Treatment of age-related cellular dysfunction

Abstract

The present disclosure relates to a method of inducing cellular rejuvenation comprising contacting a cell with an effective amount of SRSF1, SLC2A13, RNASEL, WDTC1, and / or NPM1 protein, or a nucleic acid encoding SRSF1, SLC2A13, RNASEL, WDTC1, or NPM1 protein. The present disclosure also relates to a method of inducing cellular rejuvenation comprising contacting a cell with an effective amount of an inhibitor of KAT7, ESR1, MAPK7, KDM6A, and / or CTNNB1 protein expression, or expression of a nucleic acid encoding KAT7, ESR1, MAPK7, KDM6A, and / or CTNNB1 protein.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Patent Application No. 63 / 338,967, filed May 6, 2022, U.S. Provisional Patent Application No. 63 / 357,207, filed June 30, 2022, U.S. Provisional Patent Application No. 63 / 413,818, filed October 6, 2022, and U.S. Provisional Patent Application No. 63 / 423,430, filed November 7, 2022, each of which is incorporated by reference herein in its entirety.

[0002] Reference to electronic sequence listing The contents of the electronic sequence listing (H049870765WO00-SEQ-HJD; size: 29,862 bytes; creation date: April 28, 2023) are incorporated herein by reference in their entirety. [Background technology]

[0003] background Aging is characterized by the gradual loss of function at the molecular, cellular, tissue and organismal levels. At the chromatin level, aging is associated with the progressive accumulation of epigenetic errors, which ultimately leads to aberrant gene regulation, stem cell exhaustion, aging and dysregulation of cell / tissue homeostasis. Reprogramming techniques to nuclear pluripotency via overexpression of a small number of transcription factors can revert the age and identity of any cell to that of an embryonic cell by promoting epigenetic reprogramming. Unwanted erasure of cell identity is problematic for the development of rejuvenation therapies, as it results in the destruction of tissue and organ structure, function and cell type distribution. Summary of the Invention [Means for solving the problem]

[0004] overview Aspects of the present disclosure relate to genetic intervention to reverse the age of cells, such as human dermal fibroblasts. In some embodiments, expression of serine and arginine rich splicing factor 1 (SRSF1) protein reverses the transcriptomic age of fibroblasts. In some embodiments, expression of solute carrier family 2 member 13 (SLC2A13) protein reverses the transcriptomic age of fibroblasts. In some embodiments, expression of 2-5A dependent ribonuclease (RNASEL) protein reverses the transcriptomic age of fibroblasts. In some embodiments, expression of WD and tetratricopeptide repeat protein 1 (WDTC1) protein reverses the transcriptomic age of fibroblasts. In some embodiments, expression of nucleophosmin (NPM1) protein reverses the transcriptomic age of fibroblasts.

[0005] In some embodiments, reversing cellular age is achieved by inhibiting expression of one or more pro-senescence proteins. The one or more pro-senescence proteins may be selected from the group consisting of KAT7, ESR1, MAPK7, KDM6A, and CTNNB1. In some embodiments, reversing cellular age is achieved by inhibiting expression of KAT7. In some embodiments, reversing cellular age is achieved by inhibiting expression of ESR1. In some embodiments, reversing cellular age is achieved by inhibiting expression of MAPK7. In some embodiments, reversing cellular age is achieved by inhibiting expression of KDM6A. In some embodiments, reversing cellular age is achieved by inhibiting expression of CTNNB1.

[0006] Some embodiments provide a method of inducing cellular rejuvenation comprising contacting a cell with an effective amount of SRSF1 protein or a nucleic acid encoding the SRSF1 protein. Some embodiments provide a method of inducing cellular rejuvenation comprising contacting a cell with an effective amount of SLC2A13 protein or a nucleic acid encoding the SLC2A13 protein. Some embodiments provide a method of inducing cellular rejuvenation comprising contacting a cell with an effective amount of RNASEL protein or a nucleic acid encoding the RNASEL protein. Some embodiments provide a method of inducing cellular rejuvenation comprising contacting a cell with an effective amount of WDTC1 protein or a nucleic acid encoding the WDTC1 protein. Some embodiments provide a method of inducing cellular rejuvenation comprising contacting a cell with an effective amount of NMP1 protein or a nucleic acid encoding the NPM1 protein.

[0007] Some aspects provide a method of inducing cellular rejuvenation comprising contacting a cell with an effective amount of an inhibitor of expression of one or more pro-senescence proteins. In some embodiments, the one or more pro-senescence proteins are selected from the group consisting of KAT7, ESR1, MAPK7, KDM6A, and CTNNB1. Some aspects provide a method of inducing cellular rejuvenation comprising contacting a cell with an effective amount of an inhibitor of expression of KAT7. Some aspects provide a method of inducing cellular rejuvenation comprising contacting a cell with an effective amount of an inhibitor of expression of ESR1. Some aspects provide a method of inducing cellular rejuvenation comprising contacting a cell with an effective amount of an inhibitor of expression of MAPK7. Some aspects provide a method of inducing cellular rejuvenation comprising contacting a cell with an effective amount of an inhibitor of expression of KDM6A. Some aspects provide a method of inducing cellular rejuvenation comprising contacting a cell with an effective amount of an inhibitor of expression of CTNNB1.

[0008] Another aspect provides a method of inducing cellular rejuvenation in a subject, comprising administering to the subject an effective amount of SRSF1 protein or a nucleic acid encoding SRSF1 protein. Another aspect provides a method of inducing cellular rejuvenation in a subject, comprising administering to the subject an effective amount of SLC2A13 protein or a nucleic acid encoding SLC2A13 protein. Another aspect provides a method of inducing cellular rejuvenation in a subject, comprising administering to the subject an effective amount of RNASEL protein or a nucleic acid encoding RNASEL protein. Another aspect provides a method of inducing cellular rejuvenation in a subject, comprising administering to the subject an effective amount of WDTC1 protein or a nucleic acid encoding WDTC1 protein. Another aspect provides a method of inducing cellular rejuvenation in a subject, comprising administering to the subject an effective amount of NPM1 protein or a nucleic acid encoding NPM1 protein.

[0009] Another aspect provides a method of inducing cellular rejuvenation in a subject, comprising administering to the subject an effective amount of an inhibitor of expression of one or more pro-senescence proteins. In some embodiments, the one or more pro-senescence proteins are selected from the group consisting of KAT7, ESR1, MAPK7, KDM6A, and CTNNB1. Another aspect provides a method of inducing cellular rejuvenation in a subject, comprising administering to the subject an effective amount of an inhibitor of expression of KAT7. Another aspect provides a method of inducing cellular rejuvenation in a subject, comprising administering to the subject an effective amount of an inhibitor of expression of ESR1. Another aspect provides a method of inducing cellular rejuvenation in a subject, comprising administering to the subject an effective amount of an inhibitor of expression of MAPK7. Another aspect provides a method of inducing cellular rejuvenation in a subject, comprising administering to the subject an effective amount of an inhibitor of expression of KDM6A. Another aspect provides a method of inducing cellular rejuvenation in a subject, comprising administering to the subject an effective amount of an inhibitor of expression of CTNNB1.

[0010] In some embodiments, the effective amount is sufficient to reduce cellular senescence of a cell compared to a control. In some embodiments, the effective amount is sufficient to reduce cellular senescence by at least 20%, at least 30%, at least 40%, or at least 50% compared to a control. In some embodiments, the effective amount is sufficient to reduce cellular senescence by at least 50%.

[0011] In some embodiments, an effective amount is sufficient to reduce senescence-associated β-galactosidase activity compared to a control, for example, an effective amount can be sufficient to reduce senescence-associated β-galactosidase activity by at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50%.

[0012] In some embodiments, an effective amount is sufficient to reduce proteasome activity compared to a control, for example, an effective amount can be sufficient to reduce proteasome activity by at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50%.

[0013] In some embodiments, the effective amount is sufficient to induce an average cellular rejuvenation of at least 5 years, at least 10 years, at least 15 years, at least 20 years, at least 25 years, at least 30 years, at least 35 years, at least 40 years, at least 45 years, or at least 50 years. In some embodiments, the effective amount is sufficient to induce an average cellular rejuvenation of about 5 years to about 50 years, or about 5 years to about 25 years. In some embodiments, the effective amount is sufficient to induce an average cellular rejuvenation of at least 25 years.

[0014] In some embodiments, the cell is selected from a fibroblast, a hematopoietic stem cell, an endothelial cell, a chondrocyte, a skeletal muscle stem cell, a keratinocyte, a mesenchymal stem cell, and a corneal epithelial cell. In some embodiments, the cell is a fibroblast, e.g., a human fibroblast (e.g., from the skin, bladder, lung, or reproductive system). In some embodiments, the fibroblast is a human dermal fibroblast. In some embodiments, the cell is a stem cell, e.g., a hematopoietic stem cell (HSC), such as a human HSC. In some embodiments, the cell is a cardiomyocyte. In some embodiments, the cell is a skeletal muscle stem cell.

[0015] In some embodiments, the nucleic acid comprises a heterologous promoter operably linked to the open reading frame. The heterologous promoter may be, for example, an inducible promoter.

[0016] In some embodiments, the method comprises delivering an SRSF1 protein to the cell. In some embodiments, the method comprises delivering an SLC2A13 protein to the cell. In some embodiments, the method comprises delivering an RNASEL protein to the cell. In some embodiments, the method comprises delivering a WDTC1 protein to the cell. In some embodiments, the method comprises delivering an NPM1 protein to the cell.

[0017] In some embodiments, the method comprises delivering to the cell an inhibitor of expression of one or more pro-senescence proteins, e.g., selected from the group consisting of KAT7, ESR1, MAPK7, KDM6A, and CTNNB1. In some embodiments, the method comprises delivering to the cell an inhibitor of expression of a KAT7 protein. In some embodiments, the method comprises delivering to the cell an inhibitor of expression of an ESR1 protein. In some embodiments, the method comprises delivering to the cell an inhibitor of expression of a MAPK7 protein. In some embodiments, the method comprises delivering to the cell an inhibitor of expression of a KDM6A protein. In some embodiments, the method comprises delivering to the cell an inhibitor of expression of a CTNNB1 protein.

[0018] In some embodiments, the method comprises delivering to the cell a nucleic acid comprising an open reading frame encoding a SRSF1 protein. In some embodiments, the method comprises delivering to the cell a nucleic acid comprising an open reading frame encoding a SLC2A13 protein. In some embodiments, the method comprises delivering to the cell a nucleic acid comprising an open reading frame encoding a RNASEL protein. In some embodiments, the method comprises delivering to the cell a nucleic acid comprising an open reading frame encoding a WDTC1 protein. In some embodiments, the method comprises delivering to the cell a nucleic acid comprising an open reading frame encoding a NPM1 protein.

[0019] In some embodiments, the nucleic acid is delivered on a non-viral vector (e.g., mRNA delivery by lipid nanoparticles (LNP) or electroporation). In other embodiments, the nucleic acid is delivered on a viral vector (e.g., an adeno-associated virus (AAV) vector).

[0020] In some embodiments, the contacting comprises transfecting the cell.

[0021] Some aspects provide a method of inducing cellular rejuvenation, comprising overexpressing an effective amount of SRSF1 protein in a cell. In some embodiments, the method comprises activating endogenous SRSF1 protein expression or activity at a level higher than a baseline level.

[0022] Some aspects provide a method of inducing cellular rejuvenation, comprising overexpressing an effective amount of SLC2A13 protein in the cell, hi some embodiments, the method comprises activating expression or activity of endogenous SLC2A13 protein at a level higher than a baseline level.

[0023] Some aspects provide a method of inducing cellular rejuvenation comprising overexpressing an effective amount of an RNASEL protein in a cell, hi some embodiments, the method comprises activating expression or activity of an endogenous RNASEL protein at a level higher than a baseline level.

[0024] Some aspects provide a method for inducing cellular rejuvenation, comprising overexpressing an effective amount of WDTC1 protein in a cell. In some embodiments, the method comprises activating endogenous WDTC1 protein expression or activity at a level higher than a baseline level.

[0025] Some aspects provide a method of inducing cellular rejuvenation, comprising overexpressing an effective amount of NPM1 protein in a cell, hi some embodiments, the method comprises activating expression or activity of endogenous NPM1 protein at a level higher than a baseline level.

[0026] Some embodiments provide a method of inducing cellular rejuvenation comprising delivering to a cell an effective amount of an inhibitor of expression of one or more pro-senescence proteins. For example, the one or more pro-senescence proteins may be selected from the group consisting of KAT7, ESR1, MAPK7, KDM6A, and CTNNB1. Some embodiments provide a method of inducing cellular rejuvenation comprising delivering to a cell an effective amount of an inhibitor of expression of KAT7. Some embodiments provide a method of inducing cellular rejuvenation comprising delivering to a cell an effective amount of an inhibitor of expression of ESR1. Some embodiments provide a method of inducing cellular rejuvenation comprising delivering to a cell an effective amount of an inhibitor of expression of MAPK7. Some embodiments provide a method of inducing cellular rejuvenation comprising delivering to a cell an effective amount of an inhibitor of expression of KDM6A. Some embodiments provide a method of inducing cellular rejuvenation comprising delivering to a cell an effective amount of an inhibitor of expression of CTNNB1.

[0027] In some embodiments, the method comprises reducing expression or activity of one or more endogenous pro-senescence proteins to a level lower than a baseline level. In some embodiments, the method comprises reducing expression or activity of KAT7 to a level lower than a baseline level. In some embodiments, the method comprises reducing expression or activity of ESR1 to a level lower than a baseline level. In some embodiments, the method comprises reducing expression or activity of ESR1 to a level lower than a baseline level. In some embodiments, the method comprises reducing expression or activity of MAPK7 to a level lower than a baseline level. In some embodiments, the method comprises reducing expression or activity of CTNNB1 to a level lower than a baseline level.

[0028] Some embodiments provide a cell comprising an engineered nucleic acid encoding a SRSF1 protein.

[0029] In some embodiments, the cells are fibroblasts.

[0030] In some embodiments, the fibroblasts are human dermal fibroblasts.

[0031] In some embodiments, the cells are stem cells.

[0032] In some embodiments, the stem cells are selected from hematopoietic stem cells, skeletal muscle stem cells, and mesenchymal stem cells.

[0033] In some embodiments, the stem cells are human induced pluripotent stem cells.

[0034] In some embodiments, the cells are selected from endothelial cells, chondrocytes, keratinocytes, and corneal epithelial cells.

[0035] In some embodiments, the cells express SRSF1 at a level higher than the baseline level. [Brief description of the drawings]

[0036] BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1A] A process-based transcriptomic clock reports the biological age of human fibroblasts. Schematic of transcriptomic clock training. Longitudinal transcriptomic data from human fibroblasts are used to train process-specific weak age predictors, which are then combined into an ensemble predictor. [Figure 1B] A process-based transcriptomic clock reports the biological age of human fibroblasts. Senescence scores of different clock processes across the human lifespan. Scores are calculated as a weighted average of process-specific clock gene expression and normalized across all ages (n=133). For example, the highest scores are found in the oldest old samples. Processes are divided into three clusters based on their trajectories: histone methylation shows an early logarithmic increase, junction organization, lipid transport, and sodium transport show an inflection point in midlife, and actin polymerization, ERQC, translation initiation, and WNT signaling show a late exponential increase with age. [Figure 1C] A process-based transcriptomic clock reports the biological age of human fibroblasts. Validation of the transcriptomic clock in human dermal fibroblasts from an independent dataset (n=12). The RNA clock maintains high accuracy (Pearson's r=0.93, p-value<2.2e-16; MAE=6.66) across different sequencing platforms. [Figure 1D] A process-based transcriptomic clock reports the biological age of human fibroblasts. Response of the RNA clock to different age-modulating in vitro interventions. Galactose has no effect on cellular age, unlike 2-deoxy-D-glucose (2DG), dexamethasone (DEX) treatment and contact inhibition (CI), which showed a significant increase in predicted age. Growth in hypoxia (3% O2) showed a partial reversal of the deleterious effects of contact inhibition. [Figure 1E]Process-based transcriptomic clocks report biological age of human fibroblasts. Response of RNA clock and DNA methylation (DNAm) clock (Horvath Skin and Blood5) to the time course of cell reprogramming. Predicted age scales across the time course, showing a steeper and faster decline in RNA compared to the DNAm clock. The two clocks reach consensus at the iPS stage, suggesting that the rejuvenating effect of reprogramming peaks 7 days after reprogramming, which is consistent with previous studies. [Figure 2A] Age-reversal screen identifies genes for cellular rejuvenation. Workflow of cDNA overexpression screen for rejuvenation intervention. 95 different plasmids containing genes of interest in an overexpression cassette and piggyBac transposase expression plasmid were electroporated into three different NHDF lines. Edited fibroblasts were induced with Dox (1ug / mL) for 3 days and assayed for age-related changes using RNA-Seq and flow cytometry assays. [Figure 2B] Age-reversal screen identifies genes for cellular rejuvenation. Heatmap of gene induction for all overexpression lines in three different NHDF backgrounds (n=2). There was a consensus in the log2 fold change levels across the three different lines, with a wide range of induction values ​​observed across all genes tested. [Figure 2C] Age-reversal screen identifies genes for cellular rejuvenation. Z-scores of age effects measured with our RNA clock in an overexpression screen across three NHDF lines (n=2). Genes with z-scores below -1 (age-reducing effects more than one standard deviation away from the mean), KAT7 (positive control for pro-senescence effects), and BFP (negative control) are labeled. [Figure 2D]Age-reversal screen identifies genes for cellular rejuvenation. Representative gene hit and control staining data (n=3) across the three assayed cellular processes in M55 and M79 strains. Data are normalized to gene-specific no doxycycline controls and strain-specific gene controls, and significance is tested using a two-tailed Student's t-test. [Figure 3A] Variability of the aging phenotype and its response to perturbations. Whole-transcriptome UMAP on overexpressing lines from the age-reversal screen. There is strong clustering based on donor cell line, with only minor perturbations overcoming line-specific differences. [Figure 3B] Variability of the senescence phenotype and its response to perturbations. Relationship between the transcriptomic variability of the intervention across cell lines and the differentially expressed clock genes induced thereby. A strong inverse correlation was observed between the average UMAP distance of biological replicates of the same gene and the number of associated clock DEGs, with OSKM, NOTCH1, SRSF1 and KDM6A having the largest effects. [Figure 3C] Variability of the aging phenotype and its response to perturbations. 3D landscape of all 480 NHDF transcriptomes from a large-scale screen. The xy axis represents UMAP coordinates from a, and the z axis represents predicted age, with the youngest sample having the maximum value. [Figure 3D] Variability of the aging phenotype and its response to perturbations. Heatmap of differential (old-young) process activity in seven pairs of isogenic cell lines. Higher activity is associated with an older phenotype, whereas lower activity represents younger cells. Based on the nature of process dysfunction, four distinct groups were observed: Group I = ERQC and lipid transport, Group II = WNT signaling and sodium transport, Group III = histone methylation, Group IV = translation initiation and sodium transport. [Figure 3E]Variability of the aging phenotype and its response to perturbations. Scaled age effect of SIRT1 overexpression across six lines on clock processes (n=2). Negative age effects represent younger gene expression profiles. No coordinated effects were observed between cell lines, however, responding cell lines (M65, M67, M68, M79) all showed moderate rejuvenation of WNT signaling. [Figure 4A] SRSF1 induces strong cell rejuvenation. Predicted age effects of hit overexpression in validation experiments with six NHDF lines (n=2). A strong age reduction was observed in SRSF1 samples, with a beneficial effect on the SIRT1 transcriptome observed in four out of six lines. The other three genes had weaker cell line-specific effects, except for WDTC1, which showed a significant rejuvenation in the M69 cell line. [Figure 4B] SRSF1 induces potent cellular rejuvenation. Staining data for SRSF1 across three assayed cellular processes in six cell lines (n=3). Data are normalized to uninduced samples and reported as relative effects to induced controls, with significance tested using two-tailed Student's t-tests. A strong and significant reduction in SA-βGal and proteasome activity was observed, but no concerted effect on mitochondrial membrane potential was seen. [Figure 4C] SRSF1 induces potent cell rejuvenation. Time course of the scratch assay, expressed as wound area relative to the 0 hour time point, for M79 lines after induction of OKSM, SRSF1, or BFP. Faster closure of the wound area was observed in SRSF1-treated cells, whereas a slower response was observed in cells induced with OSKM, compared to the BFP control. Data are averaged for replicates (n=3-5), error bars represent standard error of the mean, and significance relative to the BFP control is tested using a two-tailed Student's t-test. [Figure 4D]SRSF1 induces potent cellular rejuvenation. Scaled age effects of SRSF1 overexpression on clock processes across six lines (n=2). Negative age effects represent a younger gene expression profile. High correlations were observed between process effects across all six lines, with significant improvements in histone methylation and translation initiation. [Diagram 5] Normalized expression of SRSF1 in primary NHDFs of different age groups. The difference in expression between the young and old groups was statistically significant (two-tailed t-test p=0.002). [Figure 6A] SA-βGal activity in young (n=5) and aged (n=5) NHDF lines. There is a clear effect in the aged samples, but more replicates are needed to reach statistically significant results. [Figure 6B] Relative effect of SRSF1 mRNA transfection on SA-βGal activity. Results are normalized to the negative transfection control and displayed as the percentage difference in SA-βGal activity for 10 different strains. Ages of 10 different strains: 17, 22, 25, 29, 30, 65, 67, 68, 69, and 79 years. Overall, the effect of SRSF1 mRNA transfection significantly reduced the senescence phenotype in NHDFs (two-tailed t-test p=0.004). [Figure 7A] Collagen expression in young (n=5) and aged (n=5) NHDF lines. Aged cells have a statistically significant decrease in collagen production (two-tailed t-test p=0.02). [Figure 7B] Relative effect of SRSF1 mRNA transfection on collagen production. Results are normalized to untreated controls and displayed as percent difference in collagen staining for 10 different strains: 17, 22, 25, 29, 30, 65, 67, 68, 69, and 79 years old. Overall, the effect of SRSF1 mRNA transfection was a significant increase in collagen production in NHDFs (two-tailed t-test p<0.001). [Figure 8]Left: ROS levels upon H2O2 treatment in young (n=5) and aged (n=5) NHDF lines. ROS levels are statistically significantly elevated in aged cells (two-tailed t-test p<0.001). Right: ROS levels upon H2O2 treatment in control or aged (n=5) NHDF lines transfected with SRSF1 mRNA. Overall, the effect of SRSF1 mRNA transfection was to significantly reduce ROS levels after H2O2 treatment in aged NHDFs (two-tailed t-test p=0.002). [Figure 9] Left: Schematic of the experimental design of the in vitro scratch assay. Right: Time course of the scratch assay, expressed as wound area relative to the 0 hour time point, for M79 strains after induction of OKSM, SRSF1, or BFP. We observed a faster closure of the wound area in SRSF1-treated cells and a slower response in cells induced with OSKM compared to the BFP control. Data are averaged for replicates (n=3-5), error bars represent standard error of the mean, and significance relative to the BFP control is tested using a two-tailed Student's t-test. [Figure 10] Left: Schematic of the experimental design of the in vivo wound healing assay. Right: Excision wound area over time for aged GFP, aged SRSF1(ON-ON), and young GFP mice relative to the day 1 time point. Data are expressed as mean ± SEM. Significance was tested using a two-tailed Student's t-test (*p<.05, **p<.01) and compared to aged GFP (n=4). [Figure 11] Representative images of wound areas in aged GFP, aged SRSF1(ON-ON), and young GFP mice at day 11 after wounding. [Figure 12] Survival of wild type (N2) and raga-1(ok386) worms + / - rsp-3 RNAi (P<0.001) and (p-values ​​comparing wild type N2 on RNAi vs raga-1(ok386) on RNAi, triplicates). [Figure 13A]Percentage of HSCs in 2-week in vitro cultures of CD34+ cells from young or old donors following nucleofection with SRSF1 or GFP mRNA. The percentage of HSCs was increased in cells that received SRSF1 mRNA, suggesting a greater self-renewal capacity of the stem cell population. [Figure 13B] NK cells (%) in 2-week in vitro cultures of CD34+ cells from young or old donors after nucleofection with SRSF1 or GFP mRNA. The percentage of NK cells was higher in SRSF1-treated cells, suggesting increased lymphocyte output. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0037] Detailed Description Aging is a complex process that manifests as a progressive, multifaceted functional decline. An unstable transcriptome profile appears as a hallmark of the aging organism, and its modulation by interventions such as reprogramming has been shown to reverse signs of aging and improve function. Furthermore, aging clocks use gene expression information at the epigenetic or transcriptome level to predict the biological age of cells, but currently lack the interpretability required to assess cellular function and develop specific perturbations for cellular rejuvenation. Here, to address this issue, we developed a functionally interpretable transcriptome age predictor by integrating gene and cellular process associations with RNA-sequencing datasets. The RNA clock showed high predictive accuracy (r=0.93) when applied to multiple datasets and demonstrated robust responses to known age-modulating interventions such as reprogramming and cellular stressors. Furthermore, the clock was used as an integrative aging assay, and a transcriptome reprogramming screen for the rejuvenation of primary human fibroblasts was performed. Using the functional interpretability of the predictor, four distinct aging phenotypes were revealed based on the dysfunction of processes that affect the response to most perturbations. Nevertheless, overexpression of SRSF1 led to robust transcriptomic age reversal and functional improvement. These findings suggest a new paradigm of aging as a transcriptomic state and SRSF1 as a promising target for cellular rejuvenation.

