblast isolation and culture : Isolation in healthy patients, using a 2 mm punch biopsy from a mix of male and female patients in their 60s and 70s (aged) and 30s and 40s (young) Biopsy was performed from the mesial surface or abdomen of the mid-upper arm. Cells were cultured from these explants and maintained in Eagle's minimal essential medium supplemented with non-essential amino acids, 10% fetal bovine serum and 1% penicillin / streptomycin.

Endothelial Cell Isolation and Culture : Death from sudden head trauma in the 45s and 50s (aged) and teens (young) at the Coriell Institute, but before death from a donor who was otherwise healthy Isolation was performed from the removed iliac arteries and veins. Tissues were digested with collagenase and cells released from the lumen were used to initiate culture. 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.

Nuclear immunocytochemistry : Cells were washed with HBSS and then fixed with 15% paraformaldehyde in PBS for 15 minutes. The cells were then blocked with a blocking solution in PBS of 1% BSA and 0.3% Triton X-100 for 30 minutes. The primary antibody was then applied in 1% BSA and 0.3% Triton X-100 in PBS and incubated overnight in 4C. The next day, cells were washed with HBSS and incubated for 2 hours prior to 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.

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

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

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

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

SaβGal histochemistry : Cells were washed twice with HBSS / Ca / Mg and then fixed with 15% paraformaldehyde in PBS for 6 minutes. The cells were then rinsed 3 times with HBSS / Ca / Mg prior to staining with an X-gal chromogenic substrate cleaved by endogenous B galactosidase. Cells were maintained in staining solution and incubated overnight at 37 ° C. with external CO2. The next day, cells were washed again with HBSS / Ca / Mg before switching to 70% glycerol solution for imaging under a Leica brightfield microscope.

Cytokine profiling : This work was done with the Human Immune Monitoring Center at Stanford University. Cell medium was collected and spun at 400 rcf for 10 minutes at RT. The supernatant was then instantly frozen in liquid nitrogen until analysis. Analysis was performed using the human 63-plex kit (eBiosciences / Affymetrix). Beads were added to the 96-well plate and washed in a Biotek ELx405 washer. Samples were added to plates containing mixed antibody linked beads and incubated overnight at room temperature for 1 hour, followed by shaking at 4 ° C. A low temperature and room temperature incubation step was performed in an orbital rotary shaker at 500-600 rpm. After overnight incubation, the plates were washed in a Biotek ELx405 washer and then the biotinylated detection antibody was added with 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 reading buffer was added to the wells. Each sample was duplicated twice and measured. Plates were read using a Luminex 200 instrument with 50 beads with lower binding per cytokine per sample. Custom-made assay control beads by Radix Biosolutions are added to all wells.

Antibodies : Five primary antibodies were used for nuclear measurement: rabbit anti-histone H3K9me3 histone methylation (1: 4000), mouse anti-HP1γ heterochromatin marker (1: 200), rabbit anti-LAP2α (1: 500) nuclei. Tissued protein, mouse anti-laminin A / C nuclear envelope marker and rabbit anti-SIRT1 (1: 200).

RNA sequencing and data analysis Cells were washed and digested with TRIzol (Thermo). Total RNA was isolated using the Total RNA Purification Kit (Norgen Biotek Corp), RNA quality was evaluated by RNA analysis criteria (R6K principle, Agilent), and RNA with RIN> 9 was reverse transcribed into cDNA. A cDNA library was prepared using 1 μg of total RNA using the TruSeq RNA Sample Preparation Kit v2 (Illumina). RNA quality was assessed by Agilent Bioanalyzer 2100 and RNA with RIN> 9 was reverse transcribed into cDNA. A cDNA library was prepared using 500 ng of total RNA using the TruSeq RNA Sample Preparation Kit v2 (Illumina), which has the added benefit of molecular indexing. Prior to any PCR amplification step, the ends of all cDNA fragments were randomly ligated to an adapter pair containing a unique molecular index of 8 bp. The molecularly indexed cDNA library was then PCR amplified (15 cycles) and then QC was performed using Bioanalyzer and Qubit. After successful QC, this was sequenced on the Illumina Nextseq platform to obtain 80-bp single-ended read data. The read data was trimmed by 2 nucleotides at each end to remove low quality moieties and improve mapping to the genome. The resulting 78 nucleotide read data was compressed by deduplication and tracked how many times each sequence occurred in each sample in the database. The exact match was then used to map the unique read data to the human genome. This misreads exon-exon boundary crossings and read data with errors and SNPs / mutations, but has no substantial effect on the estimation of the level of expression of each gene. Next, each of the mapped read data was assigned an annotation derived from the underlying genome. For multiple annotations (eg, miRNAs that occur in gene introns), heuristic-based hierarchies were used to give each read data a unique identity. It was then used to identify read data belonging to each transcript and establish coverage over each position of the transcript. Since this coverage is non-uniform and sharp, Applicants used this median coverage as an estimate of gene expression. Quantile normalization was used to compare expression in different samples. Further data analysis was performed in MATLAB®. Next, the ratio of expression levels was calculated to estimate the logarithm of multiple changes (base 2). Student's t-test was used to determine significance with a p <0.05 cutoff. ENCODE gene analysis, developed by Butte Lab of Stanford Center for Biomedical Information Research Research and made publicly available, was used for transcription factor identification.

