Methods and compositions for inhibiting cell senescence

Abstract

The disclosure relates to methods of inhibiting cellular senescence involving expressing Activating Transcription Factor 3 (ATF3) in a cell at a level sufficient to inhibit senescence of the cell. In some embodiments, ATF3 is delivered to a cell via administration of a messenger ribonucleic acid comprising an open reading frame encoding ATF3.

Description

[0001] Attorney Docket No. H0498.70834WO00

[0002] METHODS AND COMPOSITIONS FOR INHIBITING CELL SENESCENCE

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS

[0004] This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 63 / 696,224, filed September 18, 2024, which is hereby incorporated by reference in its entirety.

[0005] REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0006] The content of the electronic sequence listing (H049870834WO00-SEQ-KVC.xml; Size: 19,859 bytes; and Date of Creation: September 17, 2025) is herein incorporated by reference in its entirety.

[0007] BACKGROUND

[0008] Aging is an inherent biological phenomenon characterized by a gradual decline in cellular function due to the accumulation of molecular damage over time. Human skin aging is a complex process influenced by both intrinsic factors, such as the accumulation of genetic changes, and extrinsic factors such as environmental-driven damage. As a result, aging skin undergoes significant structural and physiological transformations, making it more susceptible to various disorders. These changes underscore the need for effective strategies to combat skin aging and mitigate its adverse effects.

[0009] Several treatment options are available for skin rejuvenation and related disorders. These options include chemical, physical, and biological methods. Chemical treatments, such as retinoic acid, have been widely utilized. Physical treatments are diverse, including intense pulsed light, lasers, energy-based devices, ultrasound, and nitrogen plasma skin regeneration (PSR). Biological treatments encompass platelet-rich plasma treatment, the up-regulation of HIF-la functionality, and the application of mesenchymal stem cells. Despite these efforts, no single approach has successfully addressed the aging mechanisms of basal stem cells and simultaneously combined the effects of chronological aging and photoaging. This limitation is likely due to a lack of understanding of the molecular-level changes in each skin cell type.

[0010] SUMMARY

[0011] The inventors of the disclosure have identified Activating Transcription Factor 3 (ATF3) as a key regulator of skin aging, cellular senescence, and collagen production (e.g., in

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[0013] #14392914vl Attorney Docket No. H0498.70834WO00 fibroblasts). Motivated by that discovery, the inventors have developed a novel mechanism designed to increase expression of ATF3 in cells to slow and / or mitigate the effects of skin aging, inhibit cellular senescence, and increase collagen production.

[0014] Aspects of the present disclosure relate to a method of inhibiting cellular senescence, the method comprising expressing ATF3 in a cell at a level sufficient to inhibit senescence of the cell.

[0015] Aspects of the present disclosure relate to a method of inhibiting cellular senescence, the method comprising: delivering a messenger ribonucleic acid (mRNA) comprising an open reading frame encoding ATF3 to a skin cell in an amount effective to inhibit senescence of the skin cell.

[0016] Other aspects of the present disclosure relate to a method of inhibiting cellular senescence in a subject in need thereof, the method comprising administering to the subject a composition comprising a lipid nanoparticle (LNP) and an mRNA comprising an open reading frame encoding ATF3. In some embodiments, the mRNA is expressed in a skin cell of the subject. In some embodiments, expression of the mRNA in a skin cell of the subject inhibits senescence of the skin cell.

[0017] In some embodiments, the cell is a dermal cell or an epidermal cell. In some embodiments, the cell is a keratinocyte.

[0018] In some embodiments, the cell is a hair follicle cell (e.g., hair follicle stem cell).

[0019] In some embodiments, ATF3 is expressed in the cell at a level sufficient to reduce expression of one or more senescence-associated secretory phenotype (SASP) factors.

[0020] In some embodiments, the SASP factors are selected from cytokines, chemokines, growth factors, proteases, lipid metabolites and extracellular vesicles. In some embodiments, the SASP factors are selected from interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin- 1 beta (IL- 1 P), interferon beta (IFN-P), tumor necrosis factor alpha (TNF-a), cyclin-dependent kinase inhibitor 2A (CDKN2A), CXCL family members, C-X-C motif chemokine ligand 2 (CXCL2), matrix metalloproteinases (MMPs), serine proteases, cathepsin, cyclooxygenase, prostaglandin E2 (PGE2), and leukotriene D.

[0021] In some embodiments, ATF3 is expressed in the cell at a level sufficient to reduce expression of senescence-associated P-galactosidase. In some embodiments, ATF3 is expressed in the cell at a level sufficient to increase the rate of proliferation of the cell.

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[0024] In some embodiments, the open reading frame encodes a wild-type ATF3 protein. In some embodiments, the open reading frame comprises a nucleic acid sequence that is at least 90%, 95%, 97%, or 100% identical to SEQ ID NO: 1.

[0025] In some embodiments, the ATF3 has an amino acid sequence that is at least 90%, 95%, 97%, or 100% identical to SEQ ID NO: 2.

[0026] In some embodiments, the mRNA comprises at least one uridine nucleoside comprising N1 -methylpseudouridine triphosphate. In some embodiments, the mRNA comprises a 5' human alpha-globulin- 1 (HBA1) untranslated region (UTR) and / or a 3' HBA1 UTR. In some embodiments, the mRNA comprises a nucleic acid sequence that is at least 90%, 95%, 97%, or 100% identical to any one of SEQ ID NOs: 3-8. In some embodiments, the mRNA is delivered using a viral vector, transfection reagent, lipid nanoparticle (LNP), microneedle, and / or electroporation.

[0027] In some embodiments, the viral vector is an adeno-associated viral, adenoviral, or lentiviral vector.

[0028] In some embodiments, the transfection reagent is a cationic lipid, a polymer-based reagent, a calcium phosphate-based reagent, or a dendrimer-based reagent.

[0029] In some embodiments, the lipid nanoparticle comprises ionizable lipids and / or polyethylene glycol (PEG)-lipid conjugates.

[0030] Aspects of the present disclosure relate to a method of inhibiting cellular senescence, the method comprising: delivering an mRNA comprising an open reading frame encoding ATF3 to a subject in an amount effective to inhibit cellular senescence in the subject.

[0031] Aspects of the present disclosure relate to a method of increasing cellular collagen and / or elastin production, the method comprising: delivering mRNA comprising an open reading frame encoding ATF3 to a subject in an amount effective to increasing cellular collagen and / or elastin production in the subject.

[0032] In some embodiments, the increased cellular collagen and / or elastin production is produced by dermal fibroblasts.

[0033] In some embodiments, the open reading frame encodes a wild-type ATF3 protein. In some embodiments, the open reading frame comprises a nucleic acid sequence that is at least 90%, 95%, 97%, or 100% identical to SEQ ID NO: 1.

[0034] In some embodiments, the ATF3 has an amino acid sequence that is at least 90%, 95%, 97%, or 100% identical to SEQ ID NO: 2.

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[0037] In some embodiments, the mRNA comprises at least one uridine nucleoside comprising N1 -methylpseudouridine triphosphate. In some embodiments, the mRNA comprises a 5' HBA1 UTR and / or a 3' HBA1 UTR. In some embodiments, the mRNA comprises a nucleic acid sequence that is at least 90%, 95%, 97%, or 100% identical to any one of SEQ ID NOs: 3-8. In some embodiments, the mRNA is delivered using topical, intravenous, intramuscular, intradermal, transdermal, oral, or subcutaneous administration. In some embodiments, the mRNA is delivered to the subject using a viral vector, LNP, or microneedle.

[0038] In some embodiments, the viral vector is an adeno-associated viral, adenoviral, or lentiviral vector.

[0039] In some embodiments, the lipid nanoparticle comprises ionizable lipids and / or polyethylene glycol (PEG)-lipid conjugates.

[0040] In some embodiments, the subject has a skin disorder and / or an autoimmune disorder. In some embodiments, the subject has psoriasis, atopic dermatitis, lupus, lichen planus, epidermolysis bullosa, scleroderma, alopecia, acne vulgaris, actinic keratosis, bruises, burns, dermatitis herpetiformis, hidradenitis suppurativa, hypertrophic scars, keloids, prurigo, pyoderma gangrenosum, or vitiligo. In some embodiments, the subject has impaired wound healing, hyperpigmentation disorders, or a chronic inflammatory condition.

[0041] Aspects of the present disclosure relate to a method of treating a skin disorder in a subject, the method comprising: delivering an mRNA comprising an open reading frame encoding ATF3 to a subject in an amount effective to treat the skin disorder.

[0042] In some embodiments, the skin disorder is psoriasis, atopic dermatitis, lupus, lichen planus, epidermolysis bullosa, scleroderma, alopecia, acne vulgaris, actinic keratosis, bruises, bums, dermatitis herpetiformis, hidradenitis suppurativa, hypertrophic scars, keloids, prurigo, pyoderma gangrenosum, or vitiligo.

[0043] In some embodiments, alopecia is androgenetic alopecia, alopecia areata, telogen effluvium, traction alopecia, cicatricial alopecia, alopecia barbae, ophiasis alopecia, alopecia universalis, or pseudopelade. In some embodiments, alopecia is alopecia areata.

[0044] In some embodiments, the subject has impaired wound healing, hyperpigmentation disorders, or a chronic inflammatory condition.

[0045] Aspects of the present disclosure relate to a skin cell comprising an engineered mRNA comprising an open reading frame encoding ATF3, wherein the ATF3 is expressed by the cell at a level sufficient to inhibit senescence of the skin cell. In some embodiments, the skin cell is a dermal cell or an epidermal cell, optionally a keratinocyte.

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[0048] BRIEF DESCRIPTION OF THE DRAWINGS

[0049] FIGs. 1A-1F. Deconstructing human skin during the aging process. (A) The Overview of the Study. Human skin samples from individuals of varying ages and locations were collected for analysis. Single-cell RNA sequencing (scRNA-seq) was performed to construct a comprehensive cellular atlas. Potential therapeutic targets were subsequently identified from this atlas. The identified targets were validated using small interfering RNA (siRNA) techniques. Following validation, a messenger RNA (mRNA) treatment was developed based on these findings. (B) Uniform manifold approximation and projection (UMAP) plot showing the cell types of human skin. Color denotes the cell type derived from sc-RNA. BC: basal cell; SC: Spinous cell; GC, granular cell; ME: melanocyte; HF, hair follicle; EC, endothelial cell; FB, fibroblast; PC, pericyte; IC, immune cell. (C) UMAP plots show the representative genes of each cell type in human skin. The color key from gray to blue indicates low to high gene expression levels. KRT15, basal stem cells; PDGFRA, fibroblasts; CLDN5, endothelial cell; PTPRC, immune cells. (D) Dot plot showing the expression of representative genes for each cell type. (E) Enriched GO terms for each cell type in combined aging and chronological aging. (F) Bar charts illustrate the proportions of different cell states in human skin across young, middle, and old age groups.

[0050] FIGs 2A-2F. Changes in cellular states during combined aging, chronological aging, and photoaging. (A) Illustration of combined aging (photoaging and chronological aging) and chronological aging. Combined aging is assessed using arm tissues from young, middle-aged, and old individuals, while chronological aging is assessed using back tissues from young, middle-aged, and old individuals. (B) Bar plot depicting the number of differentially expressed genes (DEGs) in various cell types during combined aging and chronological aging. (C) Number of cell type-shared DEGs and cell-specific DEGs in different cell types during combined aging. (D) Number of cell type- shared DEGs and cell- specific DEGs in different cell types during chronological aging. (E) Venn diagram illustrating DEGs across combined aging, chronological aging, and photoaging, with circle size proportional to the number of genes. (F) Top enriched Gene Ontology (GO) Biological Process terms from enrichment analysis of genes selected by combined aging and chronological aging.