[0038] Serine- and arginine-rich splicing factor 1 (SRSF1) Aspects of the present disclosure relate, at least in part, to methods and compositions for contacting cells with a serine and arginine splicing factor 1 (SRSF1) protein or a nucleic acid encoding a SRSF1 protein to induce cellular rejuvenation and / or reverse or inhibit cellular senescence. As used herein, "SRSF1" refers to a full-length or truncated SRSF1 protein, a fragment of the SRSF1 protein, or a nucleic acid encoding the protein. Thus, the SRSF1 protein can be a wild-type, naturally occurring protein, or can be a variant of the wild-type SRSF1 protein (e.g., having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the wild-type SRSF1 protein). Experiments described herein have identified SRSF1 as a gene involved in reversing transcriptome age and reducing cellular senescence in cells, such as fibroblasts, from middle-aged and elderly donors. The SRSF1 gene encodes a member of the arginine / serine-rich splicing factor protein family. The encoded SRSF1 protein can activate or repress splicing depending on its phosphorylation state and its interaction partners. Multiple transcript variants of this gene have been found, and a pseudogene of this gene exists on chromosome 13. SRSF1 plays a role in preventing exon skipping, ensuring splicing accuracy, and controlling alternative splicing. The following SRSF1 sequences may be used according to any of the embodiments provided herein.

[0039] A non-limiting example of a human SRSF1 nucleic acid coding sequence is provided in SEQ ID NO:1. [ka]

[0040] A non-limiting example of a human SRSF1 protein sequence is provided in SEQ ID NO:2, which corresponds to the sequence provided in UniProtKB Accession No. Q07955. [ka]

[0041] Solute carrier family 2 member 13 (SLC2A13) Aspects of the present disclosure relate, at least in part, to methods and compositions for contacting cells with a solute carrier family 2 member 13 (SLC2A13) protein or a nucleic acid encoding a SLC2A13 protein to induce cellular rejuvenation and / or reverse or inhibit cellular senescence. As used herein, "SLC2A13" refers to a full-length or truncated SLC2A13 protein, a fragment of a SLC2A13 protein, or a nucleic acid encoding the protein. Thus, the SLC2A13 protein can be a wild-type, naturally occurring protein, or can be a variant of the wild-type SLC2A13 protein (e.g., having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the wild-type SLC2A13 protein). Experiments described herein have also identified SLC2A13 as a gene involved in reversing transcriptome age and reducing cellular senescence in cells, such as fibroblasts, from middle-aged and elderly donors. SLC2A13 is involved in the transport of myo-inositol and the positive regulation of amyloid-β formation. The following SLC2A13 sequences may be used in accordance with any of the embodiments provided herein:

[0042] A non-limiting example of a human SLC2A13 protein sequence is provided in SEQ ID NO:5, which corresponds to the sequence provided in UniProtKB Accession No. Q96QE2. [ka]

[0043] 2-5A-dependent ribonuclease (RNASEL) Aspects of the present disclosure relate, at least in part, to methods and compositions for contacting cells with a 2-5A-dependent ribonuclease (RNASEL) protein or a nucleic acid encoding an RNASEL protein to induce cellular rejuvenation and / or reverse or inhibit cellular senescence. As used herein, "RNASEL" refers to a full-length or truncated RNASEL protein, a fragment of an RNASEL protein, or a nucleic acid encoding the protein. Thus, an RNASEL protein can be a wild-type, naturally occurring protein, or can be a variant of a wild-type RNASEL protein (e.g., having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a wild-type RNASEL protein). Experiments described herein have also identified RNASEL as a gene involved in reversing transcriptome age and reducing cellular senescence in cells, such as fibroblasts, from middle-aged and elderly donors. RNASEL is an endoribonuclease that functions in the interferon (IFN) antiviral response. RNASEL-mediated apoptosis is the result of a JNK-dependent stress response pathway that leads to cytochrome c release from mitochondria and caspase-dependent apoptosis. The following RNASEL sequences may be used in accordance with any of the embodiments provided herein.

[0044] A non-limiting example of a human RNASEL protein sequence is provided in SEQ ID NO:6, which corresponds to the sequence provided in UniProtKB Accession No. Q05823. [ka]

[0045] WD and tetratricopeptide repeat protein 1 (WDTC1) Aspects of the present disclosure relate, at least in part, to methods and compositions for inducing cellular rejuvenation and / or reversing or inhibiting cellular senescence by contacting cells with WD and tetratricopeptide repeat protein 1 (WDTC1) protein or a nucleic acid encoding a WDTC1 protein. As used herein, "WDTC1" refers to a full-length or truncated WDTC1 protein, a fragment of a WDTC1 protein, or a nucleic acid encoding the protein. Thus, a WDTC1 protein can be a wild-type, naturally occurring protein, or can be a variant of a wild-type WDTC1 protein (e.g., having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a wild-type WDTC1 protein). Experiments described herein have also identified WDTC1 as a gene involved in reversing transcriptome age and reducing cellular senescence in cells, such as fibroblasts, from middle-aged and elderly donors. WDTC1 is predicted to be involved in ubiquitination of pathway proteins and to enable enzyme inhibitory activity, histone binding activity, and histone deacetylase binding activity. The following WDTC1 sequences may be used in accordance with any of the embodiments provided herein.

[0046] A non-limiting example of a human WDTC1 protein sequence is provided in SEQ ID NO:7, which corresponds to the sequence provided in UniProtKB Accession No. Q8N5D0-4. [ka]

[0047] Nucleophosmin 1 (NPM1) Aspects of the present disclosure relate, at least in part, to methods and compositions for inducing cellular rejuvenation and / or reversing or inhibiting cellular senescence by contacting cells with nucleophosmin 1 (NPM1) protein or a nucleic acid encoding an NPM1 protein. As used herein, "NPM1" refers to a full-length or truncated NPM1 protein, a fragment of the NPM1 protein, or a nucleic acid encoding the protein. Thus, the NPM1 protein can be a wild-type, naturally occurring protein, or can be a variant of the wild-type NPM1 protein (e.g., having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the wild-type NPM1 protein). Experiments described herein have also identified NPM1 as a gene involved in reversing transcriptome age and reducing cellular senescence in cells, such as fibroblasts, from middle-aged and elderly donors. NPM1 is predicted to be involved in ubiquitination of pathway proteins and to enable enzyme inhibitory activity, histone binding activity, and histone deacetylase binding activity. The following NPM1 sequences may be used in accordance with any of the embodiments provided herein.

[0048] A non-limiting example of a human NPM1 protein sequence is provided in SEQ ID NO:8, which corresponds to the sequence provided in UniProtKB Accession No. P06748. [ka]

[0049] Histone acetyltransferase KAT7 (KAT7) Aspects of the present disclosure relate, at least in part, to methods and compositions for contacting a cell with an inhibitor of histone acetyltransferase KAT7 (KAT7) protein or an inhibitor of a nucleic acid encoding a KAT7 protein to reduce the expression level of KAT7 protein and / or nucleic acid in the cell, induce cellular rejuvenation, and / or reverse or inhibit cellular senescence. The inhibitor can be a direct or indirect inhibitor of KAT7. In some embodiments, the inhibitor of KAT7 is a protein-based inhibitor, such as an antibody. In some embodiments, the inhibitor of KAT7 is a programmable gene editing system, including, for example, a transcription activator-like effector nuclease (TALEN), a zinc finger nuclease (ZFN), or a CRISPR-based system, such as a CRISPR-Cas9 system, a CRISPRi system, or a CRISPRoff system. In some embodiments, the inhibitor of KAT7 is an RNA interference (RNAi) molecule, such as an shRNA, an siRNA, or an miRNA. In some embodiments, the inhibitor is a small molecule drug inhibitor (having a molecular weight of 1000 Da or less). As used herein, "KAT7" refers to a full-length or truncated KAT7 protein, a fragment of the KAT7 protein, or a nucleic acid encoding the protein. Thus, the KAT7 protein can be a wild-type, naturally occurring protein, or a variant of the wild-type KAT7 protein (e.g., having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the wild-type KAT7 protein). Experiments described herein also identified KAT7 as a gene involved in reversing transcriptome age and reducing cellular senescence in cells such as fibroblasts from middle-aged and elderly donors. KAT7 is predicted to be involved in ubiquitination of pathway proteins and enable enzyme inhibitory activity, histone binding activity, and histone deacetylase binding activity. The following KAT7 sequences may be used according to any of the embodiments provided herein.

[0050] A non-limiting example of a human KAT7 protein sequence is provided in SEQ ID NO:9, which corresponds to the sequence provided in UniProtKB Accession No. O95251. [ka]

[0051] Estrogen receptor 1 (ESR1) Aspects of the present disclosure relate, at least in part, to methods and compositions for contacting a cell with an inhibitor of estrogen receptor (ESR1) protein or a nucleic acid encoding an ESR1 protein to reduce the expression level of ESR1 protein and / or nucleic acid in the cell, induce cellular rejuvenation, and / or reverse or inhibit cellular senescence. The inhibitor can be a direct inhibitor or an indirect inhibitor of ESR1. In some embodiments, the inhibitor of ESR1 is a protein-based inhibitor, such as an antibody. In some embodiments, the inhibitor of ESR1 is a programmable gene editing system (e.g., including a transcription activator-like effector nuclease (TALEN), a zinc finger nuclease (ZFN), or a CRISPR-based system (e.g., a CRISPR-Cas9 system, a CRISPRi system, or a CRISPRoff system)). In some embodiments, the inhibitor of ESR1 is an RNA interference (RNAi) molecule (e.g., an shRNA, an siRNA, or an miRNA). In some embodiments, the inhibitor is a small molecule drug inhibitor (having a molecular weight of 1000 Da or less). As used herein, "ESR1" refers to a full-length or truncated ESR1 protein, a fragment of the ESR1 protein, or a nucleic acid encoding the protein. Thus, the ESR1 protein can be a wild-type, naturally occurring protein, or can be a variant of the wild-type ESR1 protein (e.g., having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the wild-type ESR1 protein). The experiments described herein also identified ESR1 as a gene involved in reversing transcriptome age and reducing cellular senescence in cells such as fibroblasts from middle-aged and elderly donors. ESR1 is predicted to be involved in ubiquitination of pathway proteins and enable enzyme inhibitory activity, histone binding activity, and histone deacetylase binding activity. The following ESR1 sequences may be used according to any of the embodiments provided herein.

[0052] A non-limiting example of a human ESR1 protein sequence is provided in SEQ ID NO: 10, which corresponds to the sequence provided in UniProtKB Accession No. P03372. [ka]

[0053] Mitogen-activated protein kinase 7 (MAPK7) Aspects of the present disclosure relate, at least in part, to methods and compositions for contacting a cell with an inhibitor of mitogen-activated protein kinase 7 (MAPK7) protein or a nucleic acid encoding a MAPK7 protein to reduce the expression level of MAPK7 protein and / or nucleic acid in the cell, induce cellular rejuvenation, and / or reverse or inhibit cellular senescence. The inhibitor can be a direct or indirect inhibitor of MAPK7. In some embodiments, the inhibitor of MAPK7 is a protein-based inhibitor, such as an antibody. In some embodiments, the inhibitor of MAPK7 is a programmable gene editing system (e.g., including a transcription activator-like effector nuclease (TALEN), a zinc finger nuclease (ZFN), or a CRISPR-based system (e.g., a CRISPR-Cas9 system, a CRISPRi system, or a CRISPRoff system). In some embodiments, the inhibitor of MAPK7 is an RNA interference (RNAi) molecule (e.g., an shRNA, an siRNA, or an miRNA). In some embodiments, the inhibitor is a small molecule drug inhibitor (having a molecular weight of 1000 Da or less). As used herein, "MAPK7" refers to a full-length or truncated MAPK7 protein, a fragment of a MAPK7 protein, or a nucleic acid encoding the protein. Thus, the MAPK7 protein may be a wild-type, naturally occurring protein or a variant of the wild-type MAPK7 protein (e.g., having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the wild-type MAPK7 protein). Experiments described herein have also identified MAPK7 as a gene involved in reversing transcriptome age and reducing cellular senescence in cells such as fibroblasts from middle-aged and elderly donors. MAPK7 is predicted to be involved in ubiquitination of pathway proteins, enabling enzyme inhibitory activity, histone binding activity, and histone deacetylase binding activity. The following MAPK7 sequences may be used according to any of the embodiments provided herein.

[0054] A non-limiting example of a human MAPK7 protein sequence is provided in SEQ ID NO: 11, which corresponds to the sequence provided in UniProtKB Accession No. Q13164. [ka]

[0055] Lysine-specific demethylase 6A (KDM6A) Aspects of the present disclosure relate, at least in part, to methods and compositions for contacting a cell with an inhibitor of Lysine-specific demethylase 6A (KDM6A) protein or a nucleic acid encoding a KDM6A protein to reduce the expression level of KDM6A protein and / or nucleic acid in the cell, induce cellular rejuvenation, and / or reverse or inhibit cellular senescence. The inhibitor can be a direct or indirect inhibitor of KDM6A. In some embodiments, the inhibitor of KDM6A is a protein-based inhibitor, such as an antibody. In some embodiments, the inhibitor of KDM6A is a programmable gene editing system, including, for example, a transcription activator-like effector nuclease (TALEN), a zinc finger nuclease (ZFN), or a CRISPR-based system, such as a CRISPR-Cas9 system, a CRISPRi system, or a CRISPRoff system. In some embodiments, the inhibitor of KDM6A is an RNA interference (RNAi) molecule, such as an shRNA, an siRNA, or an miRNA. In some embodiments, the inhibitor is a small molecule drug inhibitor (having a molecular weight of 1000 Da or less). As used herein, "KDM6A" refers to a full-length or truncated KDM6A protein, a fragment of the KDM6A protein, or a nucleic acid encoding the protein. Thus, the KDM6A protein can be a wild-type, naturally occurring protein, or can be a variant of the wild-type KDM6A protein (e.g., having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the wild-type KDM6A protein). Experiments described herein have also identified KDM6A as a gene involved in reversing transcriptome age and reducing cellular senescence in cells such as fibroblasts from middle-aged and elderly donors. KDM6A is predicted to be involved in ubiquitination of pathway proteins and enable enzyme inhibitory activity, histone binding activity, and histone deacetylase binding activity. The following KDM6A sequences may be used in accordance with any of the embodiments provided herein.

[0056] A non-limiting example of a human KDM6A protein sequence is provided in SEQ ID NO: 12, which corresponds to the sequence provided in UniProtKB Accession No. O15550. [ka]

[0057] Catenin beta 1 (CTNNB1) Aspects of the present disclosure relate, at least in part, to methods and compositions for contacting a cell with an inhibitor of catenin beta 1 (CTNNB1) protein or a nucleic acid encoding a CTNNB1 protein to reduce the expression level of the CTNNB1 protein and / or nucleic acid in the cell, induce cellular rejuvenation, and / or reverse or inhibit cellular senescence. The inhibitor can be a direct or indirect inhibitor of CTNNB1. In some embodiments, the inhibitor of CTNNB1 is a protein-based inhibitor, such as an antibody. In some embodiments, the inhibitor of CTNNB1 is a programmable gene editing system (e.g., including a transcription activator-like effector nuclease (TALEN), a zinc finger nuclease (ZFN), or a CRISPR-based system (e.g., a CRISPR-Cas9 system, a CRISPRi system, or a CRISPRoff system). In some embodiments, the inhibitor of CTNNB1 is an RNA interference (RNAi) molecule (e.g., an shRNA, an siRNA, or an miRNA). In some embodiments, the inhibitor is a small molecule drug inhibitor (having a molecular weight of 1000 Da or less). As used herein, "CTNNB1" refers to a full-length or truncated CTNNB1 protein, a fragment of the CTNNB1 protein, or a nucleic acid encoding the protein. Thus, the CTNNB1 protein can be a wild-type, naturally occurring protein, or a variant of the wild-type CTNNB1 protein (e.g., having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the wild-type CTNNB1 protein). Experiments described herein have also identified CTNNB1 as a gene involved in reversing transcriptome age and reducing cellular senescence in cells such as fibroblasts from middle-aged and elderly donors. CTNNB1 is predicted to be involved in ubiquitination of pathway proteins and enable enzyme inhibitory activity, histone binding activity, and histone deacetylase binding activity. The following CTNNB1 sequences may be used according to any of the embodiments provided herein.

[0058] A non-limiting example of a human CTNNB1 protein sequence is provided in SEQ ID NO: 13, which corresponds to the sequence provided in UniProtKB Accession No. P35222. [ka]

[0059] protein Aspects of the present disclosure relate, at least in part, to proteins that have been found to induce cellular rejuvenation and / or reverse or inhibit cellular senescence. As used herein, the term "protein" refers to a primary amino acid structure, a secondary amino acid structure, a non-naturally folded amino acid structure, a folded tertiary amino acid structure, or a folded quaternary amino acid structure. In some embodiments, the protein is chemically synthesized. In some embodiments, the protein is translated from a nucleic acid structure. In some embodiments, the amino acid structure of the protein is mutated to include substitutions or deletions of specific amino acid residues.

[0060] Aspects of the present disclosure relate, at least in part, to proteins that have been found to induce senescence, reduce cellular rejuvenation, and / or accelerate cellular senescence. In some embodiments, inhibiting expression of the protein induces cellular rejuvenation and / or reverses or inhibits cellular senescence. In some embodiments, the protein is inhibited by a protein-based inhibitor, such as an antibody. In some embodiments, the protein is inhibited by a programmable gene editing system, including, for example, a transcription activator-like effector nuclease (TALEN), a zinc finger nuclease (ZFN), or a CRISPR-based system, such as a CRISPR-Cas9 system, a CRISPRi system, or a CRISPRoff system. In some embodiments, the protein is inhibited by an RNA interference (RNAi) molecule, such as an shRNA, an siRNA, or an miRNA. In some embodiments, the protein is inhibited by a small molecule drug inhibitor (having a molecular weight of 1000 Da or less).

[0061] The cells of the present disclosure, in some embodiments, comprise a protein encoded by an engineered nucleic acid. The term "protein" encompasses full-length functional SRSF1 protein, and full-length or truncated functional variants of the protein, unless otherwise specified. Thus, the term "protein" encompasses full-length functional SRSF1, SLC2A13, RNASEL, WDTC1, NPM1, KAT7, ESR1, MAPK7, KDM6A, and CTNNB1 proteins, and full-length or truncated functional variants of SRSF1, SLC2A13, RNASEL, WDTC1, NPM1, KAT7, ESR1, MAPK7, KDM6A, and CTNNB1 proteins, unless otherwise specified. Thus, in some embodiments, the SRSF1 protein comprises the sequence of SEQ ID NO:2 or is encoded by a nucleic acid comprising the protein coding sequence of SEQ ID NO:1. In other embodiments, the SRSF1 protein comprises a sequence having at least 70% identity to the sequence of SEQ ID NO:2 or is encoded by a nucleic acid comprising a sequence having at least 70% identity to the protein coding sequence of SEQ ID NO:1. In some embodiments, the SLC2A13 protein comprises a sequence of SEQ ID NO:5. In some embodiments, the RNASEL protein comprises a sequence of SEQ ID NO:6. In some embodiments, the WDTC1 protein comprises a sequence of SEQ ID NO:7. In some embodiments, the NPM1 protein comprises a sequence of SEQ ID NO:8. In some embodiments, the KAT7 protein comprises a sequence of SEQ ID NO:9. In some embodiments, the ESR1 protein comprises a sequence of SEQ ID NO:10. In some embodiments, the MAPK7 protein comprises a sequence of SEQ ID NO:11. In some embodiments, the KDM6A protein comprises a sequence of SEQ ID NO:12. In some embodiments, the CTNNB1 protein comprises a sequence of SEQ ID NO:13.

[0062] In some embodiments, the amino acid structure of a protein described herein has at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity (e.g., as determined by global alignment) to the amino acid sequence of any one of SEQ ID NOs: 2, 5, 6, 7, 8, 9, 10, 11, 12, or 13.

[0063] Functional variants The terms "identity" or "sequence identity" (used interchangeably herein) refer to the relationship between the amino acid sequences of two or more peptides or polypeptides, as determined by comparing the sequences. Identity also refers to the degree of sequence relatedness between or within sequences, as determined by the number of matches between strings of two or more amino acid residues. Identity measures the percentage of identical matches between the smaller of two or more sequences, with gap alignment (if any) being accommodated by a particular mathematical model or computer program. The identity of related peptides can be readily calculated by known methods. "Percent identity" as applied to peptide sequences is defined as the percentage of amino acid residues in a first sequence that are identical to the amino acid residues in a second sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent identity. Methods and computer programs for alignment are well known in the art. It is understood that identity depends on the calculation of percent identity, but may vary depending on gaps and penalties introduced in the calculation. In some embodiments, computer programs including Clustal Omega (Sievers et al., Mol Syst Biol. 2011 Oct 11;7:539) may be used for sequence alignment. In some embodiments, computer programs including BLAST®, NBLAST®, XBLAST®, or Gapped BLAST® may be used for sequence alignment. In some embodiments, percent identity is determined by aligning a sequence to a reference sequence.

[0064] In some embodiments, the SRSF1 protein comprises a sequence having at least 70% identity to the sequence of SEQ ID NO:2 and maintains a function described herein (e.g., capable of inducing cellular rejuvenation, e.g., reducing senescence-associated β-galactosidase activity, and / or reducing cellular proteasome activity). For example, a functional SRSF1 protein may comprise a sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence of SEQ ID NO:2.

[0065] In some embodiments, the functional SRSF1 protein is encoded by a nucleic acid comprising a sequence having at least 70% identity to the protein coding sequence of SEQ ID NO: 1. For example, the functional SRSF1 protein can be encoded by a nucleic acid comprising a sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the protein coding sequence of SEQ ID NO: 1.

[0066] In some embodiments, the SLC2A13 protein comprises a sequence having at least 70% identity to the sequence of SEQ ID NO:5 and maintains a function described herein (e.g., capable of inducing cellular rejuvenation of a cell, e.g., reducing senescence-associated β-galactosidase activity, and / or reducing proteasome activity of a cell). For example, a functional SLC2A13 protein can comprise a sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence of SEQ ID NO:5.

[0067] In some embodiments, the RNASEL protein comprises a sequence having at least 70% identity to the sequence of SEQ ID NO:6 and maintains a function as described herein (e.g., capable of inducing cellular rejuvenation of a cell, e.g., reducing senescence-associated β-galactosidase activity, and / or reducing proteasome activity of a cell). For example, a functional RNASEL protein may comprise a sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence of SEQ ID NO:6.

[0068] In some embodiments, the WDTC1 protein comprises a sequence having at least 70% identity to the sequence of SEQ ID NO:7 and maintains a function described herein (e.g., capable of inducing cellular rejuvenation of a cell, e.g., reducing senescence-associated β-galactosidase activity, and / or reducing proteasome activity of a cell). For example, a functional WDTC1 protein can comprise a sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence of SEQ ID NO:7.

[0069] In some embodiments, the NPM1 protein comprises a sequence having at least 70% identity to the sequence of SEQ ID NO:8 and maintains a function described herein (e.g., capable of inducing cellular rejuvenation of a cell, e.g., reducing senescence-associated β-galactosidase activity, and / or reducing proteasome activity of a cell). For example, a functional NPM1 protein can comprise a sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence of SEQ ID NO:8.