Mice : C57BL / 6 male mice and NSG mice were obtained from 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 Adaptive Panel of the Laboratory Animal Care at Stanford University.

Human skeletal muscle specimens : Subjects ranged from 10 to 78 years of age. Human muscle biopsy specimens were taken after obtaining patient informed consent as part of a human study study protocol approved by the Stanford University Institutional Review Board. All experiments were performed using fresh muscle specimens according to the availability of clinical procedures. Specimen isolation Within 1-12 hours, sample processing for cell analysis was initiated. In all studies, the standard deviation reflects variability in the data derived from the study using true biological replication (ie, a unique donor). The data did not correlate with donor identity.

MuSC isolation and purification : Muscle was collected from the hind limbs and mechanically dissociated to give a fragmented muscle suspension. This was followed by digestion in F10 solution of collagenase II-Ham (500 units per ml; Invitrogen) for 45-50 minutes. After washing, a second digestion was performed with collagenase II (100 units per ml) and dispase (2 units per ml; Thermo Fisher) for 30 minutes. The resulting cell suspension was washed, filtered and diluted 1: 100 with VCAM-bioscience (clone 429; BD Bioscience), CD31-FITC (clone MEC13.3; BD Bioscience), CD45-APC (clone). It was stained with 30-F11; BD Bioscience) and Sca-1-Pacific-Blue (clone D7; Biolegend) antibodies. Human MuSC was purified from fresh surgical samples 50, 51. Surgical samples were carefully dissected from adipose and fibrous tissue to prepare dissociated muscle suspensions as described for mouse tissue. The resulting cell suspension was then washed and filtered to anti-CD31-Alexa Fluor 488 (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). The unbound primary antibody was then washed and the cells were incubated in streptavidin-PE / Cy7 (BioLegend) for 15 minutes at 4 ° C. to detect NCAM-biotin. Cell selection was performed on a calibrated BD-FACS Maria II® 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 sown and stained with Pax7 and MyoD to assess the purity of the sorted population. See supplemental information for FACS gating strategies.

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

Bioluminescence image analysis : Each image was analyzed using Living Image software, version 4.0 (Caliper Life Sciences). The manually generated circle was placed over the area of interest and resized to completely surround the recipient mouse's limbs or designated area. Similarly, the desired background area was placed in the mouse area outside the transplanted leg.

Tissue collection : TA muscles were carefully dissected away from bone, weighed and placed in 0.5% PFA solution overnight for fixation. The muscle was then 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. Section preparation was performed on a Leica CM3050S cryostat set to generate 10 μm sections. Sections were mounted on Fisherbrand Colorfrost slides. These slides were stored at −20 ° C. until immunohistochemical tests could be performed.

Histology : Using 0.5% electron microscope grade paraformaldehyde, TA muscles were fixed for 5 hours and then transferred to 20% sucrose overnight. The muscles were then frozen in OCT to make frozen sections to 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).

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 washing 4 times with 0.3% PBST, a fluorescently conjugated secondary antibody was added and incubated at 0.3% PBST for 1 hour at room temperature. After 3 additional rinses, each slide was mounted using Fluoview mount medium.

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

Imaging : Samples were imaged using either a standard fluorescence microscope and a 10x or 20x air objective. The Volicity Imaging software was used to tune the excitation and emission filters and come with a pre-programmed AlexaFluor filter setting, which was used whenever possible. The total exposure time was optimized in the first round of imaging and then kept constant throughout the subsequent imaging.