[0051] FIGs. 3A-3F. Human epidermal keratinocyte subtypes based on their transcriptional profiles. (A) Illustration of Human Skin Aging. Human skin aging is characterized by distinct morphological and functional changes in both the epidermis and dermis. The epidermis becomes noticeably thinner in aged skin due to a reduction in keratinocyte proliferation and an increase in 5

[0052] #14392914vl Attorney Docket No. H0498.70834WO00 apoptosis. In the dermis, a significant hallmark of aging is the decreased production of collagen by fibroblasts. (B) UMAP visualizations reveal the categorization of human keratinocytes into 11 subtypes. Basal cells (BC), spinous cells (SC), and granule cells (GC) are indicated based on the expression matrix and their specific markers. Each cell type is distinguished by a unique color. (C) Expression profiles of specified marker genes are depicted across different subtypes. Gradations from gray to blue denote varying levels of gene expression, whereas deeper blue signifies higher expression. Granular cells (GC) are characterized by FLG expression, spinous cells (SC) by KRT10, and basal cell subtype 2 (BC2) by KRT5 and KRT14. (D) Heatmap presenting the scaled expression levels of genes prominently expressed exclusively within all BC, SC, and GC subtypes. The color spectrum, progressing from white to blue, signifies low to elevated gene expression levels. (E) Visualization of the lineage development of keratinocytes along differentiation trajectories. (F) Pseudo-time distribution of cells within lineage S0-S1-S3 for both young and old samples, separated by combined aging and chronological aging scenarios.

[0053] FIGs. 4A-4K. The development of mRNA therapy for human skin rejuvenation. (A) Overview summarizing the development of mRNA therapy for skin rejuvenation. (B) Identification of two distinct "gene modules" emerging during the aging process based on singlecell gene expression clustering: one characterized by overexpression and the other by underexpression. Genes within these modules are spatially mapped in the skin atlas and integrated into a gene-gene network. (C) Visualization of gene expression patterns within the identified modules using Uniform Manifold Approximation and Projection (UMAP). The gene-gene interaction networks display connectivity, with node size reflecting the number of connections. Bar plots depict expression changes of hub genes across aging stages. (D) Workflow illustrating siRNA- mediated knockdown of ATF3. (E) Immunofluorescence staining of Ki67 in human epidermal keratinocytes following siRNA-mediated ATF3 knockdown. Scale bar, 125 pm. Data are presented as mean ± SEM. n = 6 per group. *p < 0.05. (F) Quantitative PCR (qPCR) analysis of Senescence-associated Secretory Phenotype (SASP) in human epidermal keratinocytes upon siRNA-mediated ATF3 knockdown. Data are presented as mean ± SEM. (G) Senescence- associated P-galactosidase (SA-P-gal) staining of human epidermal keratinocytes following siRNA-mediated ATF3 knockdown. Scale bar, 125 pm. Data are presented as mean ± SEM. n = 3 per group. **p < 0.01. (H) Workflow depicting mRNA treatment by overexpressing the ATF3 gene, designed with in-house untranslated regions (UTRs). (I) Immunofluorescence staining of Ki67 in human epidermal keratinocytes following mRNA treatment of ATF3. Scale bar, 125 pm. Data are presented as mean ± SEM. n = 6 per group. *p < 0.05. (J) qPCR analysis of SASP in 6

[0054] #14392914vl Attorney Docket No. H0498.70834WO00 human epidermal keratinocytes upon mRNA treatment of ATF3. Data are presented as mean ± SEM. (K) SA-P-gal staining of human epidermal keratinocytes following mRNA treatment of ATF3. Scale bar, 125 pm. Data are presented as mean ± SEM. n = 3 per group. ***p < 0.001.

[0055] FIGs. 5A-5F. (A) Mechanistic insights into skin aging mediated by secreted proteins from keratinocytes to fibroblasts. (B) Network representation illustrating the crosstalk between different cell types, with edge width proportional to the number of identified ligand-receptor pairs. Numbers on edges indicate significant ligand-receptor interactions between cell populations. (C) Significant ligand-receptor pairs contributing to signaling from keratinocytes to fibroblasts were assessed using one-sided permutation tests (p-values). (D) Expression profiles of secreted proteins from keratinocytes involved in signaling to fibroblasts. (E) Experimental setup involving culture of fibroblasts with conditioned medium from young and old keratinocytes. Evaluation includes Ki67 staining, senescence staining, qPCR of collagen expression, and ELISA of collagen in fibroblasts. Scale bar, 125 pm. Data are presented as mean ± SEM. n = 3 per group. *p < 0.05, **p < 0.01, ****p < 0.0001. (F) Experimental setup involving culture of fibroblasts with conditioned medium from control and ATF3 treated keratinocytes. Evaluation includes Ki67 staining, senescence staining, qPCR of collagen, and ELISA of collagen in fibroblasts. Scale bar, 125 pm. Data are presented as mean ± SEM. n = 3 per group. *p < 0.05, **p < 0.01, ***p < 0.001.

[0056] FIGs. 6A-6E. (A) UMAP plot of all cells of human skin, colored by sample origin. (B) UMAP plot of all cells of human skin by removing the stress genes, colored by sample origin.

[0057] (B) Top 20 cell subtypes exhibiting differential gene expression between old and young cells.

[0058] (C) Expression differences among young, middle-aged, and old samples by measuring the percentage of neighbor cells. (D) Dot plot identifying top pathways upregulated in the basal stem cells. The size of the dots shows the number of genes while the color of the dot indicates adjusted p-value. (E) Pathway enrichment analysis for basal stem cells, with pathways ordered by adjusted p-value and dots colored by aging type. The plot displays the top 10 pathways specific to each aging type.

[0059] FIGs. 7A-7G. (A) Line plot across ages showing trends in 6 out of 60 cell-type- shared DEGs expression in combined aging. (B) Line plot across ages showing trends in 6 cell-type- shared DEGs expression in chronological aging. (C-F). Gene set scoring for GenAge (C), DNA repair (D), hallmark EMT (E), and NFKB (F) related genes across cell states. Violin plots display gene set scores; the color displays aging type. Adjusted Bonferroni p-values from a Wilcox test are displayed under significant differences at each age. (G) Bar plot illustrating gene 7

[0060] #14392914vl Attorney Docket No. H0498.70834WO00 expression alterations, identified by aggregating the log2 fold changes of differentially expressed genes unique to each cell state across age. The top 10 most discrepant enriched and depleted cell states between Chronological and Combined aging are shown, bars are also colored by aging type.

[0061] FIGs. 8A-8E. (A-B) Gene expression profiles of NFKB and genAge genes across three age groups and two aging types (combined aging and chronological aging). (C) Analysis of hair follicle subtypes, including subtype categorization, developmental trajectory, and gene expression profiles of marker genes across different samples. (D) Analysis of fibroblast subtypes, including subtype categorization, developmental trajectory, and gene expression profiles of marker genes across different samples. (E) Analysis of endothelial subtypes, including subtype categorization, developmental trajectory, and gene expression profiles of marker genes across different samples.

[0062] FIGs. 9A-9G. (A) Expression levels of ATF3 in young and old skin samples from our dataset. (B) Expression levels of ATF3 in young and old skin samples from other datasets. (C) Immunofluorescence staining of ATF3 in human skin. Scale bar, 200 pm; n = 3 for each group. (D) Quantitative PCR (qPCR) analysis of Senescence-associated Secretory Phenotype (SASP) and senescence-associated P-galactosidase (SA-P-gal) staining of human epidermal keratinocytes upon siRNA-mediated gene knockdown. Data are presented as mean ± SEM. n = 3 per group. **p < 0.01. (E) Comparison of senescence levels in human epidermal keratinocytes treated with mCherry Control and ATF3. The experiments were conducted in triplicate. Senescence- associated P-galactosidase (SA-P-gal) staining using C12FDG revealed significantly lower senescence levels in ATF3-treated cells compared to mCherry-treated controls. Data are presented as mean ± SD, with statistical significance determined by t-test. (F) Dose-response curve of ATF3 mRNA on senescence levels in human epidermal keratinocytes. Five concentrations were tested, and SA-P-gal activity was measured. Data are presented as mean ± 95% confidence interval. (G) mRNA treatment with the ATF3-P2A-RFP structure. Senescence levels and delivery rate were assessed in human epidermal keratinocytes treated with RFP controls and ATF3-P2A-RFP. Senescence levels were evaluated using SA-P-gal staining with C12FDG. Data are presented as mean ± SD and statistical significance was determined by t-test. ****p < 0.0001.

[0063] FIGs. 10A-10E. (A) The circo plot illustrates potential cell interactions predicted by CellphoneDB among nine major cell types in both combined aging and chronological aging. These cell types include keratinocytes, fibroblasts, endothelial cells, hair follicles, sebocytes, 8

[0064] #14392914vl Attorney Docket No. H0498.70834WO00 melanocytes, pericytes, immune cells, and Langerhans cells. The size of each node represents the number of interactions, while the width of each edge indicates the number of significant ligandreceptor pairs between the two cell types. (B) The gene-gene network displays potential ligandreceptor pairs between keratinocytes and fibroblasts. (C) The network of gene-gene interactions among down-regulated genes and secreted genes in keratinocytes, as well as the predicted genegene connections between keratinocytes and fibroblasts from CellphoneDB. (D) The gene expression profiles of secreted proteins in keratinocytes, involved in the predicted significant ligand-receptor pairs between keratinocytes and fibroblasts, are shown in different samples. (E) Workflow of the Crosstalk Experiment. HEK cells were cultured in a basal medium for 24 hours to produce a conditioned medium. This conditioned medium was then mixed 1 : 1 with fibroblast growth medium and used to culture HDF for 72 hours, with fresh conditioned medium replaced every 24 hours. Young KCM, old KCM, control mRNA-treated KCM, and ATF3 mRNA-treated KCM were prepared and utilized for fibroblast culture. After 72 hours, measurements were taken for P-galactosidase activity, collagen and elastin mRNA levels by qPCR, and collagen protein levels by ELISA.

[0065] FIGs. 11A-11D. ATF3 rejuvenates aged human skin. (A) Schematic of the experimental workflow: 62-year-old human skin explants were treated with RFP mRNA (control), platelet-rich plasma (PRP, positive control), or ATF3 mRNA via microneedle delivery and analyzed 96 h post-treatment. (B) Representative Masson’s trichrome staining and quantification of total collagen. (C) Representative Verhoeff-Van Gieson staining and quantification of elastin. (D) Immunofluorescence staining of type III collagen (C0L3A1, green) with quantification of staining intensity. ATF3 treatment significantly increased collagen, elastin, and C0L3A1 deposition compared with controls. Data are shown as mean ± s.e.m.; one-way ANOVA, P < 0.05, P < 0.01, P < 0.001. Scale bars, 100 pm (B, C), 250 pm (D).

[0066] FIGs. 12A-12C. ATF3 promotes basal keratinocyte and hair follicle stem cell renewal. (A) Schematic of human epidermal and hair follicle architecture, highlighting basal keratinocytes in the epidermis and bulge-region stem cells. (B) Quantification of KRT15+cell layer thickness in human skin explants treated with RFP mRNA, PRP, or ATF3 mRNA. (C) Representative immunofluorescence staining of KRT15, p63, and DAPI. ATF3 treatment significantly expanded the KRT15+basal / stem cell compartment compared with controls. Data are shown as mean ± s.e.m.; one-way ANOVA, P < 0.001. Scale bar, 100 pm.

[0067] FIGs. 13A-13C. ATF3 mRNA reduced scarring in a murine wound healing model. (A) Schematic illustrates the experimental design in which full-thickness excisional wounds were 9

[0068] #14392914vl Attorney Docket No. H0498.70834WO00 created on the dorsal skin of mice, followed by treatment with either control or ATF3 mRNA. White circles indicate the wound sites. In control-treated mice (B), wounds remain visible, whereas in ATF3 mRNA-treated mice (C), wound sites are largely indistinguishable from surrounding skin, indicating enhanced healing.

[0069] DETAILED DESCRIPTION

[0070] Aging is characterized by a gradual decline in function, partly due to molecular damage that accumulates over time. Human skin is susceptible to both chronological aging and environmental damage (including photoaging, which is the damage ascribed to UV exposure), resulting in detrimental structural and physiological changes with age. The accumulation of cellular damage and the subsequent induction of cellular senescence impairs skin regeneration and contributes to various skin disorders, including impaired wound healing, hyperpigmentation disorders like age spots and melasma, and chronic inflammatory conditions.

[0071] Cellular senescence is a state of permanent growth arrest that cells enter in response to various stressors, including telomere shortening, oxidative stress, and DNA damage. During senescence, cells lose the ability to proliferate, although they remain viable and metabolically active. A senescent cell, therefore, is a cell that has exited the cell cycle, displays one or more epigenetic markers consistent with senescence, and / or expresses one or more senescence cell markers.