[0070] nucleic acid Aspects of the present disclosure relate, at least in part, to nucleic acids encoding the proteins described herein. In some embodiments, the nucleic acid is engineered. In some embodiments, the nucleic acid is modified to increase expression levels. In some embodiments, the nucleic acid is mutated. In some embodiments, the nucleic acid is modified by substitution, insertion, or deletion mutation. In some embodiments, the nucleic acid is modified by truncation. In some embodiments, the truncated nucleic acid encodes a functional protein. In some embodiments, the nucleic acid is fused to a signal sequence. In some embodiments, the nucleic acid is operably linked to a promoter.

[0071] The cell of the present disclosure, in some embodiments, comprises an engineered nucleic acid. For example, the engineered nucleic acid may code for a protein having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with a functional SRSF1 protein comprising the sequence of SEQ ID NO:2. In some embodiments, the engineered nucleic acid codes for a protein having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with a functional SLC2A13 protein comprising the sequence of SEQ ID NO:5. In some embodiments, the engineered nucleic acid encodes a protein having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to a functional RNASEL protein comprising the sequence of SEQ ID NO: 6. In some embodiments, the engineered nucleic acid encodes a protein having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to a functional WDTC1 protein comprising the sequence of SEQ ID NO: 7. In some embodiments, the engineered nucleic acid encodes a protein having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to a functional NPM1 protein comprising the sequence of SEQ ID NO: 8.

[0072] An engineered nucleic acid is a polynucleotide that does not occur in nature (e.g., at least two nucleotides are covalently linked together, optionally containing a phosphodiester bond, referred to as a phosphodiester backbone). Engineered nucleic acids include recombinant and synthetic nucleic acids. Recombinant nucleic acids are molecules constructed by combining nucleic acids (e.g., isolated nucleic acids, synthetic nucleic acids, or combinations thereof) from two different organisms (e.g., human and mouse). Synthetic nucleic acids are molecules that have been amplified or synthesized chemically or by other means. Synthetic nucleic acids include those that have been chemically or otherwise modified but are still capable of base pairing (binding) with naturally occurring nucleic acid molecules. Recombinant and synthetic nucleic acids also include molecules resulting from replication of any of the above.

[0073] Engineered nucleic acids can be composed of DNA (e.g., genomic DNA, cDNA, or a combination of genomic DNA and cDNA), RNA, or hybrid molecules, e.g., the nucleic acid includes any combination of deoxyribonucleotides and ribonucleotides (e.g., artificial or natural) and any combination of two or more bases, including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine, hypoxanthine, isocytosine, and isoguanine.

[0074] In some embodiments, the nucleic acid is complementary DNA (cDNA), which is synthesized from a single-stranded RNA (e.g., messenger RNA (mRNA) or microRNA (miRNA)) template in a reaction catalyzed by reverse transcriptase.

[0075] The engineered nucleic acids of the present disclosure may be produced using standard molecular biology methods (see, e.g., Green and Sambrook, Molecular Cloning, A Laboratory Manual, 2012, Cold Spring Harbor Press). In some embodiments, the nucleic acids are produced using GIBSON ASSEMBLY® cloning (see, e.g., Gibson, DG et al. Nature Methods, 343-345, 2009; and Gibson, DG et al. Nature Methods, 901-903, 2010, each of which is incorporated herein by reference). GIBSON ASSEMBLY® typically uses three enzyme activities in a single tube reaction: a 5' exonuclease, a 3' extension activity of a DNA polymerase, and a DNA ligase activity. The 5' exonuclease activity degrades the 5' end sequence, exposing complementary sequences for annealing. The polymerase activity then fills in the gaps in the annealed domains. DNA ligase then seals the nicks and covalently joins the DNA fragments. The overlapping sequences of adjacent fragments are much longer than those used in Golden Gate Assembly, resulting in a higher rate of correct assembly. MegaGate molecular cloning methods may be used. MegaGate is a toxin-free Gateway technology that removes the ccdb toxin used in Gateway recombinase cloning, instead utilizing meganuclease-mediated digestion to remove background vector during cloning (see, for example, Kramme C. et al. STAR Protoc. 2021 Oct 22;2(4):100907, incorporated herein by reference). Other methods of producing engineered polynucleotides may be used in accordance with the present disclosure.

[0076] In some embodiments, the engineered nucleic acid comprises a promoter operably linked to an open reading frame. A promoter is a nucleotide sequence to which RNA polymerase binds to the initial transcript (e.g., ATG). A promoter is typically located immediately upstream (5' end) of the transcription start site. In some embodiments, the promoter is a heterologous promoter. A heterologous promoter is not naturally associated with the open reading frame to which it is operably linked.

[0077] In some embodiments, the promoter is an inducible promoter. Inducible promoters can be controlled in vivo, for example, by chemicals, temperature, or light. Inducible promoters allow for, for example, temporal and / or spatial control of gene expression. Inducible promoters for use according to the present disclosure include any inducible promoter described herein or known to one of skill in the art. Examples of inducible promoters include chemically / biochemically regulated promoters and physically regulated promoters, such as alcohol-regulated promoters, tetracycline-regulated promoters (e.g., anhydrotetracycline (aTc)-responsive promoters and other tetracycline-responsive promoter systems including tetracycline repressor protein (tetR), tetracycline operator sequence (tetO), and tetracycline transactivator fusion protein (tTA)), steroid-regulated promoters (e.g., rat glucocorticoid receptor, human estrogen receptor 1 (EGFR), human estrogen receptor 2 (ER), human estrogen receptor 3 (ER), human estrogen receptor 4 (ER), human estrogen receptor 5 (ER), human estrogen receptor 6 (ER), human estrogen receptor 7 (ER), human estrogen receptor 8 (ER), human estrogen receptor 9 (ER), human estrogen receptor 10 (ER), human estrogen receptor 11 (ER), human estrogen receptor 12 (ER), human estrogen receptor 13 (ER), human estrogen receptor 14 (ER), human estrogen receptor 15 (ER), human estrogen receptor 16 (ER), human estrogen receptor 17 (ER), human estrogen receptor 18 (ER), human estrogen receptor 19 (ER), human estrogen receptor 20 (ER), human estrogen receptor 21 (ER), human estrogen receptor 22 (ER), human estrogen receptor 23 (ER), human estrogen receptor 24 (ER), human estrogen receptor 25 (ER), human estrogen receptor 26 (ER), human estrogen receptor 27 (ER), human In some embodiments, the inducible promoter is a tetracycline-inducible promoter. In some embodiments, the inducible promoter is a doxycycline-inducible promoter. In other embodiments, the inducible promoter is a constitutive promoter (active in vivo, unregulated).

[0078] An open reading frame is a contiguous sequence of codons that begins with a start codon (e.g., ATG) and ends with a stop codon (e.g., TAA, TAG, TGA, etc.) and encodes a polypeptide, such as a protein. An open reading frame is operably linked to a promoter if the promoter controls transcription of the open reading frame.

[0079] Vectors used for delivery of engineered nucleic acids include viral vectors and non-viral vectors. Non-limiting examples of viral vectors include retroviruses, adenoviruses, adeno-associated viruses (AAV), and herpes simplex viruses. Non-limiting examples of non-viral vectors include minicircles, plasmids, bacterial artificial chromosomes (BAC), and yeast artificial chromosomes. Transposon-based systems, such as the piggyBac™ system (e.g., Chen et al. Nature Communications. 2020; 11(1): 3446), can be used as vector systems for delivering engineered nucleic acids. Other non-limiting examples include nanoparticle-based systems, such as lipid nanoparticles.

[0080] Cell Rejuvenation Some embodiments provide a method of inducing cellular rejuvenation, for example to counter the effects of aging. Mammalian aging has been summarized and classified into nine "hallmarks" of aging: genomic instability, telomere attrition, epigenetic changes, loss of proteostasis, dysregulated nutrient sensing, mitochondrial dysfunction, cellular senescence, stem cell exhaustion, and altered intracellular communication (Lopez-Otin C et al. Cell 2013; 153: 1194-1217). Cellular rejuvenation is a process that not only slows down aging, but also reverses aging and leads to younger cells. Cellular rejuvenation can reduce or remove age-related damage and aging hallmarks accumulated during the lifespan of a cell. Thus, "inducing cellular rejuvenation" refers to a process (method) that causes the reversal of aging hallmarks. Induction of cellular rejuvenation can be assessed, for example, by assessing the transcriptome profile, gene expression of one or more nuclear and / or epigenetic markers, proteolytic activity, mitochondrial health and / or function, expression of one or more SASP cytokines, or the methylation landscape of a cell compared to a control (e.g., a reference value obtained from a young or senescent cell).

[0081] Aspects of the disclosure relate to a method of inducing cellular rejuvenation, comprising contacting a cell with an effective amount of a serine- and arginine-rich splicing factor 1 (SRSF1) protein or a nucleic acid encoding the SRSF1 protein, a solute carrier family member 13 (SLC2A13) protein or a nucleic acid encoding the SLC2A13 protein, a 2-5A-dependent ribonuclease (RNASEL) protein or a nucleic acid encoding the RNASEL protein, a WD- and tetratricopeptide repeat protein 1 (WDTC1) protein or a nucleic acid encoding the WDTC1 protein, or a NPM1 protein or a nucleic acid encoding the NPM1 protein. In some embodiments, the disclosure relates to a method of inducing cellular rejuvenation, comprising contacting a cell with an effective amount of a serine- and arginine-rich splicing factor 1 (SRSF1) protein or a nucleic acid encoding the SRSF1 protein.

[0082] Aspects of the present disclosure relate to a method of inducing cellular rejuvenation in a subject, comprising administering to the subject an effective amount of a serine- and arginine-rich splicing factor 1 (SRSF1) protein or a nucleic acid encoding the SRSF1 protein, a solute carrier family member 13 (SLC2A13) protein or a nucleic acid encoding the SLC2A13 protein, a 2-5A-dependent ribonuclease (RNASEL) protein or a nucleic acid encoding the RNASEL protein, a WD- and tetratricopeptide repeat protein 1 (WDTC1) protein or a nucleic acid encoding the WDTC1 protein, or a NPM1 protein or a nucleic acid encoding the NPM1 protein. In some embodiments, the present disclosure relates to a method of inducing cellular rejuvenation in a subject, comprising administering to the subject an effective amount of a SRSF1 protein or a nucleic acid encoding the SRSF1 protein.

[0083] Aspects of the disclosure relate to a method of inducing cellular rejuvenation, comprising contacting a cell with an effective amount of an inhibitor of expression of a KAT7 protein or a nucleic acid encoding a KAT7 protein, expression of an ESR1 protein or a nucleic acid encoding an ESR1 protein, expression of a MAPK7 protein or a nucleic acid encoding a MAPK7 protein, expression of a KDM6A protein or a nucleic acid encoding a KDM6A protein, or expression of a CTNNB1 protein or a nucleic acid encoding a CTNNB1 protein.

[0084] Aspects of the present disclosure relate to a method of inducing cellular rejuvenation in a subject, comprising administering to the subject an effective amount of an inhibitor of expression of KAT7 protein or of a nucleic acid encoding the KAT7 protein, expression of ESR1 protein or of a nucleic acid encoding the ESR1 protein, expression of MAPK7 protein or of a nucleic acid encoding the MAPK7 protein, expression of KDM6A protein or of a nucleic acid encoding the KDM6A protein, or expression of CTNNB1 protein or of a nucleic acid encoding the CTNNB1 protein.

[0085] Aspects of the present disclosure relate to a method of inducing cellular rejuvenation in a subject, comprising administering to the subject an effective amount of one or more anti-senescence proteins and an effective amount of an inhibitor of one or more pro-senescence proteins. In some embodiments, the one or more anti-senescence genes are selected from the group consisting of SRSF1, SLC2A13, RNASEL, WDTC1, and NPM1. In some embodiments, the one or more pro-senescence proteins are selected from the group consisting of KAT7, ESR1, MAPK7, KDM6A, and CTNNB1.

[0086] In some embodiments, the effective amount is sufficient to induce an average cellular rejuvenation of at least 5 years, at least 10 years, at least 15 years, at least 20 years, at least 25 years, at least 30 years, at least 35 years, at least 40 years, at least 45 years, or at least 50 years. In some embodiments, the effective amount is sufficient to induce an average cellular rejuvenation of at least 25 years.

[0087] Aspects of the present disclosure relate to a method for inducing cellular rejuvenation, comprising overexpressing an effective amount of SRSF1 protein in a cell. In some embodiments, the method comprises activating endogenous SRSF1 protein expression or activity at a level higher than a baseline level.

[0088] A "rejuvenated cell" is a senescent cell that has been transiently or stably transfected with a protein or a nucleic acid encoding the protein, such that the cell retains one or more cell identity markers and has a transcriptome profile of a younger cell. Thus, a "rejuvenated cell" is a senescent cell that has been transiently or stably transfected with a SRSF1 protein or a nucleic acid encoding a SRSF1 protein, such that the cell retains one or more cell identity markers and has a transcriptome profile of a younger cell. A "rejuvenated cell" may be a senescent cell that has been transiently or stably transfected with a SLC2A13, RNASEL, WDTC1, and / or NPM1 protein, or a nucleic acid encoding a SLC2A13, RNASEL, WDTC1, and / or NPM1 protein, such that the cell retains one or more cell identity markers and has a transcriptome profile of a younger cell.

[0089] A transcriptome profile refers to the set of all RNA molecules in a cell or cell population. Depending on the particular experiment, it may be used to refer to all RNA or just mRNA. It differs from an exome in that it only contains the RNA molecules found in a particular cell population, and typically includes the amount or concentration of each RNA molecule in addition to the molecular identity. Methods for obtaining a transcriptome profile include DNA microarrays and next-generation sequencing techniques such as RNA-Seq. Transcription can also be studied at the individual cell level by single-cell transcriptomics. There are two general methods for inferring transcriptome sequences. In one approach, sequence reads are mapped to either the reference genome itself (that of the organism whose transcriptome is being studied) or to closely related species. In the other approach, de novo transcriptome assembly, software is used to infer transcripts directly from short sequence reads.

[0090] In some embodiments, the transcriptome profile of the rejuvenated cell becomes more similar to the transcriptome profile of the young cell. For example, the transcriptome profile of the rejuvenated cell is [ka] The method may include increasing or decreasing gene expression (e.g., towards levels expected in a young cell) of one or more genes selected from:

[0091] In some embodiments, the rejuvenated cells exhibit increased gene expression of one or more nuclear and / or epigenetic markers compared to a control (e.g., a reference value). For example, the markers can be selected from HP1 gamma, H3K9me3, lamina support protein LAP2 alpha, and SIRT1 protein.

[0092] In some embodiments, rejuvenated cells exhibit increased proteolytic activity compared to a control. For example, increased proteolytic activity can be measured as increased cellular autophagosome formation, increased chymotrypsin-like proteasome activity, or a combination thereof.

[0093] In some embodiments, the rejuvenated cells exhibit improved mitochondrial health and / or function compared to a control. For example, improved mitochondrial health and function can be measured as an increase in mitochondrial membrane potential, a decrease in reactive oxygen species (ROS), or a combination thereof.

[0094] In some embodiments, rejuvenated cells exhibit decreased expression of one or more SASP cytokines compared to a control, for example, the SASP cytokines include one or more of IL18, IL1A, GROA, IL22, and IL9.

[0095] In some embodiments, the rejuvenated cells exhibit a reversal of the methylation landscape, which can be measured, for example, by Horvath clock estimation.

[0096] In some embodiments, the control is a young cell or a senescent cell, or a reference value obtained from a young cell or a senescent cell.

[0097] In some embodiments, inducing cellular rejuvenation leads to a reduction or inhibition of cellular senescence. For example, senescence-associated β-galactosidase activity of a cell may be reduced. As another example, proteasome activity of a cell may be reduced.

[0098] Here, an "effective amount" of SRSF1 protein (or other proteins, such as SLC2A13, RNASEL, WDTC1, or NPM1) or a nucleic acid encoding SRSF1 protein (or other proteins, such as SLC2A13, RNASEL, WDTC1, or NPM1) is an amount sufficient to initiate a reversal of hallmarks of senescence (e.g., a younger transcriptomic profile (more similar to a younger cell), increased gene expression of one or more nuclear and / or epigenetic markers, increased proteolytic activity, improved mitochondrial health and / or function, decreased expression of one or more SASP cytokines, or a reversal of the methylation landscape of the cell) compared to a control (e.g., a reference value obtained from a younger cell or a senescent cell). An "effective amount" of SRSF1 protein (or other proteins such as SLC2A13, RNASEL, WDTC1, or NPM1) or a nucleic acid encoding SRSF1 protein (or other proteins such as SLC2A13, RNASEL, WDTC1, or NPM1) also includes an amount sufficient to reduce SA-β-gal activity and / or proteasome activity.

[0099] Here, an "effective amount" of an inhibitor of expression of a pro-senescence protein (e.g., KAT7, ESR1, MAPK7, KDM6A, and CTNNB1) is an amount sufficient to initiate a reversal of hallmarks of senescence (e.g., a younger transcriptomic profile (more similar to younger cells), increased gene expression of one or more nuclear and / or epigenetic markers, increased proteolytic activity, improved mitochondrial health and / or function, decreased expression of one or more SASP cytokines, or a reversal of the methylation landscape of the cell) compared to a control (e.g., a reference value obtained from a younger or senescent cell). An "effective amount" of an inhibitor of expression of a pro-senescence protein also includes an amount sufficient to reduce SA-β-gal activity and / or proteasome activity.

[0100] cellular aging Cellular senescence is the disruption of cellular proliferation and function. During cellular senescence, cells continue to maintain viability and metabolic activity but lose the ability to proliferate.

[0101] Many cell types undergo cellular senescence after many cell division cycles. This barrier to further proliferation that occurs after many cell division cycles is called replicative senescence. Replicative senescence is thought to occur when cells' telomeres shorten with successive cell divisions, reaching a point where the cell triggers a DNA damage response (the so-called "Hayflick limit"), ultimately leading to the induction of growth arrest and cellular senescence. Cellular senescence can be induced even without telomere loss or dysfunction. DNA damage can take the form of chromosomal dysfunction, such as aneuploidy resulting from unequal chromosome segregation during mitosis, DNA strand breaks, or chemical modification of DNA. Cellular senescence can also be induced by a DNA damage response (DDR), which may or may not reflect actual DNA damage.

[0102] In some embodiments, cellular senescence is characterized by and can be induced by changes in chromatin organization that induce changes in gene expression, such as the "senescence-associated secretory phenotype" ("SASP"), in which senescent cells secrete inflammatory cytokines and mitokines that can damage or alter surrounding tissues. Thus, the SASP is an array of diverse cytokines, chemokines, growth factors, and proteases that are characteristic of senescent cells. Senescent cells are stable, non-dividing cells that are still metabolically active and exhibit upregulation of a wide range of genes, including genes that code for secreted proteins such as inflammatory cytokines, chemokines, extracellular matrix remodeling factors, and growth factors. These secreted proteins may function physiologically within the tissue microenvironment to propagate stress responses or communicate with neighboring cells. This SASP phenotype is a key characteristic that reveals the paracrine function of senescent cells and distinguishes them from non-senescent cell cycle arrested cells, such as quiescent cells and terminally differentiated cells. SASP cytokines are cytokines that are specifically produced by senescent cells to generate a senescence-associated secretory phenotype. These cytokines include, but are not limited to, IL18, IL1A, GROA, IL22, and IL9.

[0103] Studies have shown that cellular senescence is associated with age-related symptoms, including epidermal thinning, skin wrinkling, hair loss and graying, loss of muscle thickness and strength, increased incidence of inflammation, metabolic disorders, decreased endurance, and age-related diseases.Furthermore, cellular senescence is believed to contribute to difficulties with wound healing.

[0104] Therefore, preventing ongoing cellular senescence or reversing cellular senescence in senescent cells would be advantageous in the treatment of various age-related conditions, wound healing, and cosmetic applications.

[0105] There are several assays that researchers use to detect senescence. 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (known as x-gal), a colorimetric substrate for β-gal, has long been used to detect metabolic activity in cells in vitro. Upon hydrolysis by β-gal, x-gal is converted to a blue precipitate that can be detected using a microscope. Although the x-gal assay is seen as the "gold standard" method, it is limited in that it is a colorimetric assay. C12FDG is a fluorescent alternative to x-gal. It also functions as a β-galactosidase substrate, but has the disadvantage of leaking out of the cell after a short period of time. In flow cytometry, a combination of antibody markers such as p16ARF and p21 can be used. An alternative is CellEvent™ Senescence Green Reagent. It provides a highly sensitive fluorescent substrate for β-gal that can be used to detect senescent cells in flow cytometry assays and imaging procedures. Not only is it a fluorescent substrate for β-gal, but it also offers the advantage of being able to bind covalently to intracellular proteins and therefore not leak out of cells over time.

[0106] Aspects of the disclosure relate to contacting a cell with an effective amount of SRSF1 protein (or other proteins such as SLC2A13, RNASEL, WDTC1, or NPM1) or a nucleic acid encoding SRSF1 protein (or other proteins such as SLC2A13, RNASEL, WDTC1, or NPM1), where the effective amount is sufficient to reduce cellular senescence compared to a control cell. In some embodiments, the effective amount is sufficient to reduce cellular senescence of the cell by at least 20%, at least 30%, at least 40%, or at least 50%. In some embodiments, the effective amount is sufficient to reduce cellular senescence of the cell by at least 50%. In some embodiments, the effective amount is sufficient to reduce cellular senescence of the cell by at least 50%. In some embodiments, the effective amount is sufficient to reduce senescence-associated β-galactosidase activity of the cell compared to a control. In some embodiments, an effective amount is sufficient to reduce senescence-associated β-galactosidase activity of a cell by at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50%. In some embodiments, an effective amount is sufficient to reduce proteasome activity of a cell compared to a control. In some embodiments, an effective amount is sufficient to reduce proteasome activity of a cell by at least 25%, at least 30%, at least 40%, at least 45%, or at least 50%.

[0107] Aspects of the disclosure relate to contacting a cell with an effective amount of an inhibitor of expression of one or more senescence-promoting genes (e.g., KAT7, ESR1, MAPK7, KDM6A, and CTNNB1), where the effective amount is sufficient to reduce cellular senescence compared to a control cell. In some embodiments, the effective amount is sufficient to reduce cellular senescence of the cell by at least 20%, at least 30%, at least 40%, or at least 50%. In some embodiments, the effective amount is sufficient to reduce cellular senescence of the cell by at least 50%. In some embodiments, the effective amount is sufficient to reduce senescence-associated β-galactosidase activity of the cell compared to a control. In some embodiments, the effective amount is sufficient to reduce senescence-associated β-galactosidase activity of the cell by at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50%. In some embodiments, an effective amount is sufficient to reduce proteasome activity in a cell relative to a control, hi some embodiments, an effective amount is sufficient to reduce proteasome activity in a cell by at least 25%, at least 30%, at least 40%, at least 45%, or at least 50%.

[0108] Senescence-associated β-galactosidase activity Some embodiments provide a method of inducing cellular rejuvenation, comprising contacting a cell with an effective amount of SRSF1 protein (or other proteins such as SLC2A13, RNASEL, WDTC1, or NPM1) or a nucleic acid encoding SRSF1 protein (or other proteins such as SLC2A13, RNASEL, WDTC1, or NPM1), where the effective amount is sufficient to reduce senescence-associated beta-galactosidase (SA-βgal) activity. Detectable SA-βgal activity at pH 6.0 allows identification of senescent cells in culture and mammalian tissues. SA-βgal activity can be assessed, for example, using cytochemistry protocols suitable for histochemical detection of individual senescent cells in both culture and tissue biopsies. As another example, a method based on alkalinization of lysosomes followed by use of 5-dodecanoylaminofluorescein di-β-D-galactopyranoside (C12FDG), a fluorescent substrate for βgal activity, can be used. For exemplary protocols, see, e.g., Debacq-Chainiaux, F et al. Nature Protocols 2009; 4: 1798-1806. Cytochemical methods can be applied to tissue sections and require simple reagents and equipment. Fluorescence-based methods have the advantage of being more quantitative and sensitive.

[0109] In some embodiments, an effective amount is sufficient to reduce SA-βgal activity by at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100% relative to a control, hi some embodiments, an effective amount is sufficient to reduce SA-βgal activity in cells by about 50% to about 100%.