Image analysis : Using ImageJ, by using the color threshold plug-in, the percentage of the area composed of collagen was calculated to create a mask with only collagen-positive areas. The area was then divided by the total area of the sample found using the free drawing tool. All other analyzes were performed using the Volocity software, the fibers were manually counted using the free drawing tool, and the numbers of nuclei, eMHC + fibers, neuromuscular junctions and blood vessels were manually counted.

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

MitoTracker staining and flow cytometric analysis : MuSCs and controls undergoing reprogramming were washed twice with pure HamsF10 (without serum or pen / strip). MuSC was then stained with 0.5 μM MitoTracker Green FM (Thermo Fisher, M7514) and DAPI at 37 ° C. for 30 minutes, washed 3 times with pure Hams F10 and analyzed using a BD FACSAria III flow cytometer.

Statistical analysis : Unless otherwise stated, full statistical analysis was performed using MATLAB R2017a (MathWorks software) or GraphPad Prism 5 (GraphPad software). The t-test was used for statistical analysis. All error bars are s. e. m. Represents; * p <0.05; ** p <0.001; *** p <0.0001.

Although the preferred embodiments of the present disclosure have been described and described, it is acknowledged that various changes may be made therein without departing from the spirit and scope of the present disclosure.

P embodiment

Embodiment P1. A way to rejuvenate cells
a) A step of transfecting a cell with one or more non-integrated messenger RNAs encoding one or more cell reprogramming factors, for which the transfection step is at least 2 days and 4 days or less. Steps performed once daily; and b) are steps that translate one or more non-integrated messenger RNAs to produce one or more cell reprogramming factors in the cell, resulting in transient reprogramming of the cell. A method involving steps that rejuvenates cells without dedifferentiating into stem cells.

Embodiment P2.1 The method according to embodiment P1, wherein the cell reprogramming factor of 2.1 or more is selected from the group consisting of OCT4, SOX2, KLF4, c-MYC, LIN28 and NANOG.

Embodiment P3. The method according to embodiment P2, wherein the cell reprogramming factor comprises OC4, SOX2, KLF4, c-MYC, LIN28 and NANOG.

Embodiment P4. The method according to embodiment P1, wherein the cell is a mammalian cell.

Embodiment P5. The method according to embodiment P4, wherein the cell is a human cell.

Embodiment P6. The method according to embodiment P1, wherein the cells are derived from an elderly subject.

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.

Embodiment P8. Transient reprogramming results in increased expression of HP1 gamma, H3K9me3, lamina-supporting proteins LAP2alpha and SIRT1 proteins, decreased nuclear folds, decreased vesicle formation, increased cellular autophagosome formation, and increased chymotrypsin-like proteasome activity. The method according to embodiment P1, which results in increased mitochondrial membrane potential or decreased reactive oxygen species (ROS).

Embodiment P9. The method of embodiment P1, wherein the cells are in a tissue or organ.

Embodiment P10. The method of embodiment P9, wherein transient reprogramming reduces the number of aged cells in a tissue or organ.

Embodiment P11. The method of embodiment P9, wherein transient reprogramming reduces the expression of GMSCF, IL18 and TNFα.

Embodiment P12. The method according to embodiment P9, wherein the treatment restores the function of cells in a tissue or organ, increases differentiation potential, enhances survival rate, or increases replication capacity or longevity.

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

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

Embodiment P15. A method for treating an age-related disease or condition of interest: a) one or more non-integrated messenger RNAs encoding one or more cell reprogramming factors in vivo into cells in need of rejuvenation. Alternatively, a step of transfecting with Exvivo, wherein the transfecting step is performed once daily for at least 2 and 4 days; and b) one or more cell reprogramming factors in the cells. A method comprising a step of expressing and resulting in transient reprogramming of the cell, wherein the cell is rejuvenated without dedifferentiating into a stem cell.

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

Embodiment P17. 16. The method of embodiment P16, wherein the cell reprogramming factor comprises OCT4, SOX2, KLF4, c-MYC, LIN28 and NANOG.

Embodiment P18. The method according to embodiment P15, further comprising transplanting a rejuvenated cell into a subject.

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

Embodiment P20. The method of embodiment P15, wherein the age-related disease or condition is a neurodegenerative disease or musculoskeletal disorder.