[0072] Among the various molecular players involved in cellular aging and senescence, Activating Transcription Factor 3 (ATF3) has been identified herein as an important factor. ATF3 is a member of the ATF / CREB family of transcription factors, which play roles in regulating gene expression in response to various physiological and pathological stimuli. ATF3 is involved in a wide range of cellular processes, including the modulation of cellular senescence and proliferation. As disclosed in some embodiments herein, the upregulation of ATF3 in epidermal cells (e.g., keratinocytes) inhibits cellular senescence and promotes cell proliferation.

[0073] The mechanism by which ATF3 inhibits cellular senescence is multifaceted. ATF3 functions as a transcriptional regulator, modulating the expression of genes involved in stress response and cell cycle regulation. When expressed at elevated levels, ATF3 can suppress the activity of senescence-inducing pathways, such as the p53 / p21 and pl6INK4a / Rb pathways, thereby preventing the cells from entering a senescent state or reducing the rate at which the cells senesce. ATF3 can protect cells from UV-induced apoptosis by inducing pl5PAFexpression or through facilitating the recruitment of DNA-repair proteins to damaged DNA sites.

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[0076] Additionally, upregulation of ATF3 in epidermal cells induces the secretion of factors that further modulate skin rejuvenation. These factors, including growth factors, cytokines, and extracellular matrix proteins, play a role in cellular communication and tissue homeostasis. For example, epidermal cells upregulated with ATF3 can secrete growth factors such as Epidermal Growth Factor (EGF), Keratinocyte Growth Factor (KGF), and Transforming Growth Factor-P (TGF-P). These factors inhibit cellular senescence and promote proliferation in dermal cells (e.g., fibroblasts, macrophages, and / or mast cells), as well as induce production of elastin, an extracellular matrix protein that imparts elasticity and resilience to the skin.

[0077] Epidermal cells upregulated with ATF3 can also secrete chemokines, which are signaling proteins that attract immune cells and promote tissue repair. One such chemokine is CXCL2, which plays a role in recruiting neutrophils to sites of injury and inflammation. CXCL2 can also enhance the production of proteins associated with collagen production in dermal cells.

[0078] Therefore, presented herein are methods of inhibiting cellular senescence and / or promoting cellular proliferation in epidermal and / or dermal cells. Further, presented herein are methods of enhancing the production of extracellular matrix components, such as collagen and elastin, in dermal cells. As a result, the methods described in herein provide a comprehensive approach to treating age-related skin conditions and other dermatological issues associated with cellular senescence and impaired proliferation.

[0079] Activating Transcription Factor 3 (ATF3)

[0080] Some aspects of the present disclosure relate to methods involving a nucleic acid encoding an ATF3 protein. The wild-type amino acid sequence of human ATF3 may be found at NCBI Reference Sequence No. NP_001025458.1 and is reproduced below as SEQ ID NO: 2. ATF-3 isoform 1 [Homo sapiens] NP_001025458.1 MMLQHPGQVSASEVSASAIVPCLSPPGSLVFEDFANLTPFVKEELRFAIQNKHLCHRMSS ALESVTVSDRPLGVSITKAEVAPEEDERKKRRRERNKIAAAKCRNKKKEKTECLQKESE KLESVNAELKAQIEELKNEKQHLIYMLNLHRPTCIVRAQNGRTPEDERNLFIQQIKEGTL QS (SEQ ID NO: 2)

[0081] In some embodiments, the nucleic acid encodes an ATF3 protein. In some embodiments, the nucleic acid encodes a wild-type ATF3 protein. In some embodiments, the nucleic acid encodes the amino acid sequence of SEQ ID NO: 2. In some embodiments, the ATF3 has the amino acid sequence of SEQ ID NO: 2. In some embodiments, the nucleic acid encodes an amino acid sequence that is at least 90%, 95%, 97%, or 100% identical to SEQ ID NO: 2. In

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[0083] #14392914vl Attorney Docket No. H0498.70834WO00 some embodiments, the ATF3 has an amino acid sequence that is at least 90%, 95%, 97%, or 100% identical to SEQ ID NO: 2.

[0084] In some embodiments, the nucleic acid comprises an open reading frame (ORF). An ORF is a continuous stretch of codons that begins with a start codon (e.g., ATG), ends with a stop codon (e.g., TAA, TAG, or TGA), and encodes a polypeptide, for example, a protein. An example ORF encoding ATF3 may be found at GenBank Accession No. BT006996.1. As another example, an ORF sequence encoding ATF3 may correspond to SEQ ID NO: 1, below.

[0085] ATF-3 ORF [Homo sapiens]

[0086] ATGATGCTTCAACACCCAGGCCAGGTCTCTGCCTCGGAAGTGAGTGCTTCTGCCATC GTCCCCTGCCTGTCCCCTCCTGGGTCACTGGTGTTTGAGGATTTTGCTAACCTGACGC CCTTTGTCAAGGAAGAGCTGAGGTTTGCCATCCAGAACAAGCACCTCTGCCACCGG ATGTCCTCTGCGCTGGAATCAGTCACTGTCAGCGACAGACCCCTCGGGGTGTCCATC ACAAAAGCCGAGGTAGCCCCTGAAGAAGATGAAAGGAAAAAGAGGCGACGAGAAA GAAATAAGATTGCAGCTGCAAAGTGCCGAAACAAGAAGAAGGAGAAGACGGAGTG CCTGCAGAAAGAGTCGGAGAAGCTGGAAAGTGTGAATGCTGAACTGAAGGCTCAGA TTGAGGAGCTCAAGAACGAGAAGCAGCATTTGATATACATGCTCAACCTTCATCGG CCCACGTGTATTGTCCGGGCTCAGAATGGGAGGACTCCAGAAGATGAGAGAAACCT

[0087] CTTTATCCAACAGATAAAAGAAGGAACATTGCAGAGCTAA (SEQ ID NO: 1)

[0088] In some embodiments, the nucleic acid comprises a sequence corresponding to SEQ ID NO: 1. In some embodiments, the nucleic acid comprises a sequence that is at least 90%, 95%, 97%, or 100% identical to SEQ ID NO: 1. In some embodiments, the ORF comprises a nucleic acid sequence corresponding to SEQ ID NO: 1. In some embodiments, the ORF comprises a nucleic acid sequence that is at least 90%, 95%, 97%, or 100% identical to SEQ ID NO: 1.

[0089] In some embodiments, the ORF encodes an ATF3 protein. In some embodiments, the ORF encodes a wild-type ATF3 protein. In some embodiments, the ORF encodes the amino acid sequence of SEQ ID NO: 2. In some embodiments, the ORF encodes an amino acid sequence that is at least 90%, 95%, 97%, or 100% identical to SEQ ID NO: 2.

[0090] In some embodiments, the nucleic acid comprises a sequence corresponding to any one of SEQ ID NOs: 3-5. In some embodiments, the nucleic acid comprises a sequence corresponding to the RNA version of any one of SEQ ID NOs: 3-5. An RNA version of SEQ ID NOs: 3, 4,

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[0092] #14392914vl Attorney Docket No. H0498.70834WO00 and / or 5 are nucleic acids in which thymine (T) nucleobases are replaced by uracil (U) nucleobases).

[0093] Table 1. Nucleic acid sequences

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[0102] In some embodiments, the nucleic acid comprises a sequence corresponding to SEQ ID NO: 3. In some embodiments, the nucleic acid comprises a sequence corresponding to SEQ ID NO: 4. In some embodiments, the nucleic acid comprises a sequence corresponding to SEQ ID NO: 5. In some embodiments, the nucleic acid comprises a sequence corresponding to SEQ ID NO: 6. In some embodiments, the nucleic acid comprises a sequence corresponding to SEQ ID NO: 7. In some embodiments, the nucleic acid comprises a sequence corresponding to SEQ ID NO: 8. In some embodiments, the nucleic acid comprises a sequence that is at least 90%, 95%, 97%, or 100% identical to SEQ ID NO: 3. In some embodiments, the nucleic acid comprises a sequence that is at least 90%, 95%, 97%, or 100% identical to SEQ ID NO: 4. In some embodiments, the nucleic acid comprises a sequence that is at least 90%, 95%, 97%, or 100% identical to SEQ ID NO: 5. In some embodiments, the nucleic acid comprises a sequence that is at least 90%, 95%, 97%, or 100% identical to SEQ ID NO: 6. In some embodiments, the nucleic acid comprises a sequence that is at least 90%, 95%, 97%, or 100% identical to SEQ ID NO: 7. In some embodiments, the nucleic acid comprises a sequence that is at least 90%, 95%, 97%, or 100% identical to SEQ ID NO: 8. In some embodiments, the nucleic acid comprises a sequence that is at least 90%, 95%, 97%, or 100% identical to any one of SEQ ID NOs: 3-8.

[0103] #14392914vl Attorney Docket No. H0498.70834WO00

[0104] ATF3 Expression

[0105] Some aspects of the present disclosure relate to methods comprising expressing ATF3 in a cell, population of cells, tissue, or a subject, by delivering a nucleic acid encoding ATF3 to the cell, population of cells, tissue, or a subject.

[0106] “Increased expression,” as used herein, refers to a level of ATF3 expression in the cell, population of cells, tissue, and / or subject, that is higher than the level of ATF3 expression in the cell, population of cells, tissue, and / or subject prior to delivery of the nucleic acid. Methods for determining the level of expression of a protein in a cell, population of cells, tissue and / or subject are disclosed herein and known in the art.

[0107] In some embodiments, delivering a nucleic acid encoding ATF3 to a cell results in increased expression of ATF3 in the cell. In some embodiments, the cell is a skin cell. In some embodiments, the cell is a dermal cell or an epidermal cell. In some embodiments, the cell is an epidermal cell (i.e., a cell of the epidermis.). In some embodiments, the cell is a hair follicle cell. In some embodiments, the cell is a hair follicle stem cell (HFSC).

[0108] The epidermis is the outermost layer of the skin and serves as the primary barrier between the body and the external environment. It is composed of several distinct cell types, including keratinocytes, melanocytes, Langerhans cells, and Merkel cells. In some embodiments, the cell is a keratinocyte. Keratinocytes are the predominant cell type, comprising about 90% of the cells in the epidermis. Impaired keratinocyte function, such as increases in keratinocyte senescence and decreased keratinocyte proliferation can result in compromised barrier integrity, leading to increased susceptibility to infections and irritants, as well as to age-related skin disorders or diseases.

[0109] Hair follicles are tunnel-like organs within the skin that produce hair by pushing out keratinized cells from the root. Hair follicles are connected to blood vessels, a sebaceous gland, and a small muscle (arrector pili), all of which work together to facilitate hair growth in a cycle of growth, rest, and shedding. A hair follicle cell includes any cell found throughout the hair follicle (matrix, outer root sheath, inner root sheath, dermal papilla, etc.). Hair follicle stem cells (HFSCs) include multipotent cells (e.g., adult stem cells) located in the bulge region of a hair follicle that generate new hair and skin cells.

[0110] In some embodiments, delivering a nucleic acid encoding ATF3 to a population of cells results in increased expression of ATF3 in one or more cells of the population. In some embodiments, a nucleic acid encoding ATF3 is delivered to a population of cells. In some embodiments, the population of cells comprises skin cells. In some embodiments, the population 18

[0111] #14392914vl Attorney Docket No. H0498.70834WO00 of cells comprises epidermal cells. In some embodiments, the epidermal cells comprise keratinocytes. In some embodiments, the population of cells comprises keratinocytes. In some embodiments, the population of cells comprises dermal cells. In some embodiments, the dermal cells comprise fibroblasts, macrophages, and / or mast cells. In some embodiments, the population of cells comprises fibroblasts, macrophages, and / or mast cells. In some embodiments, the population of cells comprises hair follicle stem cells (HFSCs).

[0112] In some embodiments, delivering a nucleic acid encoding ATF3 to a subject results in increased expression of ATF3 in the subject. In some embodiments, a nucleic acid encoding ATF3 is delivered to a subject. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human.

[0113] In some embodiments, delivering the nucleic acid to the subject results in increased expression of ATF3 in one or more cells of a skin tissue, optionally cells of the epidermis, optionally keratinocytes. In some embodiments, a nucleic acid encoding ATF3 is delivered to tissue of a subject. In some embodiments, the tissue is skin tissue. In some embodiments, the tissue comprises one or more cells of the skin tissue. In some embodiments, the tissue comprises one or more cells of the epidermis. In some embodiments, the tissue comprises dermal cells and / or epidermal cells. In some embodiments, the tissue comprises keratinocytes. In some embodiments, the tissue comprises fibroblasts, macrophages, and / or mast cells.