[0110] Some embodiments provide a method of inducing cellular rejuvenation, comprising contacting a cell with an effective amount of an inhibitor of expression of one or more pro-senescence proteins (e.g., KAT7, ESR1, MAPK7, KDM6A, and CTNNB1) or expression of one or more nucleic acids encoding one or more pro-senescence proteins, where the effective amount is sufficient to reduce senescence-associated beta-galactosidase (SA-βgal) activity. Detectable SA-βgal activity at pH 6.0 can identify senescent cells in culture and mammalian tissues. SA-βgal activity can be assessed, for example, using cytochemistry protocols suitable for histochemical detection of individual senescent cells in both culture and tissue biopsies. As another example, a method based on alkalinization of lysosomes followed by the use of 5-dodecanoylaminofluorescein di-β-D-galactopyranoside (C12FDG), a fluorescent substrate for βgal activity, can be used. For exemplary protocols, see, e.g., Debacq-Chainiaux, F et al. Nature Protocols 2009; 4: 1798-1806. Cytochemical methods can be applied to tissue sections and require simple reagents and equipment. Fluorescence-based methods have the advantage of being more quantitative and sensitive.

[0111] In some embodiments, an effective amount is sufficient to reduce SA-βgal activity by at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100% relative to a control, hi some embodiments, an effective amount is sufficient to reduce SA-βgal activity in cells by about 50% to about 100%.

[0112] Proteasome activity Some aspects provide a method of inducing cellular rejuvenation, comprising contacting a cell with an effective amount of SRSF1 protein (or other proteins such as SLC2A13, RNASEL, WDTC1, or NPM1) or a nucleic acid encoding SRSF1 protein (or other proteins such as SLC2A13, RNASEL, WDTC1, or NPM1), where the effective amount is sufficient to reduce cellular proteasome activity. Proteasome activity refers to the degradation of unnecessary or damaged proteins by the protein complex proteasome through proteolysis, a chemical reaction that breaks peptide bonds. Chymotrypsin-like proteasome activity is a unique catalytic activity of the proteasome. In some embodiments, proteasome activity is measured using fluorescently labeled peptides. In some embodiments, proteasome activity is determined by measuring the release of a fluorophore from a peptide substrate. In some embodiments, proteasome activity is measured by a staining assay. In some embodiments, the staining assay measures a cellular characteristic associated with senescence. In some embodiments, the staining assay measures the activity of the 20S proteasome. In some embodiments, the staining assay measures the activity of the 26S proteasome.

[0113] In some embodiments, an effective amount is sufficient to reduce proteasome activity in a cell by at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% compared to a control, hi some embodiments, an effective amount is sufficient to reduce proteasome activity in a cell by about 50% to about 100%.

[0114] Some aspects provide a method of inducing cellular rejuvenation, comprising contacting a cell with an effective amount of an inhibitor of expression of one or more pro-senescence proteins (e.g., KAT7, ESR1, MAPK7, KDM6A, and CTNNB1) or expression of one or more nucleic acids encoding one or more pro-senescence proteins, where the effective amount is sufficient to reduce cellular proteasome activity. Proteasome activity refers to the degradation of unnecessary or damaged proteins by the proteasome, a protein complex, through proteolysis, a chemical reaction that breaks peptide bonds. Chymotrypsin-like proteasome activity is a unique catalytic activity of the proteasome. In some embodiments, proteasome activity is measured using fluorescently labeled peptides. In some embodiments, proteasome activity is determined by measuring the release of a fluorophore from a peptide substrate. In some embodiments, proteasome activity is measured by a staining assay. In some embodiments, the staining assay measures a cellular characteristic associated with senescence. In some embodiments, the staining assay measures the activity of the 20S proteasome. In some embodiments, the staining assay measures the activity of the 26S proteasome.

[0115] In some embodiments, an effective amount is sufficient to reduce proteasome activity in a cell by at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% compared to a control, hi some embodiments, an effective amount is sufficient to reduce proteasome activity in a cell by about 50% to about 100%.

[0116] Rejuvenated cells and production methods Some aspects of the disclosure relate to a method of inducing rejuvenation of a cell (one or more cells) by overexpression of SRSF1 protein. Other aspects relate to a method of inducing rejuvenation of a cell (one or more cells) by overexpression of SLC2A13 protein. Still other aspects relate to a method of inducing rejuvenation of a cell (one or more cells) by overexpression of RNASEL protein. Still other aspects relate to a method of inducing rejuvenation of a cell (one or more cells) by overexpression of WDTC1 protein. Other aspects relate to a method of inducing rejuvenation of a cell (one or more cells) by overexpression of NPM1 protein. Some aspects relate to a method of inducing rejuvenation of a cell (one or more cells) by contacting the cell with an inhibitor of expression of one or more pro-senescence proteins (e.g., KAT7, ESR1, MAPK7, KDM6A, and CTNNB1) or one or more nucleic acids encoding one or more pro-senescence proteins. In some embodiments, function is restored in the cell. Functions include, for example, mitochondrial function, proteolytic activity, heterochromatin levels, histone methylation, nuclear lamina polypeptides, cytokine secretion, or senescence. In some embodiments, SRSF1 protein or nucleic acid (DNA or RNA, e.g., RNA) encoding SRSF1 protein can be used to rejuvenate various cell types, including fibroblasts, hematopoietic stem cells, endothelial cells, chondrocytes, skeletal muscle stem cells, keratinocytes, mesenchymal stem cells, and corneal epithelial cells, optionally while retaining the identity of the cells. In some embodiments, SLC2A13 protein or nucleic acid (DNA or RNA, e.g., RNA) encoding SLC2A13 protein can be used to rejuvenate various cell types, including fibroblasts, hematopoietic stem cells, endothelial cells, chondrocytes, skeletal muscle stem cells, keratinocytes, mesenchymal stem cells, and corneal epithelial cells, optionally while retaining the identity of the cells.In some embodiments, the RNASEL protein or a nucleic acid (DNA or RNA, e.g., RNA) encoding the RNASEL protein can be used to rejuvenate a variety of cell types, including fibroblasts, hematopoietic stem cells, endothelial cells, chondrocytes, skeletal muscle stem cells, keratinocytes, mesenchymal stem cells, and corneal epithelial cells, optionally while preserving the identity of the cells. In some embodiments, the WDTC1 protein or a nucleic acid (DNA or RNA, e.g., RNA) encoding the WDTC1 protein can be used to rejuvenate a variety of cell types, including fibroblasts, hematopoietic stem cells, endothelial cells, chondrocytes, skeletal muscle stem cells, keratinocytes, mesenchymal stem cells, and corneal epithelial cells, optionally while preserving the identity of the cells. In some embodiments, the NPM1 protein or a nucleic acid (DNA or RNA, e.g., RNA) encoding the NPM1 protein can be used to rejuvenate a variety of cell types, including fibroblasts, hematopoietic stem cells, endothelial cells, chondrocytes, skeletal muscle stem cells, keratinocytes, mesenchymal stem cells, and corneal epithelial cells, optionally while preserving the identity of the cells.

[0117] Some aspects provide a method of inducing cellular rejuvenation, comprising transfecting a cell with a gene associated with inducing cellular rejuvenation, thereby inducing cellular rejuvenation. In some embodiments, a rejuvenated cell is produced.

[0118] In some embodiments, the rejuvenated cells have a phenotypic or activity profile similar to that of young cells, including one or more of the following: 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.

[0119] In some embodiments, the rejuvenated cells have a transcriptomic profile that is more similar to the transcriptomic profile of a young cell. In some embodiments, the transcriptomic profile of a rejuvenated cell includes: [ka] The present invention includes an increase or decrease in gene expression (e.g., toward levels expected in a young cell) of one or more genes selected from the group consisting of:

[0120] In some embodiments, the rejuvenated cells exhibit increased gene expression of one or more nuclear and / or epigenetic markers compared to a control (e.g., a reference value). In some embodiments, the one or more nuclear and / or epigenetic markers are selected from HP1 gamma, H3K9me3, lamina support protein LAP2 alpha, and SIRT1 protein. In some embodiments, the rejuvenated cells exhibit increased gene expression of HP1 gamma. In some embodiments, the rejuvenated cells exhibit increased gene expression of H3K9me3. In some embodiments, the rejuvenated cells exhibit increased gene expression of lamina support protein LAP2 alpha. In some embodiments, the rejuvenated cells exhibit increased gene expression of SIRT1 protein. In some embodiments, the rejuvenated cells exhibit increased gene expression of HP1 gamma, H3K9me3, lamina support protein LAP2 alpha, and SIRT1 protein.

[0121] In some embodiments, rejuvenated cells have proteolytic activity more similar to that of young cells. In some embodiments, proteolytic activity is measured as an increase in cellular autophagosome formation, an increase in chymotrypsin-like proteasome activity, or a combination thereof. In some embodiments, proteolytic activity is measured as an increase in cellular autophagosome formation. Autophagosomes are spherical structures with a bilayer membrane. They are key structures in macroautophagy, an intracellular degradation system for cytoplasmic contents (e.g., abnormal intracellular proteins, excess or damaged organelles) and invading microorganisms. After formation, autophagosomes deliver cytoplasmic components to lysosomes. The outer membrane of the autophagosome fuses with the lysosome to form the autolysosome. Lysosomal hydrolases degrade the contents delivered to the autophagosome and its inner membrane. In some embodiments, proteolytic activity is measured as an increase in chymotrypsin-like proteasome activity. In some embodiments, proteolytic activity is measured as an increase in cellular autophagosome formation and an increase in chymotrypsin-like proteasome activity.

[0122] In some embodiments, the rejuvenated cells exhibit improved mitochondrial health and function compared to a control (e.g., a reference value). In some embodiments, the improved mitochondrial health and function is measured as an increase in mitochondrial membrane potential, a decrease in reactive oxygen species (ROS), or a combination thereof. In some embodiments, the improved mitochondrial health and function is measured as an increase in mitochondrial membrane potential. In some embodiments, the improved mitochondrial health and function is measured as a decrease in reactive oxygen species (ROS). In some embodiments, the improved mitochondrial health and function is measured as an increase in mitochondrial membrane potential and a decrease in reactive oxygen species (ROS).

[0123] In some embodiments, the rejuvenated cells exhibit decreased expression of one or more SASP cytokines compared to a control (e.g., a reference value). In some embodiments, the one or more SASP cytokines include IL18, IL1A, GROA, IL22, and IL9. In some embodiments, the rejuvenated cells exhibit decreased expression of IL18. In some embodiments, the rejuvenated cells exhibit decreased expression of IL1A. In some embodiments, the rejuvenated cells exhibit decreased expression of GROA. In some embodiments, the rejuvenated cells exhibit decreased expression of IL22. In some embodiments, the rejuvenated cells exhibit decreased expression of IL9. In some embodiments, the rejuvenated cells exhibit decreased expression of IL18, IL1A, GROA, IL22, and IL9.

[0124] In some embodiments, the rejuvenated cells exhibit a reversal of the methylation landscape, in some embodiments, the reversal of the methylation landscape is measured by Horvath clock estimation.

[0125] In some embodiments, the reference value is obtained from young cells or senescent cells.

[0126] It should be understood that an "increase" or "decrease" (e.g., a decrease or reduction) in a property and / or function exhibited by a cell is relative or comparative to a control, such as a reference value (e.g., a value obtained from a young cell or an aged cell).

[0127] Cell Transfection Methods Aspects of the present disclosure relate to contacting a cell with an effective amount of SRSF1 protein (or SLC2A13, RNASEL, WDTC1, or NPM1 protein) or a nucleic acid (e.g., DNA) encoding the SRSF1 protein (or SLC2A13, RNASEL, WDTC1, or NPM1 protein). In some embodiments, the method comprises delivering the SRSF1 protein to the cell. In some embodiments, the method comprises delivering a nucleic acid comprising an open reading frame encoding the SRSF1 protein to the cell. In some embodiments, the nucleic acid is delivered on a non-viral vector (e.g., a plasmid) or a viral vector (e.g., an rAAV vector). In some embodiments, the contacting comprises transfecting the cell (e.g., using electroporation or a chemical transfection agent).

[0128] Transfection refers to the uptake of exogenous (e.g., engineered) nucleic acid (e.g., DNA or RNA) by a cell. A cell is "transfected" when an exogenous nucleic acid is introduced into the cell membrane. Many transfection techniques are generally known in the art. See, for example, Graham et al. (1973) Virology, 52:456, Sambrook et al. (2001) Molecular Cloning, a laboratory manual, 3rd edition, Cold Spring Harbor Laboratories, New York, Davis et al. (1995) Basic Methods in Molecular Biology, 2nd edition, McGraw-Hill, and Chu et al. (1981) Gene 13: 197. Such techniques can be used to introduce one or more engineered nucleic acids into a cell. The term refers to both stable and transient uptake of nucleic acid (e.g., DNA or RNA). For example, transfection can be used to transiently introduce mRNA encoding SRSF1, SLC2A13, RNASEL, WDTC1, and / or NPM1 into cells in need of rejuvenation.

[0129] In some embodiments, cells are transfected with engineered nucleic acids by nucleofection. As used herein, the term "nucleofection" refers to an electroporation-based transfection method that allows nucleic acids such as DNA and RNA to be introduced into cells by applying a specific voltage and using specific reagents. See, for example, Distler et al. (2005) Exp Dermatol, 14(4):315-20.

[0130] In embodiments, transfection of cells with SRSF1 protein (or SLC2A13, RNASEL, WDTC1, or NPM1 protein) or a nucleic acid encoding SRSF1 protein (or SLC2A13, RNASEL, WDTC1, or NPM1 protein) may be achieved by a transfection method selected from electroporation, nucleofection, lipofectamine and LT-1 mediated transfection, dextran mediated transfection, calcium phosphate precipitation, polybrene mediated transfection, encapsulation of nucleic acid (e.g., mRNA) in liposomes, and direct microinjection. In some embodiments, transfection of cells with protein or nucleic acid is achieved by lipofectamine and LT-1 mediated transfection. In some embodiments, transfection of cells with protein or nucleic acid is achieved by dextran mediated transfection. In some embodiments, transfection of cells with protein or nucleic acid is achieved by calcium phosphate precipitation. In some embodiments, transfection of cells with protein or nucleic acid is achieved by polybrene mediated transfection. In some embodiments, transfection of cells with proteins or nucleic acids is achieved by electroporation. In some embodiments, transfection of cells with proteins or nucleic acids is achieved by encapsulating mRNA in liposomes. In some embodiments, transfection of cells with proteins or nucleic acids is achieved by direct microinjection.

[0131] In some embodiments, the cells express SRSF1 at a level higher than the baseline level.

[0132] Methods for inhibiting protein and / or nucleic acid expression Aspects of the present disclosure relate, at least in part, to the identification of proteins associated with inhibiting cellular rejuvenation and / or accelerating cellular senescence. In some embodiments, these proteins are referred to as "pro-senescence" proteins. In some embodiments, the pro-senescence protein is KAT7. In some embodiments, the pro-senescence protein is ESR1. In some embodiments, the pro-senescence protein is MAPK7. In some embodiments, the pro-senescence protein is KDM6a. In some embodiments, the pro-senescence protein is CTNNB1.

[0133] Aspects of the present disclosure relate, at least in part, to methods of inhibiting expression of a pro-senescence protein or expression of a nucleic acid encoding a pro-senescence protein. In some embodiments, expression of a pro-senescence protein or expression of a nucleic acid encoding a pro-senescence protein is achieved using a clustered regularly interspaced short palindromic repeats (CRISPR) system. In some embodiments, expression of a pro-senescence protein or expression of a nucleic acid encoding a pro-senescence protein is achieved using a CRISPR-Cas9 system. In some embodiments, expression of a pro-senescence protein or expression of a nucleic acid encoding a pro-senescence protein is achieved using a CRISPR interference (CRISPRi) system. In some embodiments, inhibition of expression of a pro-senescence protein or expression of a nucleic acid encoding a pro-senescence protein is achieved using a CRISPRoff system. In some embodiments, inhibition of expression of a pro-senescence protein or expression of a nucleic acid encoding a pro-senescence protein is achieved using an shRNA. In some embodiments, inhibition of expression of a pro-senescence protein or expression of a nucleic acid encoding a pro-senescence protein is achieved using an siRNA. In some embodiments, inhibition of expression of a pro-senescence protein or expression of a nucleic acid encoding a pro-senescence protein is achieved using an miRNA. In some embodiments, inhibition of expression of a pro-senescence protein or of a nucleic acid encoding a pro-senescence protein is achieved using a small molecule inhibitor, hi some embodiments, inhibition of expression of a pro-senescence protein or of a nucleic acid encoding a pro-senescence protein is achieved using an antibody.

[0134] In some embodiments, the CRISPR system is a genome editing system. In some embodiments, a CRISPR guide RNA (gRNA) is associated with a CRISPR endonuclease. In some embodiments, the gRNA guides the endonuclease to a site in the genome. In some embodiments, the endonuclease cleaves genomic DNA. In some embodiments, cleaving the genomic DNA inhibits expression of the gene. In some embodiments, inhibiting expression of the gene inhibits expression of a protein encoded by the gene. In some embodiments, the endonuclease is a Cas9 endonuclease. In some embodiments, the endonuclease is a Cas12a endonuclease. In some embodiments, the endonuclease is a Cas13 endonuclease. In some embodiments, the CRISPR system comprises a gRNA and an endonuclease. In some embodiments, the CRISPR-Cas9 system comprises a gRNA and a Cas9 endonuclease. See, e.g., Doudna JA, Charpentier E. Science 346, 6213 (2014), the entire contents of which are incorporated herein by reference.

[0135] In some embodiments, the CRISPR system is a CRISPRi system. In some embodiments, the CRISPRi system includes a gRNA and a nuclease-inactive endonuclease. In some embodiments, the nuclease-inactive endonuclease is a nuclease-inactive Cas9 (dCas9). In some embodiments, the nuclease-inactive endonuclease cannot cleave DNA. In some embodiments, the nuclease-inactive endonuclease blocks transcription of a target gene. In some embodiments, the target gene is a gene determined by the gRNA. In some embodiments, the nuclease-inactive endonuclease blocks transcription by steric hindrance. In some embodiments, the CRISPRi reduces expression of the target gene and the protein encoded by the target gene compared to a control. See, e.g., Larson HM, et al. Nature Protocols 8, 2180-2196 (2013), the entire contents of which are incorporated herein by reference.

[0136] In some embodiments, the CRISPR system is a CRISPRoff system. In some embodiments, the CRISPRoff system includes a gRNA and a nuclease-inactive endonuclease. In some embodiments, the nuclease-inactive endonuclease is a nuclease-inactive Cas9 (dCas9). In some embodiments, the nuclease-inactive endonuclease cannot cleave DNA. In some embodiments, the CRISPRoff system initiates methylation at a target site in the genome. In some embodiments, the methylation blocks transcription of the target gene. In some embodiments, the CRISPRoff reduces expression of the target gene and the protein encoded by the target gene compared to a control. See, e.g., Nunez JK, et al. Cell 184, 2503-2519 (2021), the entire contents of which are incorporated herein by reference.

[0137] In some embodiments, inhibition of expression of a pro-senescence protein or expression of a nucleic acid encoding a pro-senescence protein is achieved using small hairpin RNA (shRNA). In some embodiments, the shRNA is inserted into a cell and converted into a hairpin RNA structure. In some embodiments, the shRNA binds to an mRNA and blocks translation of the mRNA. In some embodiments, the mRNA is degraded after binding to the shRNA. In some embodiments, inhibition of expression of a pro-senescence protein or expression of a nucleic acid encoding a pro-senescence protein is achieved using an siRNA. In some embodiments, the siRNA is an RNA duplex designed to target a specific mRNA sequence. In some embodiments, the siRNA binds to an mRNA and promotes degradation of the mRNA. In some embodiments, inhibition of expression of a pro-senescence protein or expression of a nucleic acid encoding a pro-senescence protein is achieved using a small molecule inhibitor. In some embodiments, the small molecule inhibitor targets a signaling molecule. In some embodiments, the small molecule inhibitor inhibits transcription. In some embodiments, the small molecule inhibitor blocks translation. In some embodiments, the small molecule inhibitor targets a DNA binding domain.

[0138] As will be appreciated by one of skill in the art, any of the inhibitory methods described herein can be delivered to a cell using any one of the delivery systems described herein, including but not limited to viral or non-viral vectors.

[0139] Cell type In some aspects, the methods are used to induce rejuvenation of cells. In some embodiments, the methods provided herein may be applied to any type of cell requiring rejuvenation. The cells may be intact living cells, natural or modified. The cells may be isolated from other cells, mixed with other cells in culture, or within a tissue (partial or intact) or organism. The methods described herein may be performed on samples including, for example, single cells, cell populations, or tissues or organs containing cells. The methods may also be used to deliver nucleic acids or proteins to cells in vivo. The cells selected for rejuvenation depend, in some embodiments, on the desired therapeutic effect for treating an age-related disease or condition.

[0140] In some embodiments, the cells are mammalian cells (e.g., cells derived from a mammalian subject suitable for transplantation into the same or a different subject). In some embodiments, the cells are human cells. In some embodiments, the cells are from an aged subject.

[0141] The cells may be xenogeneic, autologous, or allogeneic. The cells may be primary cells obtained directly from a mammalian subject. The cells may be cells obtained from the culture and expansion of cells obtained from a subject. In some embodiments, the cells are genetically engineered to express SRSF1 protein and / or a nucleic acid encoding the SRSF1 protein. In some embodiments, the cells are genetically engineered to express SLC2A13 protein and / or a nucleic acid encoding the SLC2A13 protein. In some embodiments, the cells are genetically engineered to express RNASEL protein and / or a nucleic acid encoding the RNASEL protein. In some embodiments, the cells are genetically engineered to express WDTC1 protein and / or a nucleic acid encoding the WDTC1 protein. In some embodiments, the cells are genetically engineered to express NPM1 protein and / or a nucleic acid encoding the NPM1 protein.

[0142] In some aspects, the method comprises contacting (e.g., transfecting) the cell with a therapeutically effective amount of SRSF1 protein or a nucleic acid encoding the SRSF1 protein. In other aspects, the method comprises contacting (e.g., transfecting) the cell with a therapeutically effective amount of SLC2A13 protein or a nucleic acid encoding the SLC2A13 protein. In still other aspects, the method comprises contacting (e.g., transfecting) the cell with a therapeutically effective amount of RNASEL protein or a nucleic acid encoding the RNASEL protein. In still other aspects, the method comprises contacting (e.g., transfecting) the cell with a therapeutically effective amount of WDTC1 protein or a nucleic acid encoding the WDTC1 protein. In still other aspects, the method comprises contacting (e.g., transfecting) the cell with a therapeutically effective amount of NPM1 protein or a nucleic acid encoding the protein. In some embodiments, the cell is selected from a fibroblast, a hematopoietic stem cell, an endothelial cell, a chondrocyte, a skeletal muscle stem cell, a keratinocyte, a mesenchymal stem cell, and a corneal epithelial cell. In some embodiments, the cell is a fibroblast. In some embodiments, the cell is a hematopoietic stem cell. In some embodiments, the cell is an endothelial cell. In some embodiments, the cell is a chondrocyte. In some embodiments, the cell is a skeletal muscle stem cell. In some embodiments, the cell is a keratinocyte. In some embodiments, the cell is a mesenchymal stem cell. In some embodiments, the cell is a corneal epithelial cell. In some embodiments, the cell is a cardiomyocyte.

[0143] Fibroblasts are a type of cell that contributes to the formation of connective tissue, a fibrous cellular material that supports and connects other tissues or organs in the body. Fibroblasts secrete collagen proteins that help maintain the structural framework of tissues. Dermal fibroblasts are the main cell type present in the connective tissue of the skin (dermis). Fibroblasts interact with epidermal cells during hair development and in interfollicular skin. In addition, they also play an important role in skin wound healing and skin bioengineering. Detailed procedures for establishing and maintaining primary cultures of adult human dermal fibroblasts are known (see, for example, Kisiel et al. Methods Mol Biol. 2019;1993:71-78).

[0144] In some embodiments, the rejuvenated fibroblasts exhibit a transcriptome profile similar to that of young fibroblasts. In some embodiments, the rejuvenated fibroblasts exhibit increased gene expression of one or more nuclear and / or epigenetic markers compared to the above control (e.g., reference value). In some embodiments, the rejuvenated fibroblasts have proteolytic activity more similar to that of the above young cells. In some embodiments, the rejuvenated fibroblasts exhibit improved mitochondrial health and function compared to the above control (e.g., reference value). In some embodiments, the rejuvenated fibroblasts exhibit a reversal of the methylation landscape.