Embodiment P21. A method for treating a disease or disorder associated with chondrocyte degeneration in a subject, a) one or more non-integrated messenger RNAs encoding one or more cellular reprogramming factors, chondrocytes that require rejuvenation. Steps of in vivo or exvivo transfecting cells, wherein the transfecting step is performed once daily for at least 2 and 4 days; and b) one or more cells in chondrocytes. A method comprising expressing a reprogramming factor and resulting in transient reprogramming of chondrocytes, wherein the chondrocytes are rejuvenated without dedifferentiating into stem cells.

Embodiment P21 The method according to embodiment P21, wherein the cell reprogramming factor of 22.1 or more species is selected from the group consisting of OCT4, SOX2, KLF4, c-MYC, LIN28 and NANOG.

Embodiment P23. 22. The method of embodiment P22, wherein the cell reprogramming factor comprises OC4, SOX2, KLF4, c-MYC, LIN28 and NANOG.

Embodiment P24. The method according to embodiment P21, wherein the disease or disorder associated with cartilage degeneration is arthritis.

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

Embodiment P26. 21. The method of embodiment P21, wherein the treatment reduces inflammation in the subject.

Embodiment P27. The method of embodiment P21, wherein the transfection step is performed in Exvivo and the rejuvenated chondrocytes are transplanted into the arthritic joint of interest.

Embodiment P28. The method of embodiment P27, wherein the chondrocytes are isolated from a cartilage sample obtained from the subject.

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.

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

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

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

Embodiment P33. A method for treating a disease or disorder involving muscle degeneration in a subject a) in vivo or to skeletal muscle stem cells with one or more non-integrated messenger RNAs encoding one or more cell reprogramming factors. A step of transfecting with Exvivo, wherein the transfecting step is performed once daily for at least 2 and 4 days; and b) one or more cell reprogramming factors in skeletal muscle stem cells. A method comprising the steps of expressing and resulting in transient reprogramming of skeletal muscle stem cells, wherein the skeletal muscle stem cells are rejuvenated without losing their ability to differentiate into muscle cells.

Embodiment P34.1 The method according to embodiment P33, wherein one or more cell reprogramming factors are selected from the group consisting of OCT4, SOX2, KLF4, c-MYC, LIN28 and NANOG.

Embodiment P35. The method of embodiment P34, wherein the cell reprogramming factor comprises OC4, SOX2, KLF4, c-MYC, LIN28 and NANOG.

Embodiment P36. 13. The method of embodiment P33, wherein the transfection step is performed in Exvivo and rejuvenated skeletal muscle stem cells are transplanted into muscle in need of repair or regeneration in the subject.

Embodiment P37. The method of embodiment P33, wherein skeletal muscle stem cells are isolated from a muscle sample obtained from a subject.

Embodiment P38. The method of embodiment P33, wherein the treatment results in muscle fiber regeneration.

Embodiment P39. The method according to embodiment P33, wherein the treatment restores the potency of skeletal muscle stem cells.

Embodiment P40. The method according to embodiment P33, wherein the subject is an elderly subject.

Embodiment P41. The method according to embodiment P33, wherein the subject is a mammalian subject.

Embodiment P42. The method according to embodiment P41, wherein the mammalian subject is a human subject.

Embodiment

Embodiment 1. A method of rejuvenating cells by transfecting cells with one or more non-integrated messenger RNAs encoding one or more cell reprogramming factors for up to 5 consecutive days, thereby rejuvenating. A method involving the step of producing cells.

Embodiment 2. The method according to embodiment 1, wherein the transcriptome profile of the rejuvenated cells becomes similar to the transcriptome profile of the young cells.

Embodiment 3. Transcriptome profile of rejuvenated cells increases gene expression of one or more genes selected from RPL37, RHOA, SRSF3, EPHB4, ARHGAP18, RPL31, FKBP2, MAP1LC3B2, Elf1, Phf8, Pol2s2, Taf1 and Sin3a. The method according to the second embodiment.

Embodiment 4. The method according to any one of the preceding embodiments, wherein the rejuvenated cells exhibit increased gene expression of one or more nuclei and / or epigenetic markers as compared to a reference value.

Embodiment 5. The method of embodiment 4, wherein the marker is selected from HP1 gamma, H3K9me3, lamina support protein LAP2alpha and SIRT1 protein.

Embodiment 6. The method according to any one of the preceding embodiments, wherein the rejuvenated cells show increased proteolytic activity relative to the reference value.

Embodiment 7. 13. The method of embodiment 6, wherein increased proteolytic activity is measured as increased cellular autophagosome formation, increased chymotrypsin-like proteasome activity, or a combination thereof.

Embodiment 8. The 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.