[0114] Cellular Senescence

[0115] Some aspects of the present disclosure relate to methods of inhibiting cellular senescence. “Inhibition” refers to a reduction in the activity, function, or level of a biological process or molecule. In the context of cellular senescence, “inhibition” refers to a reduction or delay in the onset or progression of senescence-associated cell cycle arrest, along with a decrease in the associated phenotypic markers that characterize senescent cells (e.g., SA-P-Gal, SASP). SA-P-Gal

[0116] One such phenotypic marker is senescence-associated P-galactosidase (SA-P-Gal). SA-P- Gal is well-known in the art as a biomarker for cellular senescence. SA-P-Gal activity is typically detected at pH 6.0 and is elevated in senescent cells. The elevated SA-P-Gal activity in senescent cells is associated with increased lysosomal mass, a hallmark of the senescent phenotype. This increase in lysosomal content is thought to result from the upregulation of lysosomal biogenesis pathways as cells enter senescence.

[0117] 19

[0118] #14392914vl Attorney Docket No. H0498.70834WO00

[0119] By measuring changes in SA-P-Gal activity, whether a given intervention effectively delays the onset of senescence or reverses senescent phenotypes can be evaluated. As one nonlimiting example, to determine if the delivery of a nucleic acid encoding ATF3 to a cell, population of cells, tissue, and / or subject inhibited senescence in the cell, population of cells, tissue, and / or subject, the level of SA-P-Gal in the cell, population of cells, tissue, and / or subject may be measured before and after the delivery of the nucleic acid. If the level of SA-P-Gal postdelivery of the nucleic acid is decreased relative to the level of SA-P-Gal pre-delivery of the nucleic acid, then the senescence in the cell, population of cells, tissue, and / or subject is “inhibited.”

[0120] Various methods exist for detecting SA-P-Gal activity. For example, histochemical staining using the chromogenic substrate X-gal (5-bromo-4-chloro-3-indolyl P-D- galactopyranoside) is a commonly used technique. In this assay, senescent cells stained with X- gal exhibit blue coloration due to the enzymatic cleavage of X-gal by SA-P-Gal, producing an insoluble blue dye. This method allows for the visualization and quantification of SA-P-Gal- positive cells under light microscopy. In an alternative example, fluorescent substrates such as C12FDG (5-dodecanoylaminofluorescein di-P-D-galactopyranoside) can be used for sensitive detection of SA-P-Gal activity. Upon cleavage by SA-P-Gal, C12FDG produces a fluorescent signal detectable by flow cytometry or fluorescence microscopy, enabling precise quantification of SA-P-Gal activity in individual cells or cell populations. However, any suitable method for determining SA-P-Gal activity known in the art may be used.

[0121] Senescence-Associated Secretory Phenotype (SASP)

[0122] Additionally, senescent cells exhibit altered gene expression profiles characterized by the secretion of pro-inflammatory cytokines, chemokines, and proteases, collectively known as the senescence-associated secretory phenotype (SASP). SASP is a complex and dynamic mixture of factors that includes cytokines (e.g., interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin- 1 beta (IL- ip), interferon beta (IFN-P), and tumor necrosis factor alpha (TNF-a)), chemokines (e.g., C- X-C motif chemokine ligand 2 (CXCL2) and other CXCL family members), growth factors (e.g., cyclin-dependent kinase inhibitor 2A (CDKN2A)), proteases (e.g., matrix metalloproteinases (MMPs), serine proteases, and cathepsins), lipid metabolites (e.g., cyclooxygenase, prostaglandin E2 (PGE2) and leukotriene D), and extracellular vesicles.

[0123] SASP is a hallmark characteristic of senescence. During senescence, cells undergo a permanent cell cycle arrest. This cell cycle arrest is accompanied by changes in gene expression 20

[0124] #14392914vl Attorney Docket No. H0498.70834WO00 that lead to the secretion of SASP factors. These secreted factors serve several functions, including reinforcing the senescent state in an autocrine manner and affecting the surrounding tissue environment in a paracrine manner. The SASP can contribute to tissue remodeling, immune surveillance, and the propagation of senescence to neighboring cells.

[0125] By measuring changes in SASP levels, whether a given intervention effectively delays the onset of senescence or reverses senescent phenotypes can be evaluated. As one non-limiting example, to determine if the delivery of a nucleic acid encoding ATF3 to a cell, population of cells, tissue, and / or subject inhibited senescence in the cell, population of cells, tissue, and / or subject, the levels of one or more SASP factors (e.g., IL-6, IL-8, IL-ip, IFN-P, TNF-a, CXCL2, CDKN2A, PGE2, leukotriene D, and cyclooxygenase) in the cell, population of cells, tissue, and / or subject may be measured before and after the delivery of the nucleic acid. If the levels of one or more SASP factor post-delivery of the nucleic acid is decreased relative to the level of one or more SASP factor pre-delivery of the nucleic acid, then the senescence in the cell, population of cells, tissue, and / or subject is “inhibited.”

[0126] Various methods exist in the art for detecting SASP activity including, but not limited to, enzyme-linked immunosorbent assays (ELISA) (i.e., to quantify specific cytokines, chemokines, and growth factors in cell culture supernatants or biological fluids), western blotting and / or immunohistochemistry (i.e., to detect and quantify protein levels of SASP factors in cell lysates and tissue sections), quantitative polymerase chain reaction (qPCR), multiplex assays, mass spectrometry and / or flow cytometry. However, any suitable method for measuring SASP activity known in the art may be used.

[0127] Methods of Inhibiting Cellular Senescence

[0128] Some aspects of the present disclosure provide a method of inhibiting cellular senescence, the method comprising expressing Activating Transcription Factor 3 (ATF3) in a cell at a level sufficient to inhibit senescence of the cell. "A level sufficient" refers to a specific threshold or amount of a substance, activity, or condition that achieves the desired effect or outcome in a given biological context. For example, in the context of expressing ATF3 in a cell, “a level sufficient to inhibit senescence” refers to the level of ATF3 expression that prevents and / or delays the onset of cellular senescence. This involves upregulating ATF3 gene expression so that the resulting protein concentration is adequate to interfere with or modulate the signaling pathways and molecular mechanisms that drive cells into a senescent state. By achieving a “sufficient level” of ATF3 expression, the cell can maintain its proliferative capacity, resist 21

[0129] #14392914vl Attorney Docket No. H0498.70834WO00 stress-induced damage, and avoid entering a state of permanent growth arrest characteristic of senescence. As disclosed herein, the inhibition of senescence may be determined utilizing any technique for determining senescence inhibition known in the art, including, but not limited to, measuring decreased levels of senescence-associated biomarkers (e.g., SASP and / or SA-P-Gal).

[0130] Some aspects of the present disclosure provide a method of inhibiting cellular senescence, the method comprising: delivering a messenger ribonucleic acid (mRNA) comprising an open reading frame encoding Activating Transcription Factor 3 (ATF3) to a skin cell in an amount effective to inhibit senescence of the skin cell. "An amount effective" refers to a specific quantity of a substance or intervention that produces a desired therapeutic or biological effect in a given context. For example, in the context of delivering an mRNA encoding ATF3 to a cell, “an amount effective to inhibit senescence” refers to the amount of mRNA encoding ATF3 that prevents and / or delays the onset of cellular senescence in the cell.

[0131] In some embodiments, the ATF3 is expressed in the cell at a level sufficient to reduce expression of one or more senescence-associated secretory phenotype (SASP) factors. In some embodiments, the SASP factors are selected from cytokines, chemokines, growth factors, proteases, lipid metabolites and extracellular vesicles. In some embodiments, the SASP factors are selected from interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-1 beta (IL-ip), interferon beta (IFN-P), tumor necrosis factor alpha (TNF-a), cyclin-dependent kinase inhibitor 2A (CDKN2A), CXCL family members, C-X-C motif chemokine ligand 2 (CXCL2), matrix metalloproteinases (MMPs), serine proteases, cathepsin, cyclooxygenase, prostaglandin E2 (PGE2), and leukotriene D. In some embodiments, ATF3 is expressed in the cell at a level sufficient to reduce expression of senescence-associated P-galactosidase. In some embodiments, ATF3 is expressed in the cell at a level sufficient to increase the rate of proliferation of the cell.

[0132] Some aspects of the present disclosure provide a method of inhibiting senescence in a population of cells, the method comprising delivering a nucleic acid encoding activating transcription factor 3 (ATF3) to the population, wherein delivering the nucleic acid to the population results in inhibition of senescence in one or more cells of the population. In some embodiments, following delivering the nucleic acid, the expression of one or more senescence- associated secretory phenotype (SASP) factors is reduced in one or more cells of the population, relative to a control. As used herein, “a control” refers to the baseline or initial level of the specific variable or parameter being measured prior to an intervention. For example, in the context of reducing SASP factor expression in one or more cells of the population relative to a

[0133] 22

[0134] #14392914vl Attorney Docket No. H0498.70834WO00 control, the “control” refers to the level of SASP factor expression in the cells of the population prior to delivery of the nucleic acid encoding ATF3.

[0135] In some embodiments, the expression of senescence-associated P-galactosidase is reduced by at least 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, or 75%, relative to a control. In some embodiments, the expression of senescence-associated P-galactosidase is reduced by 5- 100%, relative to a control. In some embodiments, the expression of senescence-associated P- galactosidase is reduced by 5-80%, 5-70%, 5-60%, 5-50%, 5-40%, 5-30%, 5-25%, 5-20%, 20- 100%, 20-90%, 20-70%, 20-50%, 25-100%, 25-75%, 25-50%, 50-100%, or 50-75%, relative to a control. In some embodiments, the expression of the one or more SASP factors is reduced by at least 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, or 75%, relative to a control. In some embodiments, the expression of the one or more SASP factors is reduced by 5-80%, 5-70%, 5- 60%, 5-50%, 5-40%, 5-30%, 5-25%, 5-20%, 20-100%, 20-90%, 20-70%, 20-50%, 25-100%, 25- 75%, 25-50%, 50-100%, or 50-75%, relative to a control. In some embodiments, the expression of CDKN2A, IFN-P, IL-ip, IL-6, and / or IL-8 is reduced by 5-80%, 5-70%, 5-60%, 5-50%, 5- 40%, 5-30%, 5-25%, 5-20%, 20-100%, 20-90%, 20-70%, 20-50%, 25-100%, 25-75%, 25-50%, 50-100%, or 50-75%, relative to a control.

[0136] Some aspects of the present disclosure provide methods of inhibiting cellular senescence in a subject comprising: delivering an mRNA comprising an open reading frame encoding ATF3 to a subject in an amount effective to inhibit cellular senescence in the subject. Some aspects of the present disclosure provide a method of inhibiting senescence in cells of a skin tissue of a subject, the method comprising administering a nucleic acid encoding ATF3 to the subject, wherein administering the nucleic acid to the subject results in inhibition of senescence in skin tissue of the subject. Some aspects of the present disclosure provide a method of inhibiting senescence in dermal cells of a subject, the method comprising administering a nucleic acid encoding ATF3 to epidermal cells of the subject, wherein administering the nucleic acid to the epidermal cells results in inhibition of senescence in the dermal cells of the subject.

[0137] “Administering” as used herein, refers to the process of providing an agent, such as a drug, compound, or composition, (e.g., a nucleic acid encoding ATF3) to a subject in a manner that is pharmacologically useful (i.e., results in the intended pharmacological or therapeutic effect). This delivery can be carried out by various routes, including oral, intravenous, intramuscular, subcutaneous, intranasal, topical, or any other route known in the art appropriate for the specific agent and condition being treated. In some embodiments, the nucleic acid is

[0138] 23

[0139] #14392914vl Attorney Docket No. H0498.70834WO00 administered via topical, intravenous, intramuscular, intradermal, transdermal, oral, or subcutaneous administration.