[0145] In some embodiments, the rejuvenated endothelial cells exhibit a transcriptomic profile similar to that of young endothelial cells. In some embodiments, the rejuvenated endothelial cells exhibit increased gene expression of one or more nuclear and / or epigenetic markers compared to the above control (e.g., reference value). In some embodiments, the rejuvenated endothelial cells have proteolytic activity more similar to that of the above young cells. In some embodiments, the rejuvenated endothelial cells exhibit improved mitochondrial health and function compared to the above control (e.g., reference value). In some embodiments, the rejuvenated endothelial cells exhibit a reversal of the methylation landscape.

[0146] In some embodiments, the rejuvenated chondrocytes exhibit decreased expression of inflammatory factors and / or increased ATP and collagen metabolism. In some embodiments, the inflammatory factors include RANKL, iNOS2, IL6, IFNa, MCP3 and MIP1A. In some embodiments, the rejuvenated chondrocytes exhibit decreased expression of RANKL. In some embodiments, the rejuvenated chondrocytes exhibit decreased expression of iNOS2. In some embodiments, the rejuvenated chondrocytes exhibit decreased expression of IL6. In some embodiments, the rejuvenated chondrocytes exhibit decreased expression of IFNa. In some embodiments, the rejuvenated chondrocytes exhibit decreased expression of MCP3. In some embodiments, the rejuvenated chondrocytes exhibit decreased expression of MIP1A. In some embodiments, the rejuvenated chondrocytes exhibit decreased expression of RANKL, iNOS2, IL6, IFNa, MCP3 and MIP1A. In some embodiments, the rejuvenated chondrocytes exhibit increased ATP and collagen metabolism. In some embodiments, ATP and collagen metabolism is measured by one or more of an increase in ATP levels, a decrease in ROS and an increase in SOD2 expression, an increase in COL2A1 expression and overall proliferation by chondrocytes. In some embodiments, ATP and collagen metabolism is measured by an increase in ATP levels. In some embodiments, ATP and collagen metabolism is measured by a decrease in ROS and an increase in SOD2 expression. In some embodiments, ATP and collagen metabolism is measured by an increase in COL2A1 expression and overall proliferation by chondrocytes.

[0147] In some embodiments, rejuvenated skeletal muscle stem cells exhibit higher proliferation capacity, enhanced ability to differentiate into myoblasts and myofibers, lower kinetics of activation from quiescence, the ability to rejuvenate the muscle microenvironment, restore muscle youth, or combinations thereof.

[0148] In some embodiments, rejuvenated keratinocytes exhibit increased proliferative capacity, reduced inflammatory phenotype, reduced expression of RNAKL and INOS2, reduced expression of cytokines MIP1A, IL6, IFNa, MCP3, increased ATP, and increased expression levels of SOD2 and COL2A1.

[0149] In some embodiments, the rejuvenated mesenchymal stem cells exhibit decreased senescence parameters, increased cell proliferation, and / or decreased ROS levels. In some embodiments, the rejuvenated mesenchymal stem cells exhibit decreased senescence parameters. In some embodiments, the senescence parameters include pl6 expression, p2l expression, and positive SA Gal staining. In some embodiments, the rejuvenated mesenchymal stem cells exhibit increased cell proliferation. In some embodiments, the rejuvenated mesenchymal stem cells exhibit decreased ROS levels. In some embodiments, the rejuvenated mesenchymal stem cells exhibit decreased senescence parameters, increased cell proliferation, and decreased ROS levels.

[0150] In some embodiments, the rejuvenated corneal epithelial cells exhibit a decrease in senescence parameters. In some embodiments, the senescence parameters include one or more of expression of p21, expression of p16, mitochondrial biogenesis PGCla, and expression of inflammatory factor IL8. In some embodiments, the senescence parameters include p21. In some embodiments, the senescence parameters include expression of p16. In some embodiments, the senescence parameters include mitochondrial biogenesis PGC1a. In some embodiments, the senescence parameters include expression of inflammatory factor IL8. In some embodiments, the senescence parameters include expression of p21, expression of p16, mitochondrial biogenesis PGC1a, and expression of inflammatory factor IL8.

[0151] In some embodiments, the rejuvenated cardiomyocytes exhibit a decrease in senescence parameters. In some embodiments, the senescence parameters include expression of cyclin-dependent kinase inhibitors (CDKIs), such as p16INK4a, or p21Cip1 and p27Kip1, and activation of DNA damage response pathways. In some embodiments, the senescence parameters include expression of p16INK4a. In some embodiments, the senescence parameters include expression of CDKIs. In some embodiments, the senescence parameters include activation of DNA damage response pathways.

[0152] In some embodiments, the cell is a stem cell. In some embodiments, the cell is a hematopoietic stem cell. Stem cells are cells that retain the ability to self-renew by mitosis and can differentiate into a wide variety of specific 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 the developing embryo, stem cells can differentiate into all specific embryonic tissues. In adult organisms, stem and progenitor cells act as the body's repair system by replenishing specific cells. Totipotent stem cells are produced from the fusion of an egg cell and a sperm cell. Cells produced by the first few divisions of a fertilized egg are also totipotent. These cells can differentiate into embryonic and extraembryonic cell types. Pluripotent stem cells are the descendants of totipotent cells and can differentiate into cells derived from any of the three germ layers. Pluripotent stem cells can only produce cells of closely related cell families (e.g., hematopoietic stem cells differentiate into red blood cells, white blood cells, platelets, etc.). Unipotent cells can only produce one cell type, but have the property of self-renewal, which distinguishes them from non-stem cells. Induced pluripotent stem cells (iPSCs) are a type of pluripotent stem cell that can be obtained from adult cells that have been reprogrammed into an embryonic-like pluripotent state. Induced pluripotent stem cells can be obtained from adult somatic cells, such as skin or blood cells, for example.

[0153] treatment Aspects of the disclosure relate to methods and compositions for delivering or administering any of the proteins or nucleic acids described herein to a subject in need thereof.

[0154] subject A "subject" to which administration is contemplated refers to a human (i.e., male or female of any age, e.g., a pediatric subject (e.g., an infant, a child, or an adolescent) or an adult subject (e.g., a young adult, a middle-aged adult, or an elderly adult)) or a non-human animal. In certain embodiments, the non-human animal is a mammal (e.g., a primate (e.g., a cynomolgus or rhesus monkey), a commercially suitable mammal (e.g., a cow, a pig, a horse, a sheep, a goat, a cat, or a dog), or a bird (e.g., a commercially suitable bird such as a chicken, a duck, a goose, or a turkey)). In certain embodiments, the non-human animal is a fish, a reptile, or an amphibian. The non-human animal may be male or female at any stage of development. The non-human animal may be a transgenic or genetically engineered animal. The term "patient" refers to a human subject in need of treatment for a disease.

[0155] In some embodiments, the subject is a human subject. In some embodiments, the subject is a young adult. A young adult subject is between the ages of 18 and 44, inclusive.

[0156] In some embodiments, the subject is a middle-aged subject. The middle-aged subject is between 45 and 65 years old, inclusive. In some embodiments, the middle-aged subject is between 50 and 65 years old, or between 55 and 65 years old.

[0157] In some embodiments, the subject is an elderly subject. An elderly subject may be over 65 years old. In some embodiments, an elderly subject is between 70 and 85 years old, or between 75 and 85 years old.

[0158] In some embodiments, the subject is at least 50 years old. In some embodiments, the subject is at least 55 years old. In some embodiments, the subject is at least 60 years old. In some embodiments, the subject is at least 65 years old. In some embodiments, the subject is at least 70 years old. In some embodiments, the subject is at least 75 years old.

[0159] formulation The pharmaceutical compositions described herein can be prepared by any method known in the pharmaceutical art. In general, such methods of preparation include combining a compound described herein (i.e., the "active ingredient") with a carrier or excipient, and / or one or more other accessory ingredients, and then, if necessary and / or desirable, shaping and / or packaging the product into the desired single or multiple dosage unit.

[0160] Pharmaceutical compositions can be prepared, packaged, and / or sold in bulk, as a single unit dose, and / or as a plurality of single unit doses. A "unit dose" is a discrete amount of a pharmaceutical composition comprising a predetermined amount of an active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient that would be administered to a subject, and / or a convenient fraction of such a dosage, such as one-half or one-third of such a dosage.

[0161] The relative amounts of active ingredient, pharma- ceutically acceptable excipient, and / or any additional ingredients in the pharmaceutical compositions described herein will vary depending on the identity, size, and / or condition of the subject being treated, as well as the route by which the composition is administered. The compositions may contain from 0.1% to 100% (w / w) active ingredient.

[0162] Pharmaceutically acceptable excipients used in the manufacture of the provided pharmaceutical compositions include inert diluents, dispersing and / or granulating agents, surfactants and / or emulsifying agents, disintegrating agents, binders, preservatives, buffers, lubricants, and / or oils. Excipients such as cocoa butter and suppository waxes, colorants, coating agents, sweeteners, flavorings, and perfuming agents may also be present in the compositions.

[0163] The proteins and nucleic acids described herein may be formulated for a particular route of administration, for example, depending on the intended treatment. In some embodiments, the SRSF1 protein (or SLC2A13, RNASEL, WDTC1, or NPM1 protein) or the nucleic acid encoding the protein may be formulated for local delivery. In some embodiments, the SRSF1 protein (or SLC2A13, RNASEL, WDTC1, or NPM1 protein) or the nucleic acid encoding the protein is formulated for subcutaneous delivery. In some embodiments, the SRSF1 protein (or SLC2A13, RNASEL, WDTC1, or NPM1 protein) or the nucleic acid encoding the protein is formulated for intravenous delivery. In some embodiments, the SRSF1 protein (or SLC2A13, RNASEL, WDTC1, or NPM1 protein) or the nucleic acid encoding the protein is formulated for intramuscular delivery. In some embodiments, the formulation includes an mRNA encoding the SRSF1 protein (or an SLC2A13, RNASEL, WDTC1, or NPM1 protein) and a lipid nanoparticle (LNP) or other lipid-based delivery system. The SRSF1 protein (or an SLC2A13, RNASEL, WDTC1, or NPM1 protein) or a nucleic acid encoding the protein is formulated for delivery by electroporation in other embodiments.

[0164] In some embodiments, proteins, nucleic acids, or inhibitors of proteins and / or nucleic acids may be formulated with a pharma- ceutically acceptable excipient that does not cause significant adverse toxic effects in a subject, such as a human subject.

[0165] Route of Administration The route of administration of the proteins, nucleic acids, inhibitors, or protein and / or nucleic acid expression described herein can vary depending on how they are formulated. Non-limiting examples of routes of administration include topical, oral, nasal, intravenous, intramuscular, subcutaneous, and intraperitoneal.

[0166] The compositions provided herein can be administered by any route, including enteral (e.g., oral), parenteral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, subcutaneous, intracerebroventricular, transdermal, interdermal, rectal, intravaginal, intraperitoneal, topical (by powder, ointment, cream, and / or drops), mucosal, nasal, buccal, sublingual; intratracheal instillation, bronchial instillation, and / or inhalation; and / or oral spray, nasal spray, and / or aerosol. Specifically contemplated routes are oral administration, intravenous administration (e.g., systemic intravenous injection), local administration via the blood and / or lymphatic supply, and / or direct administration to the affected area. In general, the most appropriate route of administration will depend on a variety of factors, including the nature of the agent (e.g., its stability in the environment of the gastrointestinal tract) and / or the condition of the subject (e.g., whether the subject can tolerate oral administration). In certain embodiments, the compounds or pharmaceutical compositions described herein are suitable for topical administration to the eye of a subject.

[0167] The terms "administer," "administering," or "administration" refer to implanting, absorbing, ingesting, injecting, inhaling, or otherwise introducing a compound described herein, or a composition thereof, into a subject.

[0168] An "effective amount" of a compound described herein refers to an amount sufficient to induce a desired biological response. An effective amount of a compound described herein may vary depending on factors such as the desired biological endpoint, side effects, severity of the disease or disorder, identity, pharmacokinetics, and pharmacodynamics of the particular compound, the condition being treated, the mode, route, and desired or required frequency of administration, the species, age, and health or general condition of the subject. In certain embodiments, the effective amount is a therapeutically effective amount. In certain embodiments, the effective amount is a prophylactic treatment. In certain embodiments, the effective amount is the amount of a compound described herein in a single dose. In certain embodiments, the effective amount is the total amount of a compound described herein in multiple doses. In certain embodiments, the desired dosage is administered three times a day, twice a day, once a day, every other day, every third day, every week, every other week, every three weeks, or every four weeks. In certain embodiments, the desired dosage is delivered using multiple administrations (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more administrations).

[0169] The terms "treatment," "treat," and "treating" refer to reversing, alleviating, delaying the onset, or inhibiting progression of a disease as described herein. In some embodiments, treatment may be administered after one or more signs or symptoms of a disease have appeared or been observed. In other embodiments, treatment may be administered in the absence of signs or symptoms of a disease. For example, a susceptible subject may be administered treatment prior to the appearance of symptoms (e.g., in light of a history of symptoms and / or exposure to a pathogen). Treatment may also be continued after symptoms have resolved, e.g., to delay or prevent recurrence.

[0170] A "therapeutically effective amount" of a compound described herein is an amount sufficient to provide a therapeutic benefit in the treatment of a condition or to delay or minimize one or more symptoms associated with the condition. A therapeutically effective amount of a compound refers to an amount of a therapeutic agent that alone or in combination with other therapies provides a therapeutic benefit in the treatment of a condition. The term "therapeutically effective amount" can include an amount that improves overall treatment, reduces or avoids symptoms, signs, or causes of a condition, and / or enhances the therapeutic effect of another therapeutic agent.

[0171] Additional Embodiments Related to SLC2A13 1. A method for inducing cellular rejuvenation, comprising contacting a cell with an effective amount of SLC2A13 protein or a nucleic acid encoding an SLC2A13 protein.

[0172] 2. A method for inducing cellular rejuvenation in a subject, comprising administering to the subject an effective amount of SLC2A13 protein or a nucleic acid encoding an SLC2A13 protein.

[0173] 3. The method of embodiment 1 or 2, wherein the effective amount is sufficient to reduce cellular senescence of the cell compared to a control.

[0174] 4. The method of embodiment 3, wherein the effective amount is sufficient to reduce cellular senescence of the cell by at least 20%, at least 30%, at least 40%, or at least 50%.

[0175] 5. The method of embodiment 4, wherein the effective amount is sufficient to reduce cellular senescence of the cell by at least 50%.

[0176] 6. The method of any one of embodiments 3-5, wherein the effective amount is sufficient to reduce senescence-associated β-galactosidase activity in a cell compared to a control.

[0177] 7. The method of embodiment 6, wherein the effective amount is sufficient to reduce senescence-associated β-galactosidase activity in the cells by at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50%.

[0178] 8. The method of any one of embodiments 3 to 7, wherein the effective amount is sufficient to reduce proteasome activity in a cell compared to a control.

[0179] 9. The method of embodiment 8, wherein the effective amount is sufficient to reduce cellular proteasome activity by at least 25%, at least 30%, at least 40%, at least 45%, or at least 50%.

[0180] 10. The method of any one of the preceding embodiments, wherein the effective amount is sufficient to induce an average of cellular rejuvenation of at least 5 years, at least 10 years, at least 15 years, at least 20 years, at least 25 years, at least 30 years, at least 35 years, at least 40 years, at least 45 years, or at least 50 years.

[0181] 11. The method of embodiment 10, wherein the effective amount is sufficient to induce an average of cellular rejuvenation of at least 25 years.

[0182] 12. The method of any one of the preceding embodiments, wherein the cells are selected from fibroblasts, hematopoietic stem cells, endothelial cells, chondrocytes, skeletal muscle stem cells, keratinocytes, mesenchymal stem cells, and corneal epithelial cells.

[0183] 13. The method of embodiment 12, wherein the cell is a fibroblast.

[0184] 14. The method of embodiment 13, wherein the fibroblasts are human dermal fibroblasts.

[0185] 15. The method of any one of the preceding embodiments, wherein the nucleic acid comprises a heterologous promoter operably linked to the open reading frame.

[0186] 16. The method of embodiment 15, wherein the heterologous promoter is an inducible promoter.

[0187] 17. The method of any one of the preceding embodiments, comprising delivering SLC2A13 protein to a cell.

[0188] 18. The method of any one of the preceding embodiments, comprising delivering to the cell a nucleic acid comprising an open reading frame encoding a SLC2A13 protein.

[0189] 19. The method of any one of the preceding embodiments, wherein the nucleic acid is delivered on a non-viral or viral vector.

[0190] 20. The method of any one of the preceding embodiments, wherein the contacting comprises transfecting the cell.

[0191] 21. A method for inducing cellular rejuvenation, comprising overexpressing an effective amount of SLC2A13 protein in the cell.

[0192] 22. The method of embodiment 21, wherein the method comprises activating expression or activity of endogenous SLC2A13 protein at a level higher than a baseline level.

[0193] Additional Embodiments Related to RNASEL 1. A method of inducing cellular rejuvenation, comprising contacting a cell with an effective amount of an RNASEL protein or a nucleic acid encoding an RNASEL protein.

[0194] 2. A method of inducing cellular rejuvenation in a subject, comprising administering to the subject an effective amount of an RNASEL protein or a nucleic acid encoding an RNASEL protein.

[0195] 3. The method of embodiment 1 or 2, wherein the effective amount is sufficient to reduce cellular senescence of the cell compared to a control.

[0196] 4. The method of embodiment 3, wherein the effective amount is sufficient to reduce cellular senescence of the cell by at least 20%, at least 30%, at least 40%, or at least 50%.

[0197] 5. The method of embodiment 4, wherein the effective amount is sufficient to reduce cellular senescence of the cell by at least 50%.

[0198] 6. The method of any one of embodiments 3-5, wherein the effective amount is sufficient to reduce senescence-associated β-galactosidase activity in a cell compared to a control.

[0199] 7. The method of embodiment 6, wherein the effective amount is sufficient to reduce senescence-associated β-galactosidase activity in the cells by at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50%.

[0200] 8. The method of any one of embodiments 3 to 7, wherein the effective amount is sufficient to reduce proteasome activity in a cell compared to a control.

[0201] 9. The method of embodiment 8, wherein the effective amount is sufficient to reduce cellular proteasome activity by at least 25%, at least 30%, at least 40%, at least 45%, or at least 50%.

[0202] 10. The method of any one of the preceding embodiments, wherein the effective amount is sufficient to induce an average of cellular rejuvenation of at least 5 years, at least 10 years, at least 15 years, at least 20 years, at least 25 years, at least 30 years, at least 35 years, at least 40 years, at least 45 years, or at least 50 years.

[0203] 11. The method of embodiment 10, wherein the effective amount is sufficient to induce an average of cellular rejuvenation of at least 25 years.

[0204] 12. The method of any one of the preceding embodiments, wherein the cells are selected from fibroblasts, hematopoietic stem cells, endothelial cells, chondrocytes, skeletal muscle stem cells, keratinocytes, mesenchymal stem cells, and corneal epithelial cells.

[0205] 13. The method of embodiment 12, wherein the cell is a fibroblast.

[0206] 14. The method of embodiment 13, wherein the fibroblasts are human dermal fibroblasts.

[0207] 15. The method of any one of the preceding embodiments, wherein the nucleic acid comprises a heterologous promoter operably linked to the open reading frame.

[0208] 16. The method of embodiment 15, wherein the heterologous promoter is an inducible promoter.

[0209] 17. The method of any one of the preceding embodiments, comprising delivering an RNASEL protein to a cell.

[0210] 18. The method of any one of the preceding embodiments, comprising delivering a nucleic acid comprising an open reading frame encoding an RNASEL protein to a cell.

[0211] 19. The method of any one of the preceding embodiments, wherein the nucleic acid is delivered on a non-viral or viral vector.

[0212] 20. The method of any one of the preceding embodiments, wherein the contacting comprises transfecting the cell.

[0213] 21. A method for inducing cellular rejuvenation, comprising overexpressing an effective amount of RNASEL protein in a cell.

[0214] 22. The method of embodiment 21, wherein the method comprises activating expression or activity of endogenous RNASEL protein at a level higher than a baseline level.

[0215] Additional Embodiments Related to WDTC1 1. A method for inducing cellular rejuvenation, comprising contacting a cell with an effective amount of WDTC1 protein or a nucleic acid encoding the WDTC1 protein.

[0216] 2. A method for inducing cellular rejuvenation in a subject, comprising administering to the subject an effective amount of WDTC1 protein or a nucleic acid encoding the WDTC1 protein.

[0217] 3. The method of embodiment 1 or 2, wherein the effective amount is sufficient to reduce cellular senescence of the cell compared to a control.

[0218] 4. The method of embodiment 3, wherein the effective amount is sufficient to reduce cellular senescence of the cell by at least 20%, at least 30%, at least 40%, or at least 50%.

[0219] 5. The method of embodiment 4, wherein the effective amount is sufficient to reduce cellular senescence of the cell by at least 50%.

[0220] 6. The method of any one of embodiments 3-5, wherein the effective amount is sufficient to reduce senescence-associated β-galactosidase activity in a cell compared to a control.

[0221] 7. The method of embodiment 6, wherein the effective amount is sufficient to reduce senescence-associated β-galactosidase activity in the cells by at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50%.

[0222] 8. The method of any one of embodiments 3 to 7, wherein the effective amount is sufficient to reduce proteasome activity in a cell compared to a control.

[0223] 9. The method of embodiment 8, wherein the effective amount is sufficient to reduce cellular proteasome activity by at least 25%, at least 30%, at least 40%, at least 45%, or at least 50%.

[0224] 10. The method of any one of the preceding embodiments, wherein the effective amount is sufficient to induce an average of cellular rejuvenation of at least 5 years, at least 10 years, at least 15 years, at least 20 years, at least 25 years, at least 30 years, at least 35 years, at least 40 years, at least 45 years, or at least 50 years.

[0225] 11. The method of embodiment 10, wherein the effective amount is sufficient to induce an average of cellular rejuvenation of at least 25 years.

[0226] 12. The method of any one of the preceding embodiments, wherein the cells are selected from fibroblasts, hematopoietic stem cells, endothelial cells, chondrocytes, skeletal muscle stem cells, keratinocytes, mesenchymal stem cells, and corneal epithelial cells.

[0227] 13. The method of embodiment 12, wherein the cell is a fibroblast.

[0228] 14. The method of embodiment 13, wherein the fibroblasts are human dermal fibroblasts.

[0229] 15. The method of any one of the preceding embodiments, wherein the nucleic acid comprises a heterologous promoter operably linked to the open reading frame.

[0230] 16. The method of embodiment 15, wherein the heterologous promoter is an inducible promoter.

[0231] 17. The method of any one of the preceding embodiments, comprising delivering WDTC1 protein to a cell.

[0232] 18. The method of any one of the preceding embodiments, comprising delivering to the cell a nucleic acid comprising an open reading frame encoding the WDTC1 protein.

[0233] 19. The method of any one of the preceding embodiments, wherein the nucleic acid is delivered on a non-viral or viral vector.

[0234] 20. The method of any one of the preceding embodiments, wherein the contacting comprises transfecting the cell.

[0235] 21. A method for inducing cellular rejuvenation, comprising overexpressing an effective amount of WDTC1 protein in a cell.

[0236] 22. The method of embodiment 21, wherein the method comprises activating expression or activity of endogenous WDTC1 protein at a level higher than a baseline level.

[0237] Additional Embodiments Related to NPM1 1. A method of inducing cellular rejuvenation comprising contacting a cell with an effective amount of NPM1 protein or a nucleic acid encoding the NPM1 protein.

[0238] 2. A method of inducing cellular rejuvenation in a subject, comprising administering to the subject an effective amount of NPM1 protein or a nucleic acid encoding the NPM1 protein.

[0239] 3. The method of embodiment 1 or 2, wherein the effective amount is sufficient to reduce cellular senescence of the cell compared to a control.

[0240] 4. The method of embodiment 3, wherein the effective amount is sufficient to reduce cellular senescence of the cell by at least 20%, at least 30%, at least 40%, or at least 50%.

[0241] 5. The method of embodiment 4, wherein the effective amount is sufficient to reduce cellular senescence of the cell by at least 50%.

[0242] 6. The method of any one of embodiments 3-5, wherein the effective amount is sufficient to reduce senescence-associated β-galactosidase activity in a cell compared to a control.