Embodiment 9. 8. The method of embodiment 8, wherein improved mitochondrial health and function are measured as increased mitochondrial membrane potential, decreased reactive oxygen species (ROS), or a combination thereof.

Embodiment 10. The method according to any one of the preceding embodiments, wherein the rejuvenated cells show reduced expression of one or more SASP cytokines as compared to a reference value.

Embodiment 11. 10. The method of embodiment 10, wherein the SASP cytokine comprises one or more of IL18, IL1A, GROA, IL22 and IL9.

Embodiment 12. The method according to any one of the preceding embodiments, wherein the rejuvenated cells exhibit a reversal of the methylated landscape.

Embodiment 13. 12. The method of embodiment 12, wherein the inversion of the methylated landscape is measured by estimation with a Horvas clock.

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

Embodiment 15. The step of transfecting cells with messenger RNA can be 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. The method according to any one of the preceding embodiments, including the method to be performed.

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.

Embodiment 17. The method of any one of the preceding embodiments, wherein one or more cell reprogramming factors include OCT4, SOX2, KLF4, c-MYC, LIN28 and NANOG.

Embodiment 18. The method according to any one of the preceding embodiments, wherein the cell is a mammalian cell.

Embodiment 19. The method according to any one of the preceding embodiments, wherein the cell is a human cell.

20. The method according to any one of the preceding embodiments, wherein the cells are derived from an elderly subject.

21. Embodiment 21. The method according to any one of the preceding embodiments, wherein the cells are selected from fibroblasts, endothelial cells, cartilage cells, skeletal muscle stem cells, keratinocytes, mesenchymal stem cells and corneal epithelial cells.

Embodiment 22. 21. The method of embodiment 21, wherein the cells are mesenchymal stem cells.

23. 22. The method of embodiment 22, wherein the rejuvenated mesenchymal stem cells show decreased aging parameters (p16, p21 and positive SAβGal staining), increased cell proliferation, and / or decreased ROS levels.

Embodiment 24. The method according to any one of the preceding embodiments, which is performed in vitro, ex vivo or in vivo.

Embodiment 25. 24. The method of embodiment 24, which is performed in vivo.

Embodiment 26. 25. The method of embodiment 25, wherein the cells are in a tissue or organ.

Embodiment 27. The method according to any one of embodiments 25-27, wherein the number of aged cells in a tissue or organ is reduced.

Embodiment 28. The method according to any one of embodiments 25-27, wherein the expression of one or more of IL18, IL1A, GROA, IL22 and IL9 is reduced.

Embodiment 29. The method according to any one of the preceding embodiments, which is a recovery of cell function, an increase in differentiation ability, an increase in survival rate, an increase in replication ability or longevity, or a combination thereof.

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

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

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

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

Embodiment 34. A method for treating a subject's age-related disease or condition, cartilage degenerative disorder, neurodegenerative disorder and / or musculoskeletal dysfunction, comprising the step of administering a therapeutically effective amount of cells, wherein the cell is one type. A method comprising one or more non-integrated messenger RNAs encoding the above cell reprogramming factors.

Embodiment 35. The method of embodiment 34, wherein one or more cell reprogramming factors are selected from OCT4, SOX2, KLF4, c-MYC, LIN28 and NANOG.

Embodiment 36.1. The method of any one of embodiments 34-35, wherein one or more cell reprogramming factors include OCT4, SOX2, KLF4, c-MYC, LIN28 and NANOG.

Embodiment 37. The method of any one of embodiments 34-36, wherein the subject has an age-related disease or condition.

Embodiment 38. 34. The method of embodiment 34, wherein the age-related disease or condition is selected from eye, skin or musculoskeletal dysfunction.

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

Embodiment 40. 39. The method of embodiment 39, wherein the disorder is selected from arthritis, achondroplasia, spondyloarthropathies, ankylosing spondylitis, erythematosus, relapsing polychondritis and Sjogren's syndrome.

Embodiment 41. The method of any one of embodiments 39 or 40, wherein the treatment reduces the expression of inflammatory factors and / or increases ATP and collagen metabolism.

Embodiment 42. 41. The method of embodiment 41, wherein the inflammatory factor is selected from RANKL, iNOS2, IL6, IFNα, MCP3 and MIP1A.

Embodiment 43. 42. The method of embodiment 42, wherein ATP and collagen metabolism is measured by one or more of increased ATP levels, decreased ROS and increased SOD2, increased COL2A1, and overall proliferation by chondrocytes.