[0140] Methods of Increasing Cell Proliferation

[0141] Some aspects of the present disclosure relate to methods of increasing the cell proliferation and / or the rate of cell proliferation in cell, population of cells, tissue, and / or a subject through delivery of a nucleic acid encoding ATF3. In some embodiments, following delivering a nucleic acid encoding ATF3 to a cell, ATF3 is expressed in the cell at a level sufficient to increase the rate of proliferation of the cell, relative to a control. In some embodiments, following delivering the nucleic acid to a population of cells, the rate of cell proliferation is increased in the population, relative to a control. In some embodiments, following delivering the nucleic acid to a tissue, the rate of cell proliferation is increased in the tissue, relative to a control. In some embodiments, following delivering the nucleic acid to a subject, the rate of cell proliferation is increased the subject, relative to a control. In some embodiments, the rate of cell proliferation is increased by at least 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, or 75%, relative to a control. In some embodiments, the rate of cell proliferation is increased by 5- 80%, 5-70%, 5-60%, 5-50%, 5-40%, 5-30%, 5-25%, 5-20%, 20-100%, 20-90%, 20-70%, 20- 50%, 25-100%, 25-75%, 25-50%, 50-100%, or 50-75%, relative to a control

[0142] An increased rate of cell proliferation can be determined by measuring the levels of proliferation markers (e.g., Kiel 67 (KI-67)) or by measuring the total number of cells, relative to a control. KI-67, an established marker of cell proliferation, is a nuclear protein that is expressed in all active phases of the cell cycle (Gl, S, G2, and mitosis), but not in resting cells (GO). Because of this, KI-67 is commonly used to assess cell proliferation. Increased levels of KI-67 expression, relative to a control, indicate an increased rate of cell division, whereas decreased levels of KI-67 expression, relative to a control, indicate a decreased rate of cell division.

[0143] In some embodiments, the expression of KI-67 is increased in cells of the population, relative to a control. In some embodiments, the expression of KI-67 is increased by at least 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, or 75%, relative to a control. In some embodiments, the expression of KI-67 is increased by 5-80%, 5-70%, 5-60%, 5-50%, 5-40%, 5-30%, 5-25%, 5- 20%, 20-100%, 20-90%, 20-70%, 20-50%, 25-100%, 25-75%, 25-50%, 50-100%, or 50-75%, relative to a control, optionally increased by 10%-30%.

[0144] The total number of cells is another potential indicator of cell proliferation. An increase in the total number of cells over time suggests that cells are actively dividing, and that the 24

[0145] #14392914vl Attorney Docket No. H0498.70834WO00 intervention (e.g., delivery of a nucleic acid encoding ATF3), successfully promoted cell proliferation. In some embodiments, following the delivery of a nucleic acid encoding ATF3 to a population, the total number of cells in the population is increased by at least 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, or 75%, relative to a control. In some embodiments, following the delivery of a nucleic acid encoding ATF3 to a population, the total number of cells in the population is increased by 5-80%, 5-70%, 5-60%, 5-50%, 5-40%, 5-30%, 5-25%, 5-20%, 20- 100%, 20-90%, 20-70%, 20-50%, 25-100%, 25-75%, 25-50%, 50-100%, or 50-75%, relative to a control.

[0146] The total number of cells can be measured through any cell counting technique known in the art, including, but not limited to, using a hemocytometer, automated cell counters, or flow cytometry.

[0147] Methods of Increasing Collagen and / or Elastin

[0148] Some aspects of the present disclosure provide a method of increasing cellular collagen and / or elastin production comprising delivering a messenger ribonucleic acid (mRNA) comprising an open reading frame encoding Activating Transcription Factor 3 (ATF3) to a subject in an amount effective to increase cellular collagen and / or elastin production in the subject.

[0149] Some aspects of the present disclosure provide a method of increasing collagen and / or elastin production in dermal cells of a subject, the method comprising administering a nucleic acid encoding activating transcription factor 3 (ATF3) to epidermal cells of the subject, wherein administering the nucleic acid to the epidermal cells results in increased collagen production in the dermal cells of the subject. In some embodiments, the production of collagen and / or elastin is increased by at least 5%, 10%, 25%, 50%, 100%, or 200%, relative to a control. In some embodiments, the production of collagen and / or elastin is increased by 5-80%, 5-70%, 5-60%, 5- 50%, 5-40%, 5-30%, 5-25%, 5-20%, 20-100%, 20-90%, 20-70%, 20-50%, 25-100%, 25-75%, 25-50%, 50-100%, or 50-75%, relative to a control. In some embodiments, following administration of the nucleic acid, the expression and / or secretion of chemokines is increased in the epidermal cells, relative to a control. In some embodiments, the chemokines comprise C-X-C Motif Chemokine Ligand 2 (CXCL2).

[0150] To determine the efficacy of a nucleic acid in increasing collagen and elastin production, various biomarkers and quantitative methods known in the art can be employed. Increased collagen production can be measured through biochemical assays that quantify the levels of 25

[0151] #14392914vl Attorney Docket No. H0498.70834WO00 specific collagen types, such as C0L1A1, COL1A2, and COL3A1. In some embodiments, following administration of the nucleic acid, the expression of proteins involved in collagen production is increased in the dermal cells, relative to a control. In some embodiments, the proteins involved in collagen production comprise COL1A1, COL1A2, and / or COL3A1. In some embodiments, expression of proteins involved in collagen production is increased by at least 5%, 10%, 25%, 50%, 100%, or 200%, relative to a control.

[0152] Similarly, elastin levels can be assessed using assays that detect elastin-specific proteins and peptides (e.g., elastin, tropoelastin, fibrillin-1, fibrillin-2, fibulin-5, lysyl oxidase, elastin microfibril interface-located protein 1 (EMILIN- 1), elastase, latent TGF-beta binding protein 2 (LTBP2), decorin, MAGP-1 (microfibril-associated glycoprotein 1). For example, the level of collagen and / or elastin in a cell, population of cells, tissue and / or subject can be measured using suitable immunohistochemistry (IHC) or EEISA techniques disclosed herein and / or known in the art.

[0153] Methods of Treating Skin Disorders

[0154] Some aspects of the present disclosure provide a method of treating a skin disorder in a subject, the method comprising delivering to the subject a messenger ribonucleic acid (mRNA) comprising an open reading frame encoding Activating Transcription Factor 3 (ATF3) to a subject in an amount effective to treat the skin disorder in the subject. Non-limiting examples of skin disorders include psoriasis, atopic dermatitis, lupus, lichen planus, epidermolysis bullosa, scleroderma, alopecia, acne vulgaris, actinic keratosis, bruises, burns, dermatitis herpetiformis, hidradenitis suppurativa, hypertrophic scars, keloids, prurigo, pyoderma gangrenosum, and vitiligo.

[0155] In some embodiments, the skin disorder is alopecia. Alopecia refers generally to the loss or absence of hair from areas of the body where hair normally grows, most commonly the scalp. It is a descriptive term encompassing a variety of conditions that result in partial or complete hair loss. Alopecia may be temporary or permanent, localized or diffuse, and may involve hair of the scalp, face (e.g., eyebrows, eyelashes, beard), or other body sites. The pathogenesis of alopecia can vary widely depending on the underlying cause, and may involve genetic predisposition, hormonal dysregulation, autoimmune mechanisms, environmental triggers, or physical trauma to the hair follicles.

[0156] In some embodiments, alopecia is a non-scarring form of hair loss in which the hair follicles remain intact and capable of regeneration. Non-scarring alopecias can include, for 26

[0157] #14392914vl Attorney Docket No. H0498.70834WO00 example, androgenetic alopecia, alopecia areata, telogen effluvium, and traction alopecia. Androgenetic alopecia is a genetically determined and hormonally influenced condition characterized by progressive thinning of hair, typically presenting in a patterned distribution. Alopecia areata is an autoimmune condition characterized by sudden, well-demarcated patches of hair loss and may progress to more extensive forms such as alopecia totalis (loss of all scalp hair) or alopecia universalis (loss of all body hair). Variants of alopecia areata can include ophiasis alopecia, which manifests as band-like hair loss around the scalp margins, and alopecia barbae, which involves hair loss localized to the beard region. Telogen effluvium is a condition in which a large number of hair follicles prematurely enter the telogen (resting) phase, resulting in diffuse hair shedding often triggered by stress, hormonal changes, illness, or certain medications. Traction alopecia results from chronic mechanical tension on the hair shafts, commonly associated with certain hairstyles, and is typically reversible if the mechanical stress is eliminated.

[0158] In other embodiments, alopecia is a scarring, or cicatricial, form of hair loss, which is characterized by irreversible destruction of hair follicles and their replacement by fibrotic scar tissue. Scarring alopecias can arise from a variety of inflammatory or autoimmune disorders, including lichen planopilaris, frontal fibrosing alopecia, and discoid lupus erythematosus. These conditions often present as patchy areas of hair loss accompanied by scalp erythema, scaling, pustules, or atrophy. Over time, the inflammatory process destroys the follicular stem cell niche, preventing any possibility of hair regrowth. One particular form of scarring alopecia is pseudopelade of Brocq, an idiopathic condition characterized by slowly progressive, smooth, atrophic patches of hair loss that coalesce over time.

[0159] Because different forms of alopecia involve distinct pathogenic mechanisms and have varying prognoses, accurate classification is important for selecting appropriate therapeutic approaches. Non- scarring forms may respond to treatments aimed at stimulating follicular regeneration or reversing immune or hormonal triggers, whereas scarring forms often require early anti-inflammatory or immunosuppressive therapy to prevent permanent follicular destruction. The compositions, methods, and systems described herein may be applied to the treatment or prevention of any of the foregoing forms of alopecia. In some embodiments, alopecia is selected from androgenetic alopecia, alopecia areata, telogen effluvium, traction alopecia, cicatricial alopecia, alopecia barbae, ophiasis alopecia, alopecia universalis, and pseudopelade. In some embodiments, alopecia is androgenetic alopecia. In some embodiments, alopecia is alopecia areata. In some embodiments, alopecia is telogen effluvium. In some 27

[0160] #14392914vl Attorney Docket No. H0498.70834WO00 embodiments, alopecia is traction alopecia. In some embodiments, alopecia is cicatricial alopecia. In some embodiments, alopecia is alopecia barbae. In some embodiments, alopecia is ophiasis alopecia. In some embodiments, alopecia is alopecia universalis. In some embodiments, alopecia is pseudopelade.

[0161] Nucleic Acids

[0162] Some aspects of the present disclosure relate to methods involving a nucleic acid. In some embodiments, the nucleic acid comprises DNA. In some embodiments, the nucleic acid comprises RNA, such as self-amplifying RNA, circular RNA, or mRNA. In some embodiments, the nucleic acid is a messenger ribonucleic acid (mRNA). mRNA is RNA that encodes a protein or a fragment thereof and can be translated to produce the encoded protein or fragment in vitro, in vivo, in situ, or ex vivo.

[0163] Unless otherwise specified, nucleic acid sequences provided here may contain "T" bases in a representative DNA sequence; however, for RNA sequences, these "T" bases will be replaced with "U" bases. Therefore, any DNA sequence disclosed and identified by a particular sequence herein also discloses the corresponding RNA sequence, with each "T" in the DNA sequence replaced by "U."

[0164] Untranslated Regions (UTRs)

[0165] In some embodiments, the nucleic acid contains one or more regions that function as untranslated regions. A "5' untranslated region" (5' UTR) is located upstream of the start codon in an mRNA and does not encode a polypeptide. Conversely, a "3' untranslated region" (3' UTR) is situated downstream of the stop codon and also does not encode a polypeptide. The 5' UTR begins at the transcription start site and extends to, but does not include, the start codon. The 3' UTR starts immediately after the stop codon and continues until a transcriptional termination signal. Various sequences for 5' UTRs and 3' UTRs are known in the art.

[0166] In some embodiments, the nucleic acid comprises a 5’ UTR. In some embodiments, the nucleic acid comprises a 5' human alpha-globulin- 1 (HBA1) UTR. In some embodiments, the nucleic acid comprises a wild-type ATF3 5’ UTR. In some embodiments, the nucleic acid comprises a 3’ UTR. In some embodiments, the nucleic acid comprises a 3' human alpha- globulin- 1 (HBA1) UTR. In some embodiments, the nucleic acid comprises a wild-type ATF3 3’ UTR. In some embodiments, the nucleic acid comprises a 5' human alpha-globulin- 1 (HBA1) untranslated region (UTR) and / or a 3' HBA1 UTR. In some embodiments, the nucleic acid

[0167] 28

[0168] #14392914vl Attorney Docket No. H0498.70834WO00 comprises a 5' untranslated region (UTR) and / or a 3' UTR, optionally wherein the 5' UTR and / or the 3' UTR is a human alpha- globulin- 1 (HBA1) UTR. In some embodiments, the nucleic acid comprises a wild-type ATF3 untranslated region (UTR). In some embodiments, the nucleic acid comprises any suitable UTR known in the art.