[0243] 7. The method of embodiment 6, wherein the effective amount is sufficient to reduce senescence-associated β-galactosidase activity in the cells by at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50%.

[0244] 8. The method of any one of embodiments 3 to 7, wherein the effective amount is sufficient to reduce proteasome activity in a cell compared to a control.

[0245] 9. The method of embodiment 8, wherein the effective amount is sufficient to reduce cellular proteasome activity by at least 25%, at least 30%, at least 40%, at least 45%, or at least 50%.

[0246] 10. The method of any one of the preceding embodiments, wherein the effective amount is sufficient to induce an average of cellular rejuvenation of at least 5 years, at least 10 years, at least 15 years, at least 20 years, at least 25 years, at least 30 years, at least 35 years, at least 40 years, at least 45 years, or at least 50 years.

[0247] 11. The method of embodiment 10, wherein the effective amount is sufficient to induce an average of cellular rejuvenation of at least 25 years.

[0248] 12. The method of any one of the preceding embodiments, wherein the cells are selected from fibroblasts, hematopoietic stem cells, endothelial cells, chondrocytes, skeletal muscle stem cells, keratinocytes, mesenchymal stem cells, and corneal epithelial cells.

[0249] 13. The method of embodiment 12, wherein the cell is a fibroblast.

[0250] 14. The method of embodiment 13, wherein the fibroblasts are human dermal fibroblasts.

[0251] 15. The method of any one of the preceding embodiments, wherein the nucleic acid comprises a heterologous promoter operably linked to the open reading frame.

[0252] 16. The method of embodiment 15, wherein the heterologous promoter is an inducible promoter.

[0253] 17. The method of any one of the preceding embodiments, comprising delivering NPM1 protein to a cell.

[0254] 18. The method of any one of the preceding embodiments, comprising delivering to the cell a nucleic acid comprising an open reading frame encoding the NPM1 protein.

[0255] 19. The method of any one of the preceding embodiments, wherein the nucleic acid is delivered on a non-viral or viral vector.

[0256] 20. The method of any one of the preceding embodiments, wherein the contacting comprises transfecting the cell.

[0257] 21. A method for inducing cellular rejuvenation, comprising overexpressing an effective amount of NPM1 protein in the cell.

[0258] 22. The method of embodiment 21, wherein the method comprises activating expression or activity of endogenous NPM1 protein at a level higher than a baseline level.

[0259] Additional Embodiments Describing Anti-Aging Proteins 1. A method for inducing cellular rejuvenation comprising contacting a cell with an effective amount of one or more anti-aging proteins or one or more nucleic acids encoding one or more anti-aging proteins.

[0260] 2. A method for inducing cellular rejuvenation in a subject, comprising administering to the subject an effective amount of one or more anti-aging proteins or one or more nucleic acids encoding one or more anti-aging proteins.

[0261] 3. The method of embodiment 1 or 2, wherein the one or more anti-aging proteins are selected from the group consisting of SRSF1, SLC2A13, RNASEL, WDTC1, and NPM1.

[0262] 4. The method of any one of the preceding embodiments, wherein the effective amount is sufficient to reduce cellular senescence of the cell as compared to a control.

[0263] 5. The method of embodiment 4, wherein the effective amount is sufficient to reduce cellular senescence of the cell by at least 20%, at least 30%, at least 40%, or at least 50%.

[0264] 6. The method of embodiment 5, wherein the effective amount is sufficient to reduce cellular senescence of the cell by at least 50%.

[0265] 7. The method of any one of embodiments 4-6, wherein the effective amount is sufficient to reduce senescence-associated β-galactosidase activity in a cell compared to a control.

[0266] 8. The method of embodiment 7, wherein the effective amount is sufficient to reduce senescence-associated β-galactosidase activity in the cells by at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50%.

[0267] 9. The method of any one of embodiments 4 to 8, wherein the effective amount is sufficient to reduce proteasome activity in a cell compared to a control.

[0268] 10. The method of embodiment 9, wherein the effective amount is sufficient to reduce cellular proteasome activity by at least 25%, at least 30%, at least 40%, at least 45%, or at least 50%.

[0269] 11. The method of any one of the preceding embodiments, wherein the effective amount is sufficient to induce an average of cellular rejuvenation of at least 5 years, at least 10 years, at least 15 years, at least 20 years, at least 25 years, at least 30 years, at least 35 years, at least 40 years, at least 45 years, or at least 50 years.

[0270] 12. The method of embodiment 11, wherein the effective amount is sufficient to induce an average of cellular rejuvenation of at least 25 years.

[0271] 13. The method of any one of the preceding embodiments, wherein the cells are selected from fibroblasts, hematopoietic stem cells, endothelial cells, chondrocytes, skeletal muscle stem cells, keratinocytes, mesenchymal stem cells, and corneal epithelial cells.

[0272] 14. The method of embodiment 13, wherein the cell is a fibroblast.

[0273] 15. The method of embodiment 14, wherein the fibroblasts are human dermal fibroblasts.

[0274] 16. The method of any one of the preceding embodiments, wherein the one or more nucleic acids comprise one or more heterologous promoters operably linked to the one or more open reading frames.

[0275] 17. The method of embodiment 16, wherein the one or more heterologous promoters are inducible promoters.

[0276] 18. The method of any one of the preceding embodiments, comprising delivering one or more anti-aging proteins to a cell.

[0277] 19. The method of any one of the preceding embodiments, comprising delivering to the cell one or more nucleic acids comprising one or more open reading frames encoding one or more anti-aging proteins.

[0278] 20. The method of any one of the preceding embodiments, wherein the one or more nucleic acids are delivered on a non-viral or viral vector.

[0279] 21. The method of any one of the preceding embodiments, wherein the contacting comprises transfecting the cell.

[0280] 22. A method for inducing cellular rejuvenation, comprising overexpressing an effective amount of one or more anti-aging proteins in the cell.

[0281] 23. The method of embodiment 22, wherein the method comprises activating expression or activity of one or more endogenous anti-aging proteins at a level higher than a baseline level.

[0282] Additional Embodiments Related to KAT7 1. A method of inducing cellular rejuvenation comprising contacting a cell with an effective amount of an inhibitor of expression of KAT7 protein or expression of a nucleic acid encoding a KAT7 protein.

[0283] 2. A method of inducing cellular rejuvenation in a subject, comprising administering to the subject an effective amount of an inhibitor of expression of KAT7 protein or expression of a nucleic acid encoding KAT7 protein.

[0284] 3. The method of embodiment 1 or 2, wherein the inhibitor is a CRSPR-Cas9 system, a CRISPRi system, a CRISPRoff system, an shRNA, an siRNA, or a small molecule inhibitor.

[0285] 4. The method of any one of the preceding embodiments, wherein the effective amount is sufficient to reduce cellular senescence of the cell as compared to a control.

[0286] 5. The method of embodiment 4, wherein the effective amount is sufficient to reduce cellular senescence of the cell by at least 20%, at least 30%, at least 40%, or at least 50%.

[0287] 6. The method of embodiment 5, wherein the effective amount is sufficient to reduce cellular senescence of the cell by at least 50%.

[0288] 7. The method of any one of embodiments 4-6, wherein the effective amount is sufficient to reduce senescence-associated β-galactosidase activity in a cell compared to a control.

[0289] 8. The method of embodiment 7, wherein the effective amount is sufficient to reduce senescence-associated β-galactosidase activity in the cells by at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50%.

[0290] 9. The method of any one of embodiments 4 to 8, wherein the effective amount is sufficient to reduce proteasome activity in a cell compared to a control.

[0291] 10. The method of embodiment 9, wherein the effective amount is sufficient to reduce cellular proteasome activity by at least 25%, at least 30%, at least 40%, at least 45%, or at least 50%.

[0292] 11. The method of any one of the preceding embodiments, wherein the effective amount is sufficient to induce an average of cellular rejuvenation of at least 5 years, at least 10 years, at least 15 years, at least 20 years, at least 25 years, at least 30 years, at least 35 years, at least 40 years, at least 45 years, or at least 50 years.

[0293] 12. The method of embodiment 11, wherein the effective amount is sufficient to induce an average of cellular rejuvenation of at least 25 years.

[0294] 13. The method of any one of the preceding embodiments, wherein the cells are selected from fibroblasts, hematopoietic stem cells, endothelial cells, chondrocytes, skeletal muscle stem cells, keratinocytes, mesenchymal stem cells, and corneal epithelial cells.

[0295] 14. The method of embodiment 13, wherein the cell is a fibroblast.

[0296] 15. The method of embodiment 14, wherein the fibroblasts are human dermal fibroblasts.

[0297] 16. The method of any one of the preceding embodiments, wherein the nucleic acid comprises a heterologous promoter operably linked to the open reading frame.

[0298] 17. The method of embodiment 16, wherein the heterologous promoter is an inducible promoter.

[0299] 18. The method of any one of the preceding embodiments, comprising delivering an inhibitor of KAT7 protein expression to the cell.

[0300] 19. The method of any one of the preceding embodiments, comprising delivering to the cell an inhibitor of expression of a nucleic acid encoding a KAT7 protein.

[0301] 20. The method of any one of the preceding embodiments, wherein the inhibitor is delivered on a non-viral or viral vector.

[0302] 21. The method of any one of the preceding embodiments, wherein the contacting comprises transfecting the cell.

[0303] 22. The method of any one of the preceding embodiments, wherein the method comprises reducing expression or activity of endogenous KAT7 protein to a level below a baseline level.

[0304] Additional Embodiments Regarding ESR1 1. A method for inducing cellular rejuvenation, comprising contacting a cell with an effective amount of an inhibitor of expression of the ESR1 protein or of the expression of a nucleic acid encoding the ESR1 protein.

[0305] 2. A method for inducing cellular rejuvenation in a subject, comprising administering to the subject an effective amount of an inhibitor of expression of ESR1 protein or expression of a nucleic acid encoding ESR1 protein.

[0306] 3. The method of embodiment 1 or 2, wherein the inhibitor is a CRSPR-Cas9 system, a CRISPRi system, a CRISPRoff system, an shRNA, an siRNA, or a small molecule inhibitor.

[0307] 4. The method of any one of the preceding embodiments, wherein the effective amount is sufficient to reduce cellular senescence of the cell as compared to a control.

[0308] 5. The method of embodiment 4, wherein the effective amount is sufficient to reduce cellular senescence of the cell by at least 20%, at least 30%, at least 40%, or at least 50%.

[0309] 6. The method of embodiment 5, wherein the effective amount is sufficient to reduce cellular senescence of the cell by at least 50%.

[0310] 7. The method of any one of embodiments 4-6, wherein the effective amount is sufficient to reduce senescence-associated β-galactosidase activity in a cell compared to a control.

[0311] 8. The method of embodiment 7, wherein the effective amount is sufficient to reduce senescence-associated β-galactosidase activity in the cells by at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50%.

[0312] 9. The method of any one of embodiments 4 to 8, wherein the effective amount is sufficient to reduce proteasome activity in a cell compared to a control.

[0313] 10. The method of embodiment 9, wherein the effective amount is sufficient to reduce cellular proteasome activity by at least 25%, at least 30%, at least 40%, at least 45%, or at least 50%.

[0314] 11. The method of any one of the preceding embodiments, wherein the effective amount is sufficient to induce an average of cellular rejuvenation of at least 5 years, at least 10 years, at least 15 years, at least 20 years, at least 25 years, at least 30 years, at least 35 years, at least 40 years, at least 45 years, or at least 50 years.

[0315] 12. The method of embodiment 11, wherein the effective amount is sufficient to induce an average of cellular rejuvenation of at least 25 years.

[0316] 13. The method of any one of the preceding embodiments, wherein the cells are selected from fibroblasts, hematopoietic stem cells, endothelial cells, chondrocytes, skeletal muscle stem cells, keratinocytes, mesenchymal stem cells, and corneal epithelial cells.

[0317] 14. The method of embodiment 13, wherein the cell is a fibroblast.

[0318] 15. The method of embodiment 14, wherein the fibroblasts are human dermal fibroblasts.

[0319] 16. The method of any one of the preceding embodiments, wherein the nucleic acid comprises a heterologous promoter operably linked to the open reading frame.

[0320] 17. The method of embodiment 16, wherein the heterologous promoter is an inducible promoter.

[0321] 18. The method of any one of the preceding embodiments, comprising delivering an inhibitor of ESR1 protein expression to the cell.

[0322] 19. The method of any one of the preceding embodiments, comprising delivering to the cell an inhibitor of expression of a nucleic acid encoding the ESR1 protein.

[0323] 20. The method of any one of the preceding embodiments, wherein the inhibitor is delivered on a non-viral or viral vector.

[0324] 21. The method of any one of the preceding embodiments, wherein the contacting comprises transfecting the cell.

[0325] 22. The method of any one of the preceding embodiments, wherein the method comprises reducing the expression or activity of endogenous ESR1 protein to a level below a baseline level.

[0326] Additional embodiments relating to MAPK7 1. A method of inducing cellular rejuvenation, comprising contacting a cell with an effective amount of an inhibitor of expression of a MAPK7 protein or expression of a nucleic acid encoding a MAPK7 protein.

[0327] 2. A method of inducing cellular rejuvenation in a subject, comprising administering to the subject an effective amount of an inhibitor of expression of MAPK7 protein or expression of a nucleic acid encoding MAPK7 protein.

[0328] 3. The method of embodiment 1 or 2, wherein the inhibitor is a CRSPR-Cas9 system, a CRISPRi system, a CRISPRoff system, an shRNA, an siRNA, or a small molecule inhibitor.

[0329] 4. The method of any one of the preceding embodiments, wherein the effective amount is sufficient to reduce cellular senescence of the cell as compared to a control.

[0330] 5. The method of embodiment 4, wherein the effective amount is sufficient to reduce cellular senescence of the cell by at least 20%, at least 30%, at least 40%, or at least 50%.

[0331] 6. The method of embodiment 5, wherein the effective amount is sufficient to reduce cellular senescence of the cell by at least 50%.

[0332] 7. The method of any one of embodiments 4-6, wherein the effective amount is sufficient to reduce senescence-associated β-galactosidase activity in a cell compared to a control.

[0333] 8. The method of embodiment 7, wherein the effective amount is sufficient to reduce senescence-associated β-galactosidase activity in the cells by at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50%.

[0334] 9. The method of any one of embodiments 4 to 8, wherein the effective amount is sufficient to reduce proteasome activity in a cell compared to a control.

[0335] 10. The method of embodiment 9, wherein the effective amount is sufficient to reduce cellular proteasome activity by at least 25%, at least 30%, at least 40%, at least 45%, or at least 50%.

[0336] 11. The method of any one of the preceding embodiments, wherein the effective amount is sufficient to induce an average of cellular rejuvenation of at least 5 years, at least 10 years, at least 15 years, at least 20 years, at least 25 years, at least 30 years, at least 35 years, at least 40 years, at least 45 years, or at least 50 years.

[0337] 12. The method of embodiment 11, wherein the effective amount is sufficient to induce an average of cellular rejuvenation of at least 25 years.

[0338] 13. The method of any one of the preceding embodiments, wherein the cells are selected from fibroblasts, hematopoietic stem cells, endothelial cells, chondrocytes, skeletal muscle stem cells, keratinocytes, mesenchymal stem cells, and corneal epithelial cells.

[0339] 14. The method of embodiment 13, wherein the cell is a fibroblast.

[0340] 15. The method of embodiment 14, wherein the fibroblasts are human dermal fibroblasts.

[0341] 16. The method of any one of the preceding embodiments, wherein the nucleic acid comprises a heterologous promoter operably linked to the open reading frame.

[0342] 17. The method of embodiment 16, wherein the heterologous promoter is an inducible promoter.

[0343] 18. The method of any one of the preceding embodiments, comprising delivering an inhibitor of MAPK7 protein expression to the cell.

[0344] 19. The method of any one of the preceding embodiments, comprising delivering to the cell an inhibitor of expression of a nucleic acid encoding a MAPK7 protein.

[0345] 20. The method of any one of the preceding embodiments, wherein the inhibitor is delivered on a non-viral or viral vector.

[0346] 21. The method of any one of the preceding embodiments, wherein the contacting comprises transfecting the cell.

[0347] 22. The method of any one of the preceding embodiments, wherein the method comprises reducing endogenous MAPK7 protein expression or activity to a level below a baseline level.

[0348] Additional embodiments related to KDM6A 1. A method of inducing cellular rejuvenation comprising contacting a cell with an effective amount of an inhibitor of KDM6A protein expression or expression of a nucleic acid encoding a KDM6A protein.

[0349] 2. A method for inducing cellular rejuvenation in a subject, comprising administering to the subject an effective amount of an inhibitor of expression of KDM6A protein or expression of a nucleic acid encoding KDM6A protein.

[0350] 3. The method of embodiment 1 or 2, wherein the inhibitor is a CRSPR-Cas9 system, a CRISPRi system, a CRISPRoff system, an shRNA, an siRNA, or a small molecule inhibitor.

[0351] 4. The method of any one of the preceding embodiments, wherein the effective amount is sufficient to reduce cellular senescence of the cell as compared to a control.

[0352] 5. The method of embodiment 4, wherein the effective amount is sufficient to reduce cellular senescence of the cell by at least 20%, at least 30%, at least 40%, or at least 50%.

[0353] 6. The method of embodiment 5, wherein the effective amount is sufficient to reduce cellular senescence of the cell by at least 50%.

[0354] 7. The method of any one of embodiments 4-6, wherein the effective amount is sufficient to reduce senescence-associated β-galactosidase activity in a cell compared to a control.

[0355] 8. The method of embodiment 7, wherein the effective amount is sufficient to reduce senescence-associated β-galactosidase activity in the cells by at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50%.

[0356] 9. The method of any one of embodiments 4 to 8, wherein the effective amount is sufficient to reduce proteasome activity in a cell compared to a control.

[0357] 10. The method of embodiment 9, wherein the effective amount is sufficient to reduce cellular proteasome activity by at least 25%, at least 30%, at least 40%, at least 45%, or at least 50%.

[0358] 11. The method of any one of the preceding embodiments, wherein the effective amount is sufficient to induce an average of cellular rejuvenation of at least 5 years, at least 10 years, at least 15 years, at least 20 years, at least 25 years, at least 30 years, at least 35 years, at least 40 years, at least 45 years, or at least 50 years.

[0359] 12. The method of embodiment 11, wherein the effective amount is sufficient to induce an average of cellular rejuvenation of at least 25 years.

[0360] 13. The method of any one of the preceding embodiments, wherein the cells are selected from fibroblasts, hematopoietic stem cells, endothelial cells, chondrocytes, skeletal muscle stem cells, keratinocytes, mesenchymal stem cells, and corneal epithelial cells.

[0361] 14. The method of embodiment 13, wherein the cell is a fibroblast.

[0362] 15. The method of embodiment 14, wherein the fibroblasts are human dermal fibroblasts.

[0363] 16. The method of any one of the preceding embodiments, wherein the nucleic acid comprises a heterologous promoter operably linked to the open reading frame.

[0364] 17. The method of embodiment 16, wherein the heterologous promoter is an inducible promoter.

[0365] 18. The method of any one of the preceding embodiments, comprising delivering an inhibitor of KDM6A protein expression to the cell.

[0366] 19. The method of any one of the preceding embodiments, comprising delivering to the cell an inhibitor of expression of a nucleic acid encoding a KDM6A protein.

[0367] 20. The method of any one of the preceding embodiments, wherein the inhibitor is delivered on a non-viral or viral vector.

[0368] 21. The method of any one of the preceding embodiments, wherein the contacting comprises transfecting the cell.

[0369] 22. The method of any one of the preceding embodiments, wherein the method comprises reducing endogenous KDM6A protein expression or activity to a level below a baseline level.

[0370] Additional Embodiments Related to CTNNB1 1. A method for inducing cellular rejuvenation, comprising contacting a cell with an effective amount of an inhibitor of expression of CTNNB1 protein or expression of a nucleic acid encoding CTNNB1 protein.

[0371] 2. A method for inducing cellular rejuvenation in a subject, comprising administering to the subject an effective amount of an inhibitor of expression of CTNNB1 protein or expression of a nucleic acid encoding CTNNB1 protein.

[0372] 3. The method of embodiment 1 or 2, wherein the inhibitor is a CRSPR-Cas9 system, a CRISPRi system, a CRISPRoff system, an shRNA, an siRNA, or a small molecule inhibitor.

[0373] 4. The method of any one of the preceding embodiments, wherein the effective amount is sufficient to reduce cellular senescence of the cell as compared to a control.

[0374] 5. The method of embodiment 4, wherein the effective amount is sufficient to reduce cellular senescence of the cell by at least 20%, at least 30%, at least 40%, or at least 50%.

[0375] 6. The method of embodiment 5, wherein the effective amount is sufficient to reduce cellular senescence of the cell by at least 50%.

[0376] 7. The method of any one of embodiments 4-6, wherein the effective amount is sufficient to reduce senescence-associated β-galactosidase activity in a cell compared to a control.

[0377] 8. The method of embodiment 7, wherein the effective amount is sufficient to reduce senescence-associated β-galactosidase activity in the cells by at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50%.

[0378] 9. The method of any one of embodiments 4 to 8, wherein the effective amount is sufficient to reduce proteasome activity in a cell compared to a control.

[0379] 10. The method of embodiment 9, wherein the effective amount is sufficient to reduce cellular proteasome activity by at least 25%, at least 30%, at least 40%, at least 45%, or at least 50%.

[0380] 11. The method of any one of the preceding embodiments, wherein the effective amount is sufficient to induce an average of cellular rejuvenation of at least 5 years, at least 10 years, at least 15 years, at least 20 years, at least 25 years, at least 30 years, at least 35 years, at least 40 years, at least 45 years, or at least 50 years.

[0381] 12. The method of embodiment 11, wherein the effective amount is sufficient to induce an average of cellular rejuvenation of at least 25 years.

[0382] 13. The method of any one of the preceding embodiments, wherein the cells are selected from fibroblasts, hematopoietic stem cells, endothelial cells, chondrocytes, skeletal muscle stem cells, keratinocytes, mesenchymal stem cells, and corneal epithelial cells.

[0383] 14. The method of embodiment 13, wherein the cell is a fibroblast.

[0384] 15. The method of embodiment 14, wherein the fibroblasts are human dermal fibroblasts.

[0385] 16. The method of any one of the preceding embodiments, wherein the nucleic acid comprises a heterologous promoter operably linked to the open reading frame.

[0386] 17. The method of embodiment 16, wherein the heterologous promoter is an inducible promoter.

[0387] 18. The method of any one of the preceding embodiments, comprising delivering an inhibitor of CTNNB1 protein expression to the cell.

[0388] 19. The method of any one of the preceding embodiments, comprising delivering to the cell an inhibitor of expression of a nucleic acid encoding a CTNNB1 protein.

[0389] 20. The method of any one of the preceding embodiments, wherein the inhibitor is delivered on a non-viral or viral vector.

[0390] 21. The method of any one of the preceding embodiments, wherein the contacting comprises transfecting the cell.

[0391] 22. The method of any one of the preceding embodiments, wherein the method comprises reducing the expression or activity of endogenous CTNNB1 protein to a level below a baseline level.

[0392] Additional Embodiments Dealing with Pro-Senescence Proteins 1. A method for inducing cellular rejuvenation comprising contacting a cell with an effective amount of an inhibitor of expression of one or more pro-senescence proteins or expression of one or more nucleic acids encoding one or more pro-senescence proteins.

[0393] 2. A method for inducing cellular rejuvenation in a subject, comprising administering to the subject an effective amount of an inhibitor of expression of one or more pro-senescence proteins or expression of one or more nucleic acids encoding one or more pro-senescence proteins.

[0394] 3. The method of embodiment 1 or 2, wherein the one or more pro-senescence proteins are selected from the group consisting of KAT7, ESR1, MAPK7, KDM6A, and CTNNB1.

[0395] 4. The method of any one of the preceding embodiments, wherein the inhibitor is a CRSPR-Cas9 system, a CRISPRi system, a CRISPRoff system, an shRNA, an siRNA, or a small molecule inhibitor.

[0396] 5. The method of any one of the preceding embodiments, wherein the effective amount is sufficient to reduce cellular senescence of the cell as compared to a control.

[0397] 6. The method of embodiment 5, wherein the effective amount is sufficient to reduce cellular senescence of the cell by at least 20%, at least 30%, at least 40%, or at least 50%.