Embodiment 44. The method according to any one of embodiments 34-36, wherein the subject has musculoskeletal dysfunction.

Embodiment 45. The method of any one of embodiments 34-44, wherein the step of administering a therapeutically effective amount of cells comprises injection or surgical implantation.

Embodiment 46. 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 any one of embodiments 34-45. The method described.

Embodiment 47. 46. The method of embodiment 46, wherein the therapeutically effective amount of rejuvenated cells is corneal epithelial cells.

Embodiment 48. 47. The method of embodiment 47, wherein the rejuvenated corneal epithelium exhibits reduced aging parameters.

Embodiment 49. 28. The method of embodiment 48, wherein the aging parameter comprises one or more of p21 and p16 expression, mitochondrial neoplasia PGC1α, and expression of the inflammatory factor IL8.

Embodiment 50. A method for treating a subject's age-related disease or condition, cartilage degeneration disorder, and / or a subject with musculoskeletal dysfunction, encoding one or more cell reprogramming factors, therapeutically effective. A method comprising the step of administering an amount of one or more non-integrated messenger RNAs.

Embodiment 51.1 The method of embodiment 50, wherein more than 51.1 cell reprogramming factors are selected from OCT4, SOX2, KLF4, c-MYC, LIN28 and NANOG.

Embodiment 52.1 The method according to any one of embodiments 50-51-48, wherein the cell reprogramming factor of 52.1 or more species comprises Oct4, SOX2, KLF4, c-MYC, LIN28 and NANOG.

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

Embodiment 54. 53. The method of embodiment 53, wherein the age-related disease or condition is selected from eye, skin or musculoskeletal dysfunction.

Embodiment 55. The method according to any one of embodiments 50-52, wherein the subject has a cartilage degeneration disorder.

Embodiment 56. 55. The method of embodiment 55, wherein the disorder is selected from arthritis, achondroplasia, spondyloarthropathies, ankylosing spondylitis, erythematosus, relapsing polychondritis and Sjogren's syndrome.

Embodiment 57. The method according to any one of embodiments 50-52, wherein the subject has musculoskeletal dysfunction.

Embodiment 58. The method of any one of embodiments 50-57, wherein the step of administering a therapeutically effective amount of one or more non-integrated messenger RNA comprises direct injection into the target cell.

Embodiment 59. 58. The method of embodiment 58, wherein the target cell is selected from epithelial cells, endothelial cells, connective tissue cells, muscle cells and neural cells.

Embodiment 60. Exvivo rejuvenation of engineered tissue by transfecting the tissue with one or more non-integrated messenger RNAs encoding one or more cellular reprogramming factors for up to 5 consecutive days. A method comprising the step of producing a rejuvenated engineered tissue thereby.

Embodiment 61. 60. The method of embodiment 60, wherein the engineered tissue exhibits aging parameters, a decrease in pro-inflammatory factors, an improvement in histological score, or a combination thereof.

Embodiment 62. The method according to any one of embodiments 60 or 61, wherein the manipulated tissue is the manipulated skin tissue.

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

Embodiment 64. The method of any one of embodiments 60-63, wherein the histological score comprises morphology, organization and / or quality.

Embodiment 65. A pharmaceutical composition comprising rejuvenated cells, wherein the rejuvenated cells transfect one or more non-integrated messenger RNAs encoding one or more cell reprogramming factors into the cells for up to 5 consecutive days. A pharmaceutical composition obtained by the step of perfection.

Embodiment 66.1 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.

Embodiment 67. Embodiment 65 or 66, wherein the cell exhibits one or more of increased expression of HP1 gamma, H3K9me3, LAP2alpha, SIRT1, increased mitochondrial membrane potential, and decreased reactive oxygen species, and decreased expression of SASP cytokines. The composition according to.

Embodiment 68. 67. The composition of embodiment 67, wherein the SASP cytokine comprises one or more of IL18, IL1A, GROA, IL22 and IL9.

Embodiment 69. Any one of embodiments 65-68, further comprising one or more additional components selected from nutrients, cytokines, growth factors, extracellular matrix (ECM) components, antibiotics, antioxidants and immunosuppressants. The composition according to the embodiment.

Embodiment 70. The composition according to any one of embodiments 65-69, further comprising a pharmaceutically acceptable carrier.

Embodiment 71. The composition according to any one of embodiments 65-70, wherein the cells are autologous or allogeneic.