[0169] PolyA Tail

[0170] In some embodiments, the nucleic acid comprises a 3’ polyA tail. In some embodiments, the polyA tail enhances the stability (e.g., by protecting the nucleic acid from enzymatic degradation) and / or translation efficiency of the nucleic acid. In some embodiments, the polyA tail comprises 10-300 (e.g., 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300) adenosines. In some embodiments, the polyA tail may be about 50, about 100, about 150, about 200, about 250, or about 300 nucleotides. In some embodiments, the polyA tail has a length of approximately 100 nucleotides. In some embodiments, the polyA tail has a length of 107 nucleotides.

[0171] Modifications

[0172] A nucleic acid contemplated by the present disclosure may contain nucleotides that are either chemically unmodified, chemically modified, or a combination of both. Chemically unmodified nucleotides include the standard ribonucleotides (e.g., adenosine, guanosine, cytidine, and uridine.) In some embodiments, the nucleic acid includes modified nucleosides and / or nucleotides. Modified nucleotides can be synthesized using various methods, including chemical, enzymatic, or recombinant techniques.

[0173] In some embodiments, a modified nucleobase in a nucleic acid comprises Nl- methylpseudouridine (mly), N1 -methylpseudouridine triphosphate, Nl-ethyl-pseudouridine (c I \| / ), 5-methoxy-uridine (mo5U), 5-methyl-uridine (m5U), 5-methyl-cytidine (m5C), and / or pseudouridine (y). In some embodiments, a modified nucleobase in a nucleic acid comprise 5- methoxymethyl uridine, 5-methylthio uridine, 1-methoxymethyl pseudouridine, 5-methyl cytidine, and / or 5-methoxy cytidine.

[0174] In some embodiments, the nucleic acid comprises at least one uridine nucleoside comprising N1 -methylpseudouridine triphosphate. In some embodiments, an mRNA comprises at least one uridine nucleoside comprising N1 -methylpseudouridine triphosphate. In some embodiments, an ORF of the mRNA comprises at least one uridine nucleoside comprising Nl- methy Ip seudouridine tripho sphate .

[0175] 29

[0176] #14392914vl Attorney Docket No. H0498.70834WO00

[0177] Nucleic Acid Delivery

[0178] Methods of delivering a nucleic acid encompass a variety of techniques disclosed herein and known in the art.

[0179] One such approach involves the use of plasmid DNA, which comprises introducing a plasmid containing the nucleic acid into the cell via transfection. This method often employs transfection reagents such as cationic lipids, polymer-based reagents, calcium phosphate-based reagent, or dendrimer-based reagent, which facilitate the entry of the plasmid into the cell. In some embodiments, the nucleic acid is delivered to a cell, population of cells, tissue and / or subject using plasmid DNA. In some embodiments, the methods contemplated by the present disclosure involve a transfection reagent. In some embodiments, the transfection reagent is a cationic lipid, a polymer-based reagent, a calcium phosphate-based reagent, or a dendrimerbased reagent.

[0180] Another approach utilizes viral vectors, such as lentiviruses, adenoviruses, or adeno- associated viruses, to deliver a nucleic acid. In some embodiments, the nucleic acid is delivered to a cell, population of cells, tissue and / or subject using a viral vector. In some embodiments, the viral vector is an adeno-associated viral, adenoviral, or lentiviral vector.

[0181] Electroporation is another technique used to introduce nucleic acids into cells by applying an electrical field that temporarily increases the permeability of the cell membrane. This creates transient pores through which genetic material can enter the cell. It is a versatile method applicable to a wide range of cell types, including difficult-to-transfect cells. Electroporation is efficient and can deliver large molecules, such as plasmids and RNA, directly into the cytoplasm. In some embodiments, the nucleic acid is delivered to a cell, population of cells, tissue and / or subject using electroporation.

[0182] Additionally, RNA-based methods, such as mRNA transfection, can be used to deliver nucleic acids. In this approach, synthetic mRNA is introduced into the cell, bypassing the need for transcription and leading directly to protein synthesis. Lipid nanoparticles (LNPs) are often employed in this method to complex with the mRNA, protecting it from degradation and facilitating its delivery into the cell. LNPs enhance cellular uptake by fusing with the cell membrane, thereby efficiently releasing the mRNA into the cytoplasm where it can be translated into a target protein. In some embodiments, the nucleic acid is delivered to a cell, population of cells, tissue and / or subject using an LNP. In some embodiments, an LNP comprises ionizable lipids and / or polyethylene glycol (PEG)-lipid conjugates.

[0183] 30

[0184] #14392914vl Attorney Docket No. H0498.70834WO00

[0185] EXAMPLES

[0186] Example 1. Discovery of a nucleic acid-based treatment for human skin rejuvenation Decomposition of aging human skin

[0187] To obtain detailed insights into the molecular changes associated with skin aging at the single-cell level for treatment development, we performed a comprehensive and in-depth analysis of healthy human adult skin samples across various ages utilizing single-cell RNA sequencing (scRNA-seq) (FIG. 1A). We collected 10 mm skin samples from young (Y), middle- aged (M), and old (O) donors. Since the skin aging processes include intrinsic (chronological) and extrinsic (photo) aging, paired samples were obtained from the arm (subject to chronological and photoaging) and the back (subject to chronological aging). The samples were separated into epidermis and dermis before dissociation to encompass a wide range of cell types, and the cells were characterized using the lOx Genomics platform. We excluded cells with fewer than 200 genes, more than 6000 genes, over 10% mitochondrial gene expression, and doublets, resulting in a total of 61,106 high-quality skin cells. To mitigate batch biases, we integrated data using Seurat and observed uniform sample distribution in the low-dimensional space (FIG. 6A). Subsequently, we identified 31 cell subtypes and 11 cell types through graph-based Leiden clustering based on specific marker gene expression. These included all major expected skin cell types: basal cells (BC), spinous cells (SC), granular cells (GC), melanocytes (ME), Langerhans cells (LH), hair follicle cells (HF), endothelial cells (EC), fibroblasts (FB), pericytes (PC), immune cells (IC), and sebocytes (FIGs. IB- ID). Further, to ensure our analysis is not biased by the stress response associated with tissue dissociation, we removed the known stress response genes, and our 31 cell states remained discernible, suggesting a robust cell characterization Analysis of the top 30 marker genes for each cell type revealed unique transcriptional features and enriched pathways relevant to their distinct functions (FIG. IE). For example, Gene Ontology (GO) terms including epidermal / epithelial development and ECM organization were enriched for basal cells and fibroblasts, respectively (FIG. IE). Additionally, genes associated with aging and skin disorders (e.g., skin carcinoma, melanoma) were mainly identified in melanocytes. This collective single-cell dataset of human skin provides a foundational resource for studying skin aging processes.

[0188] To investigate the molecular mechanisms underlying commonalities and differences between combined aging (chronological and photoaging) and chronological aging, we initially characterized skin cell composition changes in both types of aging (FIG. IF and FIG. 6C). Although both chronological and combined aging exhibit cellular changes, the magnitude and 31

[0189] #14392914vl Attorney Docket No. H0498.70834WO00 trends of these changes differ between the two aging types. For example, a change shared between both types of aging is the increase of immune cells (IC-1 and IC-2). This is consistent with the previous observation that aged skin showed chronic, low-grade skin inflammation. In particular, the increase of immune cell proportion was larger in the combined aging, indicating that photoaging could cause stronger skin inflammation (FIG. IF). We also observed a reduction in the populations of basal stem cells BC-2, BC-3, BC-4, and BC-6 (characterized by the expression of the KRT15 marker). Given that basal stem cells play critical roles in cell adhesion, extracellular matrix organization, cell proliferation, and cellular development and differentiation (FIG. 6D), the depletion of these cells in aged skin suggests a diminished capacity for cell renewal during the aging process. By conducting the pathway analysis on the basal stem cells, we found that the combined aging was enriched for pathways involved in cellular migration and skin development, in contrast, the chronological aging was enriched for pathways involving morphogenesis and cellular regulation (FIG. 6E). Together, these findings suggest that different genes are involved in the two aging processes and highlight the necessity for targeted therapeutic strategies that address the specific genetic and cellular alterations associated with both types of aging.

[0190] Cell state alterations during photoaging and combined aging

[0191] To investigate the genetic basis of combined aging and chronological aging, we calculated the differentially expressed genes (DEGs, t-test, Bonferroni adjusted p-value) for each aging type (FIGs. 2A-2B). Most cell types (7 out of 9) exhibited a greater number of DEGs in combined aging compared to chronological aging. This observation aligns with the expectation that combined aging encompasses both photoaging and chronological aging. Notably, in HF and SB, the number of DEGs is comparable between combined aging and photoaging, suggesting a similarity in the aging processes of these cell types, indicating that these genes are activated by both chronological aging and photoaging.

[0192] After identifying variations in the number of DEGs, we examined the overlap between these genes. Specifically, we analyzed cell-type- specific DEGs and shared DEGs across five predominant cell types - FB, EC, HF, KC, and ME. Interestingly, we observed a significant number of cell-type-specific DEGs in combined aging, whereas only a few cell-type- specific DEGs were present in chronological aging. However, the number of shared DEGs was similar for both combined aging and chronological aging (FIGs. 2C-2D). This suggests that the differences between combined and chronological aging, especially in photoaging, are attributable 32

[0193] #14392914vl Attorney Docket No. H0498.70834WO00 to the shared genes. We reason that photoaging induces multiple responses in the majority of cell types, as evidenced by the overlapping DEGs across various cell types.

[0194] Furthermore, we carefully investigated the shared DEGs across 5 major cell types in combined aging and chronological aging (FIGs. 7A-7B). The DEGs exhibited more notable changes in combined aging compared to chronological aging. Specifically, in combined aging, PRMT9 expression exhibited a linear decrease in endothelial, fibroblast, and melanocyte populations (FIG. 7B), this aligns with literature previous studies that indicate a loss of PRMT9 is associated with cellular senescence and aging in transgenic mice. Intriguingly, MEF2A expression displayed consistent U-shaped trends with aging across all cell types, which may be related to MEF2A's role in cellular proliferative capacity. MEF2A expression increases during growth periods, followed by a gradual reduction during cellular senescence and aging. However, due to long-term cumulative DNA damage, aberrant cellular processes can lead to increased cellular proliferation. Conversely, in chronological aging, DCD and MUCF1 genes demonstrated relatively consistent trends in gene expression over time (FIG. 7B). These findings underscore the complexity of gene expression dynamics during aging and set the stage for further exploration of their mechanistic underpinnings

[0195] To further elucidate the function of cell-type shared DEGs, we utilized pathway enrichment analysis (FIG. 2E). Specifically, the cell-type shared genes involved in photoaging excluded genes associated with chronological aging from the pool of genes identified in combined aging. Notably, the pathways linked to combined aging closely resemble those of chronological aging but differ significantly from those associated with photoaging (FIG. 2F). Subsequently, we examined the enrichment of DEGs related to aging, DNA repair, hallmark EMT, and NFKB (inflammatory) pathways in combined aging relative to chronological aging across various cell types (FIG. 2F and FIGs. 7D-G). Importantly, all cell states demonstrated enrichment of DNA repair-related gene signatures in the combined aging (FIG. 2F and FIG. 7D), particularly within the middle-aged donor, underscoring the contribution of photoaging to DNA damage.