[0398] 7. The method of embodiment 6, wherein the effective amount is sufficient to reduce cellular senescence of the cell by at least 50%.

[0399] 8. The method of any one of embodiments 5-7, wherein the effective amount is sufficient to reduce senescence-associated β-galactosidase activity in a cell compared to a control.

[0400] 9. The method of embodiment 8, wherein the effective amount is sufficient to reduce senescence-associated β-galactosidase activity in the cells by at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50%.

[0401] 10. The method of any one of embodiments 5 to 9, wherein the effective amount is sufficient to reduce proteasome activity in a cell compared to a control.

[0402] 11. The method of embodiment 10, wherein the effective amount is sufficient to reduce cellular proteasome activity by at least 25%, at least 30%, at least 40%, at least 45%, or at least 50%.

[0403] 12. The method of any one of the preceding embodiments, wherein the effective amount is sufficient to induce an average of cellular rejuvenation of at least 5 years, at least 10 years, at least 15 years, at least 20 years, at least 25 years, at least 30 years, at least 35 years, at least 40 years, at least 45 years, or at least 50 years.

[0404] 13. The method of embodiment 12, wherein the effective amount is sufficient to induce an average of cellular rejuvenation of at least 25 years.

[0405] 14. The method of any one of the preceding embodiments, wherein the cells are selected from fibroblasts, hematopoietic stem cells, endothelial cells, chondrocytes, skeletal muscle stem cells, keratinocytes, mesenchymal stem cells and corneal epithelial cells.

[0406] 15. The method of embodiment 14, wherein the cell is a fibroblast.

[0407] 16. The method of embodiment 15, wherein the fibroblasts are human dermal fibroblasts.

[0408] 17. The method of any one of the preceding embodiments, wherein the one or more nucleic acids comprise one or more heterologous promoters operably linked to one or more open reading frames.

[0409] 18. The method of embodiment 17, wherein the one or more heterologous promoters are inducible promoters.

[0410] 19. The method of any one of the preceding embodiments, comprising delivering to the cell an inhibitor of expression of one or more pro-senescence proteins.

[0411] 20. The method of any one of the preceding embodiments, comprising delivering to the cell an inhibitor of expression of one or more nucleic acids encoding one or more pro-senescence proteins.

[0412] 21. The method of any one of the preceding embodiments, wherein the inhibitor is delivered on a non-viral or viral vector.

[0413] 22. The method of any one of the preceding embodiments, wherein the contacting comprises transfecting the cell.

[0414] 23. The method of any one of the preceding embodiments, wherein the method comprises reducing expression or activity of one or more endogenous pro-senescence proteins to a level below a baseline level.

[0415] Additional Embodiments Concerning Anti-Aging and Pro-Aging Proteins 1. A method for inducing cellular rejuvenation, comprising: (i) contacting the cell with an effective amount of one or more anti-aging proteins or one or more nucleic acids encoding one or more anti-aging proteins; and / or (ii) contacting the cell with an effective amount of an inhibitor of expression of one or more pro-senescence proteins or expression of one or more nucleic acids encoding one or more pro-senescence proteins. A method comprising:

[0416] 2. A method of inducing cellular rejuvenation in a subject, comprising: (i) administering to a subject an effective amount of one or more anti-aging proteins or one or more nucleic acids encoding one or more anti-aging proteins; and / or (ii) administering to the subject an effective amount of an inhibitor of expression of one or more pro-senescence proteins or expression of one or more nucleic acids encoding one or more pro-senescence proteins. A method comprising:

[0417] 3. The method of embodiment 1 or 2, wherein the one or more anti-aging proteins are selected from the group consisting of SRSF1, SLC2A13, RNASEL, WDTC1, and NPM1.

[0418] 4. The method of embodiment 1 or 2, wherein the one or more pro-senescence proteins are selected from the group consisting of KAT7, ESR1, MAPK7, KDM6A, and CTNNB1.

[0419] 5. The method of any one of the preceding embodiments, wherein the inhibitor is a CRSPR-Cas9 system, a CRISPRi system, a CRISPRoff system, an shRNA, an siRNA, or a small molecule inhibitor.

[0420] 6. The method of any one of the preceding embodiments, wherein the effective amount is sufficient to reduce cellular senescence of the cell as compared to a control.

[0421] 7. The method of embodiment 6, wherein the effective amount is sufficient to reduce cellular senescence of the cell by at least 20%, at least 30%, at least 40%, or at least 50%.

[0422] 8. The method of embodiment 7, wherein the effective amount is sufficient to reduce cellular senescence of the cell by at least 50%.

[0423] 9. The method of any one of embodiments 6-8, wherein the effective amount is sufficient to reduce senescence-associated β-galactosidase activity in a cell compared to a control.

[0424] 10. The method of embodiment 9, wherein the effective amount is sufficient to reduce senescence-associated β-galactosidase activity in the cells by at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50%.

[0425] 11. The method of any one of embodiments 6 to 10, wherein the effective amount is sufficient to reduce proteasome activity in a cell compared to a control.

[0426] 12. The method of embodiment 11, wherein the effective amount is sufficient to reduce cellular proteasome activity by at least 25%, at least 30%, at least 40%, at least 45%, or at least 50%.

[0427] 13. The method of any one of the preceding embodiments, wherein the effective amount is sufficient to induce an average of cellular rejuvenation of at least 5 years, at least 10 years, at least 15 years, at least 20 years, at least 25 years, at least 30 years, at least 35 years, at least 40 years, at least 45 years, or at least 50 years.

[0428] 14. The method of embodiment 13, wherein the effective amount is sufficient to induce an average of cellular rejuvenation of at least 25 years.

[0429] 15. The method of any one of the preceding embodiments, wherein the cells are selected from fibroblasts, hematopoietic stem cells, endothelial cells, chondrocytes, skeletal muscle stem cells, keratinocytes, mesenchymal stem cells and corneal epithelial cells.

[0430] 16. The method of embodiment 15, wherein the cell is a fibroblast.

[0431] 17. The method of embodiment 16, wherein the fibroblasts are human dermal fibroblasts.

[0432] 18. The method of any one of the preceding embodiments, wherein the one or more nucleic acids comprise one or more heterologous promoters operably linked to one or more open reading frames.

[0433] 19. The method of embodiment 18, wherein the one or more heterologous promoters are inducible promoters.

[0434] 20. The method of any one of the preceding embodiments, comprising delivering one or more anti-aging proteins to a cell.

[0435] 21. The method of any one of the preceding embodiments, comprising delivering to the cell an inhibitor of expression of one or more pro-senescence proteins.

[0436] 22. The method of any one of the preceding embodiments, comprising delivering to the cell one or more nucleic acids comprising one or more open reading frames encoding one or more anti-aging proteins.

[0437] 23. The method of any one of the preceding embodiments, comprising delivering to the cell an inhibitor of expression of one or more nucleic acids encoding one or more pro-senescence proteins.

[0438] 24. The method of any one of the preceding embodiments, wherein the one or more nucleic acids are delivered on a non-viral or viral vector.

[0439] 25. The method of any one of the preceding embodiments, wherein the contacting comprises transfecting the cell.

[0440] 26. A method for inducing cellular rejuvenation, comprising overexpressing an effective amount of one or more anti-aging proteins in the cell.

[0441] 27. The method of embodiment 26, wherein the method comprises activating expression or activity of one or more endogenous anti-aging proteins at a level higher than a baseline level.

[0442] 28. The method of any one of the preceding embodiments, wherein the method comprises reducing expression or activity of one or more endogenous pro-senescence proteins to a level below a baseline level.

[0443] Additional Embodiments 1. A method of inducing cellular rejuvenation comprising contacting a cell with an effective amount of a protein of Table 3 or a nucleic acid encoding a protein of Table 3.

[0444] 2. A method of inducing cellular rejuvenation in a subject comprising administering to the subject an effective amount of a protein of Table 3 or a nucleic acid encoding a protein of Table 3.

[0445] 3. The method of embodiment 1 or 2, wherein the effective amount is sufficient to reduce cellular senescence of the cell compared to a control.

[0446] 4. The method of embodiment 3, wherein the effective amount is sufficient to reduce cellular senescence of the cell by at least 20%, at least 30%, at least 40%, or at least 50%.

[0447] 5. The method of embodiment 4, wherein the effective amount is sufficient to reduce cellular senescence of the cell by at least 50%.

[0448] 6. The method of any one of embodiments 3-5, wherein the effective amount is sufficient to reduce senescence-associated β-galactosidase activity in a cell compared to a control.

[0449] 7. The method of embodiment 6, wherein the effective amount is sufficient to reduce senescence-associated β-galactosidase activity in the cells by at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50%.

[0450] 8. The method of any one of embodiments 3 to 7, wherein the effective amount is sufficient to reduce proteasome activity in a cell compared to a control.

[0451] 9. The method of embodiment 8, wherein the effective amount is sufficient to reduce cellular proteasome activity by at least 25%, at least 30%, at least 40%, at least 45%, or at least 50%.

[0452] 10. The method of any one of the preceding embodiments, wherein the effective amount is sufficient to induce an average of cellular rejuvenation of at least 5 years, at least 10 years, at least 15 years, at least 20 years, at least 25 years, at least 30 years, at least 35 years, at least 40 years, at least 45 years, or at least 50 years.

[0453] 11. The method of embodiment 10, wherein the effective amount is sufficient to induce an average of cellular rejuvenation of at least 25 years.

[0454] 12. The method of any one of the preceding embodiments, wherein the cells are selected from fibroblasts, hematopoietic stem cells, endothelial cells, chondrocytes, skeletal muscle stem cells, keratinocytes, mesenchymal stem cells, and corneal epithelial cells.

[0455] 13. The method of embodiment 12, wherein the cell is a fibroblast.

[0456] 14. The method of embodiment 13, wherein the fibroblasts are human dermal fibroblasts.

[0457] 15. The method of any one of the preceding embodiments, wherein the nucleic acid comprises a heterologous promoter operably linked to the open reading frame.

[0458] 16. The method of embodiment 15, wherein the heterologous promoter is an inducible promoter.

[0459] 17. The method of any one of the preceding embodiments, comprising delivering a protein of Table 3 to a cell.

[0460] 18. The method of any one of the preceding embodiments, comprising delivering to the cell a nucleic acid comprising an open reading frame encoding a protein of Table 3.

[0461] 19. The method of any one of the preceding embodiments, wherein the nucleic acid is delivered on a non-viral or viral vector.

[0462] 20. The method of any one of the preceding embodiments, wherein the contacting comprises transfecting the cell.

[0463] 21. A method for inducing cellular rejuvenation, comprising overexpressing an effective amount of a protein of Table 3 in the cell.

[0464] 22. The method of embodiment 21, wherein the method comprises activating expression or activity of an endogenous protein of Table 3 at a level higher than a baseline level. EXAMPLES

[0465] Working Example The transcriptome is a key determinant of cell phenotype, controlling cell identity and function. Increasing evidence supports a systems-level view of cell engineering, where exogenous signals in the form of genetic perturbations can induce desired cellular states. For example, overexpression of four Yamanaka factors was shown to not only dedifferentiate somatic cells to a pluripotent state but also reverse the age-associated functional decline of aged cells, thereby supporting the view of senescence as a transcriptomic state.

[0466] There is an increased instability of gene expression throughout aging, which serves as the basis for multiple aging clocks trained either at the epigenetic or transcriptomic level. However, the epigenetic clock 5 Little is known about the functional relevance of DNA methylation sites in aging, and current RNA clocks use age classifiers that are difficult to interpret or provide limited insight into the aging process. This lack of interpretability prevents the use of these clocks as proxies for cellular function in the study of aging.

[0467] The focus of this study was to explore transcriptome reprogramming as an approach to cellular rejuvenation. The Transcriptomic Interpretable Multi-process Ensemble (TIME) predictor was developed for the first time to accurately measure the age of human fibroblasts and respond to known biological interventions. The TIME predictor was then used to perform a cDNA overexpression screen to identify rejuvenating perturbations in aged human cells. Leveraging the functional interpretability of the clock, transcriptome differences in the senescent phenotype of cells from different aged donors were described and the impact of genetic perturbations on key cellular processes in aging was analyzed. Finally, SRSF1 was discovered as a novel aging-regulated gene, whose overexpression reprogrammed the transcriptome to a younger state through differential splicing of genes involved in histone methylation and translation initiation.

[0468] Example 1 A process-based transcriptomic aging clock A publicly available RNA-sequencing (RNA-Seq) dataset from normal primary human dermal fibroblasts (NHDFs) aged 1–96 years, as well as sequencing data from similar fibroblasts, was used to train a machine learning predictor based on the chronological age of the sample donor. The approach included a layer of functional interpretability by subsetting the transcriptome into cellular processes using Molecular Biology of the Cell Ontology. To develop the clock, process-specific weak age predictors were trained using generalized linear models (GLMs) to identify the cellular processes most predictive of age (Figure 1A). This approach stably selected eight processes with different relative contributions to age prediction: WNT signaling, histone methylation, junction organization, translation initiation, actin polymerization, lipid transport, ER quality control (ERQC), and sodium transport (data not shown). Furthermore, expression levels of genes in these eight processes were used to train an ensemble predictor, which achieved accurate age prediction using only 146 genes (Table 1, data not shown). Finally, aging was observed in a process-specific manner across the human lifespan, with three distinct trajectories identified ( Figure 1B ): an early logarithmic increase (histone methylation), an inflection point in midlife (junction organization, lipid transport, sodium transport), and a late exponential increase (translation initiation, actin polymerization, ERQC, WNT signaling).

[0469] The TIME predictor was applied to 12 other different datasets and a similar performance to the widely used epigenetic clock was observed (Pearson's r = 0.93, p-value < 2.2e-16, MAE = 6.66). 5This indicates the precision and robustness of the RNA clock (Figure 1C). Furthermore, the TIME predictor showed good agreement with the epigenetic clock in measuring in vitro aging in skin fibroblasts (0.7 PD / year), but was less accurate when applied to lung fibroblasts, suggesting tissue specificity (data not shown). When the clock was applied to cells subjected to aging-modulating treatments, the expected age-related changes were observed in the transcriptomes of premature aging samples, as well as in cells exposed to rapamycin, hypoxia, and various stresses (Figure 1D).

[0470] Using the clock on a time-course dataset of fibroblast reprogramming, we observed a significant drop in predicted age after only 3 days (Figure 1E). In comparison, the widely used DNA methylation (DNAm) clock achieved similar age predictions only at day 15 of reprogramming, after which the two assays reached consensus at the iPSC stage. This suggests a short response time for the transcriptomic clock, which is consistent with the measured effects of rapamycin in young and middle-aged cells (data not shown). We attribute the observed accelerated transcriptional rejuvenation to the dynamic nature of the gene expression network compared to the more stable and slower changing DNAm pattern. Analyzing process-specific activities across the reprogramming timeline, we found that early rejuvenation was driven by changes in some genes in WNT signaling, histone methylation, and lipid transport, leading to the acquisition of a stable young state after 7 days (data not shown).

[0471] Age-reversal screen identifies genes for cellular rejuvenation The TIME predictor was then used to identify novel rejuvenation interventions by performing a cDNA overexpression screen in human cells. Using a published target identification method (Kramme, C. et al. Cell Reports Methods (2021)), the publicly available NHDFRNA-Seq dataset was analyzed to generate a ranked list of genes predicted to have a significant impact on the aging process (Table 2). 89 genes were selected to test for their impact on the aging phenotype, including KAT7 (a gene involved in fibroblast senescence) and Yamanaka factors in a polycistronic cassette (OSKM) as positive controls.

[0472] To test the potential age-modulating effects of the target genes, candidate individual overexpression lines were generated in three different primary NHDF lines from donors aged 55, 65, and 79 years. After selecting integrants, transgene expression was induced and age-associated changes were assayed using RNA-Seq and flow cytometry-based assays (Figure 2A). Among the 95 genes, a wide range of gene induction levels was observed, ranging from no overexpression to a 4000-fold increase (Figure 2B). However, the overexpression levels among the three NHDF lines appeared to be in good agreement, indicating that the induction differences were gene-specific. Expression of most transgenes and strain identity were confirmed by performing barcode searches against raw sequence reads (data not shown). The results were consistent with the fold-change data, and basal endogenous gene expression levels were found to be negatively correlated with the observed overexpression levels (data not shown), confirming previous findings that gene-specific induction limits exist.

[0473] The effect of genetic perturbations on the aging phenotype of the three NHDF strains was analyzed using the TIME predictor. The clock accurately estimated the age of the WT NHDF strain (Table 3) and was able to detect an age-modulating effect in the control (Figure 2C). OSKM had a strong rejuvenating effect in all three strains, as expected, while KAT7 had the highest pro-aging effect. Most notably, several genes were found to induce a notable shift (-20 to -50 years) toward a juvenile transcriptome across all three NHDF strains, including SIRT1, SLC2A13, SRSF1, RNASEL, and WDTC1 (Figure 2C). While the role of SIRT1 in aging has been thoroughly studied, limited information is available regarding the role of the other four hits in aging. SLC2A13 has been found to decrease in the human dorsolateral prefrontal cortex with aging and was recently identified as a risk gene for Parkinson's disease. Expression of SRSF1 is significantly decreased in older age and is associated with parental longevity in humans. RNASEL levels in human serum have been shown to be inversely correlated with metabolic syndrome and age. WDTC1 has not previously been associated with aging, but has been associated with reduced fat mass and improved insulin sensitivity in humans.

[0474] For orthogonal readouts of senescence, we performed three staining assays that report key cellular characteristics: senescence-associated β-galactosidase activity (SA-βGal activity), mitochondrial membrane potential, and proteasome activity. Data showed that OSKM, RNASEL, SLC2A13, and SRSF1 reduced the senescent phenotype in both M55 and M79 (Figure 2D). In the mitochondrial assay, opposing effects were observed in the two strains, with SIRT1 increasing the mitochondrial potential in M55 and decreasing the mitochondrial potential in M79 (Figure 2D). Both SRSF1 and OSKM decreased the potential in M55 but increased the potential in M79, although this did not reach the significance threshold. This, together with previous observations of large variability in mitochondrial membrane potential between different donors, suggests a potential homeostatic point. Finally, proteasome assays showed reduced activity in strains overexpressing OSKM and SRSF1, both in M55 and M79 (Figure 2D). This reduction in 26S proteasome activity was unexpected, but surprisingly, the known senescence-promoting gene KAT7 increased proteasome activity in the assay. It was hypothesized that rejuvenation interventions may increase protein homeostasis, thereby reducing the burden on the proteasome and decreasing its expression and measured activity. Future studies aimed at measuring proteotoxicity will be required to better explain this result. Finally, the differential responses observed between NHDF strains in both transcriptomic and functional assays (data not shown) suggest that donor cell line-specific differences in the starting transcriptome may influence the outcome of the perturbation.

[0475] Transcriptome variability in aging phenotypes To further study the differences in the senescence transcriptome among the three early cell lines, we performed uniform manifold approximation projection (UMAP) analysis and observed distinct clusters corresponding to the three NHDF donors (Figure 3A). This suggests that after the stress of the nucleofection and selection process, the differences in the transcriptomes of the three cell lines were magnified, which became more prominent than the gene expression changes induced by the perturbation. We then analyzed the differences in the WT senescence phenotype through the perspective of clock processes by focusing on isogenic samples from individuals of different ages (age difference between 2 and 19 years) across their life span. Quantifying the activities of eight clock processes, we found that NHDFs were classified into four distinct groups defined by the type of dysfunction: group I = ERQC and lipid transport, group II = WNT signaling and sodium transport, group III = histone methylation, and group IV = translation initiation and sodium transport (Figure 3D). These results contextualize the different responses to the same perturbation in multiple cell lines and demonstrate the variability of human aging, consistent with a recent study that described four distinct human aging patterns based on the types of molecular patterns that change over time.

[0476] Interestingly, although most of the gene inductions showed different age effects among the three strains (Table 3), some perturbations clustered in UMAP space (Figure 3A), indicating that they overcame strain-specific differences. It was hypothesized that these genes (KDM6A, NOTCH1, OSKM, and SRSF1) induced strong transcriptome reprogramming due to their central role in controlling gene expression, overcoming differences in the starting gene regulatory network. Consistent with this hypothesis, we observed a strong negative correlation between the number of clock genes affected by the perturbations and the average distance in UMAP space across overexpressing strains of the same genes (Pearson's r = -0.64, p-value = 1.678e-11) (Figure 3B). Furthermore, similar to the Waddington landscape similarity, a transcriptome landscape of fibroblast senescence was generated by embedding the predicted ages of samples into a UMAP plot and found that SRSF1 and OSKM have a strong rejuvenating effect, whereas KDM6A and NOTCH1 have a significant pro-senescence effect (Figure 3C). This suggests that perturbations that confer a strong effect on the senescent phenotype as measured by the RNA clock push cells into a common transcriptional space, while the effect of weaker perturbations depends on the initial transcriptional state (data not shown).

[0477] One example of a weak perturbation with cell line-dependent effects is SIRT1 overexpression. Several reports have linked SIRT1 levels to lifespan extension in yeast, but not in worms, flies, or mice, casting doubt on its role in the aging process. In an initial screen, SIRT1 showed a transcriptome rejuvenating effect in two of three strains, but only one was statistically significant. Similarly, when gene overexpression was repeated in six different strains, a strong aging-reversing effect was observed in only half of the cell lines (Figure 4A). This further confirms that SIRT1 overexpression is a weak perturbation with cell line-specific effects. When looking at the scaled age effects that SIRT1 has on each clock process, no common patterns were found among all strains. This suggests that sirtuin overexpression has a strain-specific aging effect (Figure 3E). Nevertheless, in responding cell lines (M65, M67, M68), a slight improvement in WNT signaling was observed, suggesting that SIRT1 may act on the WNT pathway in these cells.

[0478] Table 1. Genes involved in the RNA clock. Genes included in the RNA clock are listed as their Ensembl ID (column "Predictor") and gene symbol. The raw and standardized contribution of each gene to the age prediction is displayed in the "Coefficient" and "Standardized Coefficient" columns, respectively. Additionally, the process to which each gene is associated in the Molecular Biology of the Cell Ontology is also shown (column "Process").

[0479] [Table 1] [Table 2] [Table 3] [Table 4] [Table 5] [Table 6]

[0480] Table 2. Candidate genes for age-reversal screening. Library of candidate genes listed by HGNC gene symbol. DEGs show differential expression in at least one age model used in DGEA. DEG scores calculated from DGEA based on age group (ag) and decade (d) were rounded to 3 decimal places. NA in any of the DEG score columns indicates that the gene was not differentially expressed using that age model. Rank within the network score list for each age model given by ag_rank and d_rank. Network scores for binary gene networks given by ag_ns and d_ns. Summing ag_rank and d_rank gives the Borda total rank score.

[0481] [Table 7] [Table 8] [Table 9]

[0482] Table 3. Predicted ages of the sample in age-reversal screening. Predicted age of overexpression strains from the initial perturbation screen (column "Predicted Age (years)"). The overexpressed gene (column "Gene"), the number of passages before overexpression (column "Passage") and the actual age of the donor (column "Strain Age (years)") are displayed. Wild type samples are labeled "NTg" in the "Gene" column. Replicate identifiers (column "Replicate") were randomly assigned before the experiment was performed.

[0483] [Table 10] [Table 11] [Table 12] [Table 13] [Table 14] [Table 15] [Table 16] [Table 17] [Table 18] [Table 19] [Table 20] [Table 21] [Table 22] [Table 23] [Table 24] [Table 25] [Table 26] [Table 27] [Table 28] [Table 29]

[0484] Example 2 SRSF1 induces potent cellular rejuvenation To validate the initial findings and further study the effect of the hits on the aging phenotype, we repeated the experiment by generating new overexpression lines in NHDFs from six different donors (M55, M65, M67, M68, M69, M79). Consistent with the initial results, most genes were observed to have cell line-specific effects, although some of them lost their effect in the transcriptomic assay (Figure 4A, Table 3). However, SRSF1 showed strong transcriptomic age reversal in all six lines, with an average rejuvenation effect of approximately 26 years, which was as expected given its strong effect on clock genes (Figure 3B). Functional assays showed that SRSF1 induction reduced cellular senescence by more than 50% across all six lines, suggesting functional rejuvenation of these cells (Figure 4B). However, consistent with the initial screening data, no significant effect on mitochondrial potential was observed. Proteasome activity was reduced in SRSF1 samples, consistent with the observed OSKM-induced phenotype and opposite to the pro-senescence effects of KAT7 and CDKN2A (Figure 2D). To further evaluate the rejuvenation by SRSF1 induction, the efficacy of wound healing, a key function of dermal fibroblasts, was tested using an in vitro scratch assay. Data showed that SRSF1-expressing cells reached the midpoint of wound closure 50% faster than BFP controls, whereas OSKM appeared to slightly delay wound closure (Figure 4C), consistent with previous mouse wound healing results.