[0196] Additionally, we evaluated the relative transcriptional changes of each cellular state across age groups using aggregate measures of DEG fold change (Methods). The “aggregated score” for each cell state was calculated by summing the log2 fold changes of all DEGs within each cell state. Our analysis revealed that the cell types exhibiting the most substantial changes were basal cells (FIG. 7H). Basal cells are tightly correlated with skin development (FIG. 6E) and vary in function between chronological and combined aging, indicating their critical role in maintaining skin homeostasis and contributing to the overall process of skin aging. 33

[0197] #14392914vl Attorney Docket No. H0498.70834WO00

[0198] Human epidermal keratinocytes play a key role during skin aging

[0199] Using the analysis of alterations in cell proportions and examination of DEGs, basal keratinocyte cells appear to exhibit a significant association in the aging process. Here, we investigated the heterogeneous characteristics of human epidermal keratinocytes (FIG. 3A), with a focus on the basal stem cells. Our dataset provided a thorough categorization of the keratinocytes used in this study, this unveiled discrete subpopulations within the stratified epidermis. These subpopulations encompassed seven epidermal basal cells (BC1-7), three spinous subpopulations (SC 1-3), and a terminally differentiated cell population (GC) (FIG. 3B). Three SC subtypes demonstrate elevated expression levels of genes associated with barrier function and cell-cell adhesion, including KEK7 and DSC1. Among the seven subgroups of basal cells, our study successfully captured substantial basal stem cells — BC2, BC3, BC4, and BC6, confirmed through the marker gene KRT15 (FIG. 1C and FIG. 3D). In basal stem cells, sternness marker COL17A1 and inflammatory response genes (S100A9 and SI 00 A8) were highly expressed. Besides, POSTN (Periostin), KRT5, and KRT14, which are related to wound-healing processes were enriched. These basal stem cells offer a promising avenue for investigating skin rejuvenation via stem cell differentiation.

[0200] Subsequently, pseudotime analysis was performed to elucidate the response of keratinocyte subtypes during skin development (FIG. 3E). We identified four distinct cell lineages within epidermal keratinocytes. One lineage, consistent with the epidermal development, progressed from node SO to SI to S2, a differentiation trajectory from BC to SC to GC. The remaining three lineages illustrate differentiation within basal cells, specifically from quiescent basal cells to proliferating basal cells. The S0-S1-S3 lineage, which encompasses the differentiation of basal stem cells (BC4 and BC6) to BC7 cells, exhibited significant differences between combined aging and chronological aging (FIG. 3F). This lineage demonstrated a gradual deceleration of cell development with age, indicative of reduced basal stem cell renewal capacity during middle and old age. Moreover, middle-aged samples in the combined aging group showed a more pronounced slowdown in basal stem cell renewal, highlighting the significant impact of photodamage on basal stem cells during middle age (FIG. 3F). Additionally, we explored the S0-S1-S3 trajectory concerning genes involved in the GenAge, the benchmark database of genes related to aging (FIG. 8A).

[0201] Together, our analysis revealed a consistent pattern wherein these genes measured exhibited decreased expression associated with basal stem cell renewal in aged samples, underscoring their role in regulating basal cell functions across aging contexts. We also 34

[0202] #14392914vl Attorney Docket No. H0498.70834WO00 investigated the subtypes of HF, FB, and EC, which revealed that gene expression patterns underwent changes during the aging process. (FIG. 8B-8D), indicating that alterations in these genes may contribute to the reduced renewal capacity observed in aging and that basal stem cells and basal stem cells may be an effective cell type for therapeutic inventions.

[0203] Development of an mRNA treatment targeting keratinocytes for skin aging reversal

[0204] To identify a key regulator of skin aging in keratinocytes, particularly basal stem cells, we carefully examined DEGs in keratinocytes (FIG. 4A). First, genes were categorized into distinct modules based on their expression patterns across different ages in both combined and chronological aging (Methods). From the eight identified modules, we identified two gene modules: one characterized by up-regulated genes and the other by down-regulated genes during aging (FIG. 4B). By mapping these two gene modules onto the skin atlas described in FIG. 1, we observed an association between the up-regulated gene module and granular cells (FIG. 4C). Our results aligned with the roles of granular cells in epidermal turnover and the increased propensity for keratosis with age. Furthermore, the down-regulated gene module is associated with basal stem cells, suggesting that this module is implicated in stem cell depletion (FIG. 4C). To further elucidate the genes in the two modules, we conducted a gene-gene interaction network analysis (FIG. 4C). In the up-regulated gene network, key genes include JUP, PERP, DSC2, and DSC3. These genes are associated with skin integrity and are crucial for cell-cell adhesion. Dysfunction in these genes has been shown to lead to skin fragility. In the down-regulated gene network, the key genes are TXNRD1, KLF6, and ATF3. TXNRD1 is vital for selenium metabolism and UV protection. KLF6 is related to cell proliferation, which has been reported as a skin aging gene. Of note, ATF3, a transcription factor involved in skin development and stress response, was the hub gene of the downregulated gene module, indicating that ATF3 is a key age-related transcription factor in skin aging. ATF3 was found to be associated with protecting against metabolic stress, inhibiting cancer, and reducing inflammation during aging. Thus, we determined that ATF3 is a gene for developing treatments for skin aging.

[0205] Next, we conducted a siRNA knockdown study in primary human epidermal keratinocytes (HEKs) to validate the role of ATF3 in epidermal aging (FIG. 4D). Reduction of ATF3 expression resulted in decreased proliferative capability and an increased senescence- associated secretory phenotype (SASP), including 1.76-fold elevated expression of the pro- inflammatory cytokine CDKN2A (P-value < 0.0001) (FIGs. 4E-4F). The knockdown also significantly increased senescence in keratinocytes (1.21-fold increase, P-value < 0.0001), as 35

[0206] #14392914vl Attorney Docket No. H0498.70834WO00 measured by senescence-associated P-galactosidase (SA-P-gal) staining (FIG. 4G). Collectively, these data strongly confirmed that ATF3 is a key age-related gene in skin aging.

[0207] Next, we developed a novel mRNA-based therapeutic approach for reversing skin aging through upregulation of ATF3 expression using an in vitro mRNA delivery method (FIG. 4H). We engineered mRNAs encoding ATF3 fused with P2A and RFP (ATF3-P2A-RFP), as well as constructs without P2A-RFP. The mRNA constructs were designed to include the endogenous 5' and 3' untranslated regions (UTRs) of the HBA1 gene (human alpha-globin- 1) to ensure efficient translation and stability (data not shown). Following the 5' UTR, a custom human Kozak sequence was inserted, followed by the sequential integration of the ATF3 sequence, a human P2A sequence, a red fluorescent protein (RFP) sequence, a HBA1 3' UTR, and an approximately 107 -nucleotide poly-A tail (FIG. 4H). These mRNAs were transfected into HEKs to validate treatment efficacy. We assessed parameters such as the SASP, cell proliferation, and cellular senescence. After 96 hours of treatment in HEKs, a significant reduction in the SASP was observed. Specifically, IL6 levels decreased by 10% to 29% (t-test, P < 0.0001), and IL8 levels decreased by 12% to 34% (t-test, P < 0.01). Notably, there was a marked increase in cell proliferation, ranging from 20% to 25% (t-test, P < 0.01), and a significant decrease in cellular senescence, with reductions between 22% and 25% (t-test, P < 0.0001). These results indicate the potential efficacy of the ATF3 mRNA in modulating key cellular processes associated with aging and senescence, highlighting their therapeutic promise for mRNA-mediated anti-aging interventions (FIGs. 4I-4K).

[0208] The impact of mRNA therapy on fibroblasts via keratinocytes-fibroblast communication

[0209] Given that fibroblast is another key cell type associated with skin aging, we subsequently investigated the effects of delivered ATF3 mRNA in fibroblasts. Our analysis, along with several other studies, elucidated intercellular connections between keratinocytes and fibroblasts. We identified 62 significant ligand-receptor interaction pairs between keratinocytes and fibroblasts (FIG. 5A). This strong crosstalk was observed in both combined and chronological aging (FIG. 10A). Remarkably, the strong interactions of ligand-receptor pairs are COL17A1 / COL7A1 with the aipi-complex, al ipi-complex, and a2pi-complex (FIG. 5B, 10B). These complexes correspond to integrins, pivotal for collagen synthesis, and COL17A1 has been implicated with skin aging (FIG. 5C and FIG. 10C). Furthermore, the interaction between ATF3 and the secreted protein CXCL2 serves a pivotal role in mediating the intricate crosstalk between keratinocytes and fibroblasts. This dynamic interplay between ATF3 and CXCL2 orchestrates a complex 36

[0210] #14392914vl Attorney Docket No. H0498.70834WO00 signaling cascade that regulates various aspects of fibroblast behavior, including proliferation and extracellular matrix remodeling. These findings provide compelling evidence for intercellular communication mediated by signals including ATF3, originating from keratinocytes, which influence collagen production in fibroblasts.

[0211] Next, we validated the keratinocyte-mediated crosstalk influenced fibroblast aging by carefully designed experiments (FIG. 5D and FIG. 10D). Keratinocyte-conditioned medium (KCM) collected from primary human keratinocytes of varying ages was applied to cultured fibroblasts. We compared KCM from old keratinocytes and young keratinocytes. We observed a reduction in cellular aging of approximately 20% with young KCM. Young KCM exerted a pronounced rejuvenating effect, markedly upregulating the expression of collagen genes, including COL1A1, COL1A2, and COL3A1, by 2- to 4-fold, and enhancing collagen protein production by 3.5-fold (FIG. 5D). These findings demonstrate that young keratinocytes have the capability to promote a youthful state in fibroblasts by enhancing collagen and promoting a regenerated extracellular matrix environment.

[0212] Using the evidence from above, we investigated whether ATF3 mRNA-treated keratinocytes would generate a rejuvenating effect similar to that of young keratinocytes on fibroblasts (FIG. 5E). Encouragingly, ATF3 treatment of KCM significantly reduced fibroblast senescence. Notably, ATF3 treatment induced a substantial upregulation of collagen and elastin gene expression, resulting in over a 10-fold increase in COL1A1 expression, a 5-fold increase in COL1A2, a 4-fold increase in COL3A1, and a 6-fold increase in elastin expression (FIG. 5E). The aging-associated gene ATF3 regulates keratinocyte secretion production, thereby modulating fibroblast senescence and collagen synthesis. These findings strongly support the therapeutic promise of delivering ATF3 mRNA for skin rejuvenation. ATF3 emerges as a compelling candidate for enhancing skin health and vitality by augmenting collagen synthesis in fibroblasts and mitigating senescence in both fibroblasts and keratinocytes (FIG. 5F).

[0213] Example 2. ATF3 mRNA treatment efficacy validated in Ex Vivo human skin and In Vivo mouse models

[0214] ATF3 mRNA rejuvenates aged human skin and activates basal keratinocytes and hair follicle stem cells

[0215] Human skin explants (PMID: 25363465) derived from elective plastic surgery represent a physiologically relevant experimental model that preserves native tissue architecture, including resident cell types (e.g., keratinocytes, melanocytes, Langerhans cells, and fibroblasts) and 37

[0216] #14392914vl Attorney Docket No. H0498.70834WO00 extracellular matrix components (e.g., collagen, elastin, and glycosaminoglycans). We established an ex vivo culture system using full-thickness skin obtained from a 62-year-old female donor. Microneedle patches were applied to deliver either a control mRNA (encoding red fluorescent protein, RFP), platelet-rich plasma (PRP, positive control) or therapeutic mRNA (encoding ATF3). For efficient delivery, mRNA was encapsulated in lipid-based nanoparticles (NPs) and administered using a hydrogel-based microneedle array patch (MAP). This platform facilitates effective penetration of the skin barrier and targeted delivery to cells. Our proprietary MAP system has been engineered to deliver diverse therapeutic modalities, including small molecules, proteins, and nucleic acid nanoparticles, as well as to enable sampling of interstitial skin fluid for biomarker monitoring (PMID: 36593949, PMID: 38638030).

[0217] After a 4-day incubation period, collagen production was quantified using Masson's trichrome stain. As shown in FIG. 1 IB, ATF3 mRNA treatment led to a significant increase (1.25-fold change, p<0.001) in collagen levels compared to control-treated skin. Besides, histological and molecular analyses revealed that ATF3 restored collagen and elastin networks in aged human skin, eliciting broader and more robust rejuvenation compared to PRP. Verhoeff- Van Gieson staining showed increased elastin fiber density following ATF3 treatment, whereas PRP induced only modest changes (FIG. 11C). Immunofluorescence analysis confirmed elevated type III collagen (COL3A1) expression in the dermis after ATF3 delivery, surpassing the effects observed with PRP (FIG. 1 ID). Together, these data indicate that ATF3 promotes extracellular matrix renewal and more effectively restores collagen and elastin architecture in aged skin compared with a standard-of-care treatment.