[0485] We then studied the effect of SRSF1 overexpression on individual processes of TIME predictors and observed a coordinated rejuvenation across all cell lines of gene expression related to histone methylation and translation initiation (Figure 4C). Because histone methylation has a central role in regulating gene expression, reversal of senescence-associated transcriptional changes in this process is expected to have a large and powerful beneficial effect on cells. Conversely, overexpression of KDM6A was observed to have a similar coordinated but detrimental effect on histone methylation and WNT signaling processes (data not shown). Given the strong effects of both OSKM and SRSF1 on the transcriptome, and the known dedifferentiation potential of Yamanaka factors, it was also examined whether SRSF1 also causes loss of cellular identity. When performing an enrichment analysis of fibroblast, myofibroblast, and mesenchymal stem cell (MSC) identity genes, it was found that both SRSF1 and OSKM lead to a reduction in fibroblast identity when compared to WT and BFP controls (data not shown).

[0486] As SRSF1 is a known splicing factor, we performed differential splicing analysis in the overexpression strains and found a large concordance in the distribution of splicing events among the six strains, with alternative first exons accounting for more than 50% of all differential isoforms (data not shown). Interestingly, when we looked at the parental processes of alternatively spliced ​​genes, we found that histone methylation and large ribosomal subunit organization were the only processes common to all six strains (data not shown). Furthermore, alternatively spliced ​​genes were previously identified to directly interact with SRSF1, providing further evidence of the association of SRSF1 with changes in gene expression in histone methylation and translation initiation. Thus, a mechanism is proposed whereby overexpression of SRSF1 leads to differential splicing of key regulators of histone methylation and protein translation, inducing a young gene expression profile in these processes (data not shown).

[0487] Example 3. Serine- and arginine-rich splicing factor 1 (SRSF1) is an essential sequence-specific splicing factor whose expression decreases with age in primary normal human dermal fibroblasts (NHDFs) (Figure 5). SRSF1 was predicted to be an age-regulated gene by a target prediction algorithm, and the above data show that its overexpression reverses age-related changes in the transcriptome, as well as senescence and proteasome dysfunction in multiple aged NHDFs.

[0488] Cellular senescence is a known age-related cellular dysfunction that can be measured by the established senescence-associated β-galactosidase (SA-βgal) assay (Figure 6A). Furthermore, SRSF1 activation by mRNA transfection had the effect of reducing senescence in both young and aged NHDFs (Figure 6B), confirming the effect of reversing senescence in the treated cells.

[0489] Type I collagen is one of the most abundant proteins of the extracellular matrix and serves as an important structural component of multiple tissues, such as bone, skin, and heart. With aging, production of collagen by human fibroblasts decreases (Figure 7A), leading to cellular and tissue dysfunction. Using SRSF1 mRNA transfection, collagen production was increased by 10-45% in both young and aged NHDFs (Figure 7B), reversing this aging phenotype.

[0490] Lack of resilience to oxidative stress is another cellular dysfunction associated with aging, which can be assayed by measuring reactive oxygen species (ROS) levels in response to H2O2 treatment. Here, the data show that aged cells are less able to scavenge ROS upon oxidative stress than young cells (Figure 8, left), and that SRSF1 mRNA can rescue this phenotype in aged NHDFs (Figure 8, right).

[0491] NHDFs play a key role in wound healing by migrating and closing open tissue areas. This function is dysregulated in aged cells and can be measured in vitro using an established scratch assay. The data show that SRSF1-expressing cells reached the midpoint of wound closure 50% faster than BFP controls, whereas overexpressed Yamanaka factors (OSKM) appeared to slightly delay wound closure, consistent with previous mouse wound healing studies (Figure 9 and data not shown (i.e., brightfield images of SRSF1-, OSKM-, or BFP-induced cells at 0 and 6.5 hours after scratching)). These results demonstrate that activation of SRSF1 promotes wound closure in vitro.

[0492] To further validate SRSF1 activation as an effective cell rejuvenation intervention, its effect on wound healing was tested in vivo. Transgene delivery was achieved by intradermal injection of adeno-associated viruses (AAV) containing SRSF1 or GFP under the control of a tetracycline-regulated (Tet-On) inducible promoter. Mice underwent excision wounds by punch biopsy, and wound areas were measured every 2 days. In aged mice, SRSF1 overexpression during wound healing increases the rate of wound closure compared to GFP overexpression. Furthermore, aged mice overexpressing SRSF1 reached complete wound closure by the same time point as young control mice. See Figures 10-11. These results indicate that induction of SRSF1 promotes wound closure in aged mice, highlighting that SRSF1 may be a potential therapeutic target for dermal wound healing.

[0493] To assess the effect of SRSF1 on lifespan, we performed lifespan assays in wild-type and long-lived (raga-1) worm strains with and without knockdown of rsp-3, the worm homolog of SRSF1. The data showed that rsp-3 knockdown had no effect on WT worms but suppressed lifespan extension in the raga-1 strain, indicating that rsp-3 is associated with worm longevity (Figure S12).

[0494] With aging, hematopoietic stem cells (HSCs) lose their self-renewal capacity and lymphocyte output. These cellular dysfunctions lead to several age-associated hematologic malignancies. Data showed that SRSF1 mRNA electroporation increased HSC populations in vitro and improved their differentiation capacity toward lymphoid lineages (Figures 13A-13B).

[0495] method RNA-seq data processing and analysis Raw RNA-Seq reads were aligned to the GRCh38 human genome using STAR v2.5.2b and quality control was checked using FastQC v0.11.5. Alignment files were then indexed using SAMtools v1.3.1 and mapped reads were counted using featureCounts from the Subread v2.0.1 package. To reduce inter-sample variation caused by unbalanced sequencing depth, fastq files were downsampled to 20M reads using Seqtk-1.3. In the overexpression analysis, to address the high number of multi-mapped reads discarded due to sequence similarity between endogenous and barcoded genes, reads were aligned to a genome index containing transgene sequences (including their barcodes) using kallisto v.0.46.2 and TPMs were generated from estimated counts using the tximport package. Barcode searches were performed on the downsampled fastq files using the agrep tool for approximate string matching with three mismatches. Subsequent differential expression analyses were performed using DESeq2 v1.32.0 unless otherwise stated.

[0496] Differential gene expression analysis and identification of cell line identity genes Differential gene expression analysis was performed using the DESeq2 R package v1.30.1. In particular, DESeq data objects were constructed from raw untransformed read counts and design equations reflecting the comparisons attempted. In particular, to detect differentially expressed genes in response to perturbation effects in the screening data, samples from each strain were separated and processed separately. Differentially expressed genes were first run through the “DESeq” function with standard parameters, followed by the “contrast” function to obtain genes differentially expressed between overexpressed genes and wild-type or blue fluorescent protein (BFP)-transduced controls. For each gene, the calculated p-values ​​were corrected applying the Bonferroni correction, and significance was determined at the 1% level. Cell line identity genes in the large-scale screening assay in three cell lines, and in the validation assay consisting of six strains, were identified by the following steps. First, all pairwise differentially expressed genes between wild-type strains are obtained as described above. Then, a matrix consisting of the log2 fold changes across cell lines and all differentially expressed genes was constructed. Next, for each gene in all cell lines, the average log2 fold change between the cell line under study and all other lines was calculated. Finally, the identity genes of a cell line were defined as those with the lowest or highest average log2 fold change compared to the other lines.

[0497] Differential splicing analysis Differential splicing analysis was performed using SUPPA2. In particular, the "Generate Events" subcommand of SUPPA for the GENCODE V29 annotations and parameter settings "-e SE SS MX RI FL -f ioe" were invoked to generate all potential splicing events. Then, the splice-in fraction (PSI) of all potential events was generated by invoking the "psiPerEvent" subcommand on the TPM-formatted RNA-seq data of the transcript-aligned large-scale screening assay (including six NHDF strains with SRSF1 overexpression). Finally, the "diffSplice" command was invoked using an empirical model ("-m empirical") to compare the changes between wild-type and SRSF1 overexpressing strains. The "-gc" parameter applied an automatic gene correction of p-values.

[0498] Cell type-specific marker enrichment RNA-seq samples were separated into individual cell types using CibersortX. First, a droplet-based single-cell reference dataset of stromal cells was collected from Tabula Sapiens. Then, 100 cells for each population were randomly sampled and combined into a single matrix. Signature matrices containing 300–500 genes per cell type were calculated using CibersortX. RNA-seq counts from the initial perturbation screen served as input for deconvolution. CibersortX was run in "absolute mode" to allow comparisons between samples.

[0499] Identifying cellular processes that predict age The identification of processes that predict chronological age is based on the Molecular Biology of the Cell (MBotC) Ontology, a previously curated resource that links genes to cellular processes. The MBotC Ontology is composed of four distinct layers, where broader categories are iteratively divided into more specialized functions.

[0500] Thus, the third layer was empirically selected to create a set of processes that may provide information on aging. To quantify the predictability of chronological age from these processes, a "weak" age predictor for each process was constructed as follows: (1) subset the gene expression matrix to the genes in the process under consideration; (2) perform a principal component analysis (PCA) on the subsetted matrix and replace it with a matrix containing all principal components. PCA was performed using the implementation in the h2o v3.36.0.2 R package, where the disproportionate contribution of individual genes is mitigated by standardizing the data via the "transformation" parameter; (3) train a generalized linear model (GLM) with 5-fold cross-validation using the "h2o.glm" function in the h2o R package. In particular, the model is based on a Gaussian distribution with an identity link function, standardizes the inputs before training, and implements an automatic lambda search with ridge regression. (4) train a "strong" age predictor based on the in-bag predictions of all weak predictors. Similar to the weak predictor, the strong age predictor is a GLM with a Gaussian distribution, an identity link function, and 10-fold cross-validation based on input standardization. However, in contrast to the weak model, the strong predictor employs Lasso regression to select only the minimal set of processes that provide the most information on chronological age. Any combination of processes with non-zero coefficients is considered to predict age.

[0501] Importantly, to identify cellular processes that predict age, a training dataset needs to be assembled in which the age of each sample is known in advance. In this regard, both the weak and strong age predictors are not trained on age expressed in years. Instead, age is transformed by a piecewise approximate linear transformation defined as follows:

number

[0502] This function is a suitable transformation because its inverse has the following closed form:

number

[0503] Training a process-based transcription clock The transcriptomic clock introduced in this study rests on a set of cellular processes that predict age, as described in the previous subsection. In particular, training the clock follows a two-step process. First, for each process, starting with a model with only an intercept, we iteratively add or remove genes and perform a linear regression between the current set of genes and the sample age to estimate the age using the Akaike information criterion 51 The gene set with the highest predictive power is selected by scoring each iteration using . In total, 1000 iterations are performed for each process. Then, a GLM implemented in the h2o v3.36.0.2 R package is trained on all selected genes of all processes selected in the first step. Specifically, the GLM employs a Gaussian distribution with identity link function, lambda search, and ridge regression on the standardized gene expression data. Since the number of genes selected in the first step can be larger than the number of training samples, an upper limit on the number of active predictors in the GLM was set to the number of training samples.

[0504] Predicting age of non-training samples Due to the variability of raw RNA-seq data related to sequencing depth, library preparation, and other experimental confounding factors, training and non-training samples need to be pre-processed together to detect and correct for batch effects. Therefore, for the new set of non-training samples, a common pre-processing pipeline was adopted. First, the training and non-training samples were merged into one matrix and then extracted using the NOIseq v2.34.0 R package. 53TMM normalization is performed using the “tmm” function in R package . Then, if a significant batch effect is found in the first two principal components, a batch correction is applied between training and non-training samples. For the majority of the datasets in this study, a naive Removal of Unwanted Variance (RUV) normalization from the RUVnormalize v1.24.0 R package is used. 54 An algorithm was employed. RUV requires the selection of a set of appropriate control genes that are expected to be uncorrelated with the variable of interest (e.g., age). Therefore, appropriate controls were selected that had low correlation with age in the training data (Pearson's r<0.01). However, depending on the observed batch effects, other control genes specific to the dataset should be included (see individual scripts for batch correction methods and parameters used for each dataset).

[0505] After normalization and batch correction, a transcriptional clock was trained on the training data as described in the previous section. The trained model was then used to predict the age of non-training samples using the “h2o.predict” function in the H2O R package.

[0506] Calculating process activity scores to quantify age-related functional differences The estimated coefficients of the transcriptomic clock were used to quantify the activity of all eight processes associated with aging in the samples by calculating the scalar product of the model coefficients and the expression values ​​of the corresponding genes. In cases with multiple replicates, activity scores of the same process in different samples are aggregated to an arithmetic mean. Because RNA-seq data have been shown to be sensitive to the pre-processing pipeline used to transform the raw read counts, reference process activities were calculated for all samples in the training data. These reference activities define a range of values ​​for each process that corresponds to physiological aging and are used to uniformly scale the activity scores of new samples. Due to the generalized linear model underlying the clock, lower process activity scores correspond to lower transcriptional ages and higher values ​​correspond to higher transcriptional ages.

[0507] Screening library construction All ORFs were amplified from the original vector (Addgene or ORFeome) using Q5 High Fidelity 2X Master Mix (NEB M0492S) to add an attB site, a Kozak consensus sequence "GCCACC", and a WT STOP codon. Amplified fragments were gel purified (QIAGEN 28506) and shuttled into pDONR221 (ThermoFisher 12536017) using BP Clonase II enzyme mix (ThermoFisher 11789020). Reactions were transformed into 5-alpha competent E. coli (NEBC2987H), clones were selected, and sequences were confirmed using Sanger sequencing. The resulting plasmids were miniprepped (NEB T1010L) and reacted with a pool of barcoded target vectors (PB-CT3G-ERP2-MG-BC) in a MegaGate reaction. After transformation into 5-α cells, clones were selected, sequences were verified, and specific ORFs were assigned barcodes. Final plasmids were miniprepped and used for nucleofection.

[0508] tissue culture NHDF lines were cultured in Fibroblast Medium (FM): low glucose DMEM (ThermoFisher 11885-084) supplemented with 15% FBS (GenClone 25-550) and 1% penicillin-streptomycin (ThermoFisher 15140122) at 37°C, 5% CO2, 5% O2. When induced with doxycycline, cells were switched to medium made with Tet System Approved FBS (Takara 631367) to reduce background induction. Medium was changed every other day.

[0509] To generate overexpression lines, 200,000 cells were nucleofected with 50 fmol of transposon and 50 fmol of transposase (Super piggyBac Transposase-SystemBio PB210PA-1) or 300 ng of pmaxGFP using a P2 Primary Cell 4D Nucleofector kit (Lonza V4SP-2096) and a Lonza 4D-Nucleofector with DS-150 program. Cells were allowed to recover for 45 min at room temperature and then plated in 24-well plates in 500 uL FM. The day after nucleofection, dead cells were washed with PBS and medium was replenished. Selection was started 3–4 days after nucleofection (depending on cell confluency) using 400 ng / mL puromycin (ThermoFisher A1113802) in FM (PFM). Cells were selected and expanded in parallel, switching between FM and PFM every 4 days until the pmaxGFP control reached 0% viability. At the 10 cm dish stage, cells were frozen in FM containing 5% DMSO and stored in liquid nitrogen until thawed. Due to inherent sensitivity of the cell lines to the nucleofection and selection process, as well as increased toxicity of plasmids carrying larger genes, it was not possible to generate overexpression strains of all 95 genes in all three major NHDF lines.

[0510] At the time of assay, cells were thawed into two wells of a 6-well plate in FM and switched to Tet-free media the next day. Two days after thawing, cells were harvested, counted, and plated for RNA-Seq and flow cytometry. For RNA-Seq, each cell line was plated into two wells of a 6-well plate at a density of 60,000 cells / well in FM supplemented with doxycycline (1ug / mL for initial screening, 2ug / mL for subsequent experiments). For flow cytometry, each line was plated into 18 wells of a 24-well plate at a density of 10,000 cells / well in FM (+Dox wells received 1ug / mL doxycycline). 72 hours after plating, cells were washed with PBS, stained, and harvested for flow cytometry or lysed using Monarch DNA / RNA Protection Reagent (NEB T2011L) and stored at -80°C.

[0511] RNA sequencing RNA was extracted from cell lysates using the Monarch Total RNA Miniprep Kit (NEB T2010S) and its quality was spot checked for random samples using the Bioanalyzer High Sensitivity RNA Kit (Agilent 5067-1513). 100 ng of total RNA was quantified using the Qubit RNA High Sensitivity Assay (ThermoFisher Q32852) and used for library preparation by the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina and polyA mRNA workflow. Library quality was spot checked for random samples using the Bioanalyzer High Sensitivity RNA Kit (5067-4626), after which all libraries were quantified using the Qubit dsDNA High Sensitivity Assay (ThermoFisher Q33230) and pooled together. For all samples sequenced together, RNA extraction and library preparation were performed on the same day. Sequencing was performed on an Illumina NextSeq or NovaSeq instrument by the Harvard Biopolymers Facility.

[0512] Flow cytometry Cells were stained for cellular senescence, mitochondrial membrane potential, and proteasome activity. For cellular senescence, cells were incubated with 100 nM bafilomycin A1 (VWR 102513) for 2 hours and 33 nM C12FDG (5-dodecanoylaminofluorescein di-β-D-galactopyranoside; ThermoFisher D2893) for 1 hour. Mitochondrial membrane potential and proteasome activity were multiplexed by incubating cells with 20 nM or 40 nM (M65: [46, 55, 56, 57, 60, 64, 72, 77, 82, 94, 95]) TMRM (ThermoFisher M20036), 0.125x proteasome LLVY-R110 substrate, and 0.0625x assay buffer (Millipore Sigma MAK172) for 2 hours. Samples were analyzed using a Cytoflex LX or BD LSRfortessa instrument. Analyses were performed using FlowJo (version 10.8.1).

[0513] In vitro scratch assay Cells from the M79 line carrying SRSF1, OSKM, or mTagBFP2 overexpression cassettes were grown in 10 cm dishes and treated with doxycycline (1ug / mL) for 3 days. Cells were then harvested and seeded into 24-well plates at a density of 120,000 cells per well. The following morning, plates were scraped with a p200 pipette and washed with PBS. Plates were imaged at 1.5 hour intervals using a Cellcyte live cell imaging system (Cytena) with a 10x objective.

[0514] Scratch images were stitched together, processed, and analyzed as virtual stacks for each time course using Fiji. After cropping the scratch area, image background was subtracted using a rolling ball radius of 10 pixels, and contrast was enhanced to 10% saturated pixels and normalized for all images in the stack. The Python package Bowhead v1.1.3 was used to identify the largest contiguous wound area in the images using a threshold of 0.5.

[0515] statistical analysis Statistical analysis was performed using R version 4.0.3, two-tailed Student's t-test or Z-score. All statistical tests performed are indicated in the figure legends. Data are presented as means or individual points, with box plots showing medians and quartiles. Error bars represent the standard error around the mean.

[0516] Construct sequence SRSF1 sequence >pAMP531_(PB-CT3G-ERP2-MG-BC-SRSF1) [ka] [ka] >SRSF1_full [ka]

[0517] All references, patents, and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which may include the entire document in some cases.

[0518] The indefinite articles "a" and "an," as used in the specification and claims, unless clearly indicated to the contrary, should be understood to mean "at least one."

[0519] It should also be understood that, unless expressly indicated to the contrary, in methods claimed herein that include multiple steps or acts, the order of the method steps or acts is not necessarily limited to the order in which the method steps or acts are described.

[0520] In the claims and the above specification, all transitional phrases such as "comprising," "including," "carrying," "having," "containing," "involving," "holding," "consisting of," and the like, are understood to be open-ended, i.e., meaning including but not limited to. Only the transitional phrases "consisting of" and "consisting essentially of" shall be closed-ended or semi-closed-ended transitional phrases, respectively, as defined in Section 2111.03 of the United States Patent Office Manual of Patent Examining Procedures.

[0521] The terms "about" and "substantially" preceding a numerical value mean ±10% of the stated numerical value.

[0522] When a range of values ​​is provided, each value between the upper and lower limits of that range, inclusive, is specifically contemplated and described herein.

Claims

1. 1. A method of inducing rejuvenation of a cell, comprising contacting said cell with an effective amount of serine- and arginine-rich splicing factor 1 (SRSF1) protein or a nucleic acid encoding said SRSF1 protein.

2. 23. A method of inducing cellular rejuvenation in a subject, comprising administering to the subject an effective amount of SRSF1 protein or a nucleic acid encoding said SRSF1 protein.

3. 3. The method of claim 1 or 2, wherein the effective amount is sufficient to reduce cellular senescence of the cell compared to a control.

4. 4. The method of claim 3, wherein the effective amount is sufficient to reduce cellular senescence of the cell by at least 20%, at least 30%, at least 40%, or at least 50%.

5. 5. The method of claim 4, wherein the effective amount is sufficient to reduce cellular senescence of the cell by at least 50%.

6. The method of any one of claims 3 to 5, wherein the effective amount is sufficient to reduce senescence-associated β-galactosidase activity of the cells compared to a control.

7. 7. The method of claim 6, wherein the effective amount is sufficient to reduce the senescence-associated β-galactosidase activity of the cells by at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50%.

8. The method of any one of claims 3 to 7, wherein the effective amount is sufficient to reduce proteasome activity in the cell compared to a control.

9. 9. The method of claim 8, wherein the effective amount is sufficient to reduce the proteasome activity of the cell by at least 25%, at least 30%, at least 40%, at least 45%, or at least 50%.

10. 2. The method of any one of the preceding claims, wherein the effective amount is sufficient to induce an average of cellular rejuvenation of at least 5 years, at least 10 years, at least 15 years, at least 20 years, at least 25 years, at least 30 years, at least 35 years, at least 40 years, at least 45 years, or at least 50 years.

11. 11. The method of claim 10, wherein the effective amount is sufficient to induce an average of cellular rejuvenation of at least 25 years.

12. 2. The method of any one of the preceding claims, wherein the cells are selected from fibroblasts, hematopoietic stem cells, endothelial cells, chondrocytes, skeletal muscle stem cells, keratinocytes, mesenchymal stem cells and corneal epithelial cells.

13. The method of claim 12, wherein the cell is a fibroblast.

14. The method of claim 13, wherein the fibroblasts are human dermal fibroblasts.

15. 10. The method of any one of the preceding claims, wherein the nucleic acid comprises a heterologous promoter operably linked to an open reading frame.

16. The method of claim 15 , wherein the heterologous promoter is an inducible promoter.

17. 2. The method of any one of the preceding claims, comprising delivering the SRSF1 protein to a cell.

18. 2. The method of any one of the preceding claims, comprising delivering said nucleic acid comprising an open reading frame encoding said SRSF1 protein to a cell.

19. 13. The method of any one of the preceding claims, wherein the nucleic acid is delivered on a non-viral or viral vector.

20. 2. The method of any one of the preceding claims, wherein said contacting comprises transfecting said cell.

21. 13. A method for inducing rejuvenation of a cell, comprising overexpressing an effective amount of SRSF1 protein in said cell.

22. The method of claim 21 , wherein the method comprises activating expression or activity of endogenous SRSF1 protein at a level higher than a baseline level.

23. A cell comprising an engineered nucleic acid encoding a serine- and arginine-rich splicing factor 1 (SRSF1) protein.

24. The cell of claim 23 , wherein the cell is a fibroblast.

25. The cell of claim 24, wherein the fibroblast is a human dermal fibroblast.

26. The cell of claim 23 , wherein the cell is a stem cell.

27. 27. The cell of claim 26, wherein the stem cell is selected from a hematopoietic stem cell, a skeletal muscle stem cell, and a mesenchymal stem cell.

28. 27. The cell of claim 26, wherein the stem cell is a human induced pluripotent stem cell.

29. 24. The cell of claim 23, wherein the cell is selected from an endothelial cell, a chondrocyte, a keratinocyte, and a corneal epithelial cell.

30. The cell of any one of claims 23 to 29, wherein the cell expresses SRSF1 at a level higher than the baseline level.

Images