[0218] We next investigated whether ATF3 also influences epidermal and follicular stem cell compartments (FIG. 12A). KRT15 staining revealed a marked expansion of the basal keratinocyte layer in ATF3-treated skin compared with RFP and PRP (FIG. 12B). The stem cell niches increased significantly by 1.24-fold. Importantly, ATF3 robustly expanded KRT15+hair follicle bulge stem cells, a key population that maintains long-term follicular homeostasis and initiates new hair cycles. Notably, while PRP induced only modest changes relative to RFP, ATF3 elicited a robust expansion of epidermal and follicular stem cell populations. Co-staining with p63 further confirmed enhanced renewal of epidermal and follicular progenitors (FIG. 12C). Our findings highlight ATF3 as a more potent activator of both epidermal regeneration and hair follicle stem cell renewal. By simultaneously promoting dermal remodeling and expanding HFSCs, ATF3 provides a dual mechanism of skin rejuvenation and hair regrowth that extends beyond the capacity of current treatments.

[0219] 38

[0220] #14392914vl Attorney Docket No. H0498.70834WO00

[0221] ATF3 mRNA accelerates wound healing and reduces scar formation in a mouse

[0222] As aged skin shows slow wound healing compared to young skin (PMID: 3571865), we further investigated the therapeutic potential of ATF3 mRNA in promoting wound healing and reducing scar formation using a full-thickness excisional wound model (PMID: 32314222) in mice. Two circular full-thickness wounds were generated on the dorsal skin of each mouse, followed by subdermal injection of lipid nanoparticle-encapsulated in vitro transcribed ATF3 mRNA proximal to the wound site. Two experimental groups (n = 2 mice per group) were established: ATF3 mRNA-treated and control mRNA-treated (encoding red fluorescent protein, RFP). Postoperative analgesia was maintained with extended-release buprenorphine following anesthesia recovery. Wound healing was assessed by monitoring closure kinetics over time, and tissue samples were collected for histological and molecular analyses. Hematoxylin and eosin (H&E) staining, Masson’s trichrome staining, and quantitative PCR for collagen gene expression were employed to evaluate structural integrity and extracellular matrix remodeling in the regenerated tissue. As shown in FIGs. 13A-13C, ATF3 mRNA treatment resulted in reduced scar formation compared to both control groups.

[0223] Aging is a dynamic biological reality and a complex phenomenon. Like other organs, human skin experiences aging both chronologically and from environmental factors, notably photoaging. In this study, we sought to understand the intricate mechanisms underlying chronological skin aging and photoaging and develop an mRNA treatment for aging rejuvenation. We found a progressive decline in function attributed to the accumulation of molecular damage over time. Central to our study was the development of a pioneering singlecell atlas designed to uncover genes underlying skin rejuvenation. Using this innovative atlas, we explored similarities and distinctions between two forms of aging. Furthermore, we identified ATF3 as a pivotal gene involved in the skin aging process. Leveraging our findings, we have formulated an mRNA therapy that amplifies the expression of ATF3 for skin rejuvenation, potentially heralding a new era of genetics-based skin rejuvenation therapies that could benefit millions affected by skin disorders.

[0224] This study provides further evidence that photoaging and normal aging evoke similar processes driven by immune system remodeling. We completed a deep dive into the genetic landscape of aging, leveraging cutting-edge single-cell technologies to scrutinize the nuanced changes occurring within each skin cell. There are likely different molecular mechanisms inducing inflammation and immunosuppression in the accelerated photoaging and chronological aging processes. The contrast between these two aging processes alludes to the cumulative 39

[0225] #14392914vl Attorney Docket No. H0498.70834WO00 impact of chronological aging and photoaging in combined aging. When separated into photoaging and chronological aging, our results reveal photoaging induces changes across all cell types, while chronological aging elicits changes to specific cell populations. This observation sheds light on the intricate interplay between various aging mechanisms and their differential effects on cell types, potentially providing valuable insights into the underlying molecular pathways driving aging-related changes. Furthermore, there are distinct mechanisms underlying chronological aging and photoaging, indicating the overall complexity of the aging process. Unraveling these intricate molecular pathways, we found both chronological aging and photoaging are linked to inflammatory responses, cytokine stimulation, and cellular proliferation. Combined aging shows a stronger association with skeletal system development and the organization of extracellular structures, whereas photoaging is predominantly associated with cellular dysfunction, such as monocyte chemotaxis. Further exploration of these findings could deepen our understanding of the complex aging process and its implications for cellular function and health.

[0226] Importantly, the results presented herein provide evidence of basal stem cells in the aging process, shedding light on the genes orchestrating their fate. By unraveling the intricacies of these molecular pathways, an understanding of the diverse facets of aging is gained, opening multiple avenues for novel anti-aging interventions. The down-regulated genes were predominantly mapped to basal stem cells. This finding corroborates previous research indicating that aged skin is characterized by thinning of the epidermis, decreased epidermal proliferation, and an increase in basal cell apoptosis. The ATF3 gene, a hub down-regulated gene, is associated with basal stem cell depletion and emerges as an essential mediator of skin tissue homeostasis and repair, highlighting its importance in maintaining skin integrity and resilience.

[0227] The identification of the importance of ATF3 and associated nucleic acid therapies for skin rejuvenation introduce novel and innovative strategies to combat the challenges associated with skin aging and offer implications for mitigating age-related changes.

[0228] Furthermore, our study elucidates a significant mechanism of intercellular communication between keratinocytes (particularly the basal stem cell subset) and fibroblasts. ATF3 expression in basal stem cells stimulates collagen production in fibroblasts through ATF3- regulated factors, emphasizing the complexity of skin aging. It also indicates that key regulatory genes in one cell type, such as ATF3, influence both epidermal and dermal layers. Understanding intercellular communication broadens the potential for developing therapeutic interventions for

[0229] 40

[0230] #14392914vl Attorney Docket No. H0498.70834WO00 aging in various organs, as well as for addressing other skin disorders, including inflammatory conditions and alopecia.

[0231] Finally, our research highlights the promise of a first-of-its-kind mRNA therapy for skin rejuvenation. mRNA technology has been successfully employed in vaccines, demonstrating its safety and efficacy in human populations. This modality is cost-effective and presents a promising alternative for anti-aging therapies and other medical applications such as wound healing. The use of mRNA is particularly significant due to its ability to induce precise and targeted gene expression, which can directly address molecular mechanisms. Furthermore, mRNA does not induce permanent genomic modifications, which is the case with genome editing technologies. Instead, this treatment is controlled easily with dosing regiments, and delivered mRNA is temporarily expressed in treated cells. mRNA therapies also offer rapid development timelines, allowing for swift adaptation in response to emerging skin-related conditions. Given the growing interest in regenerative medicine, the potential of mRNA to enhance skin rejuvenation and improve dermal integrity is a novel and feasible treatment approach. This innovative technology could revolutionize strategies for combating aging and other disorders, paving the way for more effective therapies, and ushering in a new era of targeted interventions with far-reaching implications for human health and longevity.

[0232] Methods related to Example 1 and Example 2

[0233] Ethics statement and tissue acquisition

[0234] This study on skin aging involved the participation of individuals who graciously donated samples of their healthy adult skin. Before their involvement, these donors provided written informed consent, ensuring ethical compliance with the No.3 Zhongshan Hospital Research Ethics Committee (REC reference: 08 / H0906 / 95+5). To ensure consistency and relevance, strict criteria were applied for the selection of donors. None of the individuals had diabetes, were obese, or suffered from other skin diseases. Their body mass index (BMI) fell within the range of 23 to 28, establishing a healthy baseline for the research.

[0235] The samples collected for analysis were of dimensions 10 mm x 10 mm, and meticulous efforts were made to procure them from identical positions on the arm and back of each donor. This standardized approach allows for accurate comparisons between the three age groups: young individuals (age 23), middle-aged individuals (age 53), and elderly individuals (age 85).

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[0238] Generation of single-cell suspension and Single-cell RNA-seq

[0239] To prepare a single-cell suspension from adult skin, healthy skin samples were first cut into thin strips while immersed in phosphate-buffered saline (PBS). The top 200 pm layer was isolated using a dermatome with a Pilling Weeprep blade and a 0.008-gauge Goulian guard. Grid slits were introduced into the skin sheets for enzymatic access, followed by a 1-hour treatment with 2U / ml dispase II in RPMI at 37°C. Subsequently, the epidermis was peeled from the dermis, and both fragments were separately digested in a petri dish at 37°C 5% CO2 in RF-10 media with 1.6 mg / ml type IV collagenase (Worthington, CLS-4) for 12 hours. RF-10 media consists of Roswell Park Memorial Institute media (RPMI) (Sigma, R0883) supplemented with 10% (v / v) fetal calf serum (FCS, Life technologies, 10270106), lOOU / ml Penicillin (Sigma, P0781), 100 pg / ml Streptomycin (Sigma, P0781) and 1% (v / v) L-Glutamine (Sigma, G7513). The work was conducted within class II biological safety cabinets using autoclave- sterilized equipment. The media was collected using a serological pipette and filtered through a sterile 100 pm cell strainer (BD Falcon, 352360). After washing the petri dish and strainer with RF-10 media to gather any remaining cells, centrifugation was employed to pellet the cells at 500 x g for 5 minutes. The supernatant was discarded and the pellet was gently resuspended in 1 ml RF- 10 media through pipetting up and down. Cell counting was performed using a hemocytometer after staining 10 pl of the sample with an equal amount of 0.4% trypan blue (Sigma, T8154) to identify dead cells.

[0240] Single cells were captured using droplet emulsions, and scRNA-seq libraries were generated following the manufacturer's instructions using the Chromium lOx Single-Cell Instrument (lOx Genomics) and lOx Genomics Chromium Single Cell 3’ GEM Library and Gel Bead Kit v2. Each channel was loaded with cells, aiming for 10,000 cells per sample, with cell concentration measured by Moxi GO II (Orflo Technologies). The cDNA was amplified for 12 cycles in the Bio-Rad C1000 Touch Thermal cycler with 96 Deep-Well Reaction Module. Amplified cDNAs and final libraries were assessed using a Fragment Analyzer (AATI) with a High Sensitivity NGS Analysis Kit (Advanced Analytical) to determine the average fragment length of the lOx cDNA libraries. Quantification of the libraries was done via qPCR using the Kapa Library Quantification kit. Subsequently, the libraries were diluted to a final concentration of 2 nM and pooled together for sequencing. All libraries were sequenced using the NovaSeq 6000 Sequencing System (Illumina) with an average coverage of 50,000 raw reads per cell, sequenced in a 28 x 10 x 10 x 90 bp configuration.

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[0243] Processing raw data from scRNA-seq

[0244] Raw data from single-cell RNA sequencing (scRNA-seq) were processed using the default parameters of the Cell Ranger software suite (lOx Genomics). The quality assessment of the sample- specific FASTQ files was performed based on the Cell Ranger counts, which were aligned to the human reference genome (GRCh38) using the STAR aligner to create a gene expression matrix. Each transcript's expression level was determined by the count of unique molecular identifiers (UMIs) assigned to that transcript. The gene expression matrices were utilized for downstream analyses. scRNA-seq data analysis and cell-type identification

[0245] Filtered feature barcode matrices from Cell Ranger ARC were analyzed using the Seurat R package. Following the initial Cell Ranger metric assessment, cells with fewer than 200 genes or more than 6,000 genes and more than 10% of mitochondrial genes were excluded from downstream analyses. After quality control, a total of 61,106 cells remained and were utilized for subsequent bioinformatic analyses. Sequencing reads for each gene were normalized and scaled using the SCTransform function in Seurat. We performed data integration using Seurat to mitigate potential batch effects across samples and experiments. Subsequently, we applied dimensionality reduction and clustering techniques to analyze the integrated data. Cell clustering was performed using the 'FindClusters' function with a resolution of 0.8, utilizing the first 30 PCs to define cell identities. Dimensionality reduction was performed using the 'RunUMAP' function, and the results were visualized with Uniform Manifold Approximation and Projection (UMAP). Marker genes for each cluster were determined using the Wilcoxon rank- sum test with the 'FindAllMarkers' function. Only genes with log fold change > 0.25 and adjusted p value < 0.05 were considered as marker genes.

[0246] Identification of aging-associated differentially expressed genes (DEGs)

[0247] To identify aging-associated DEGs between the old and young groups (O / Y), middle- aged and young groups (M / Y), and old and middle-aged groups (O / M) for each cell type, we utilized the "FindMarkers" function in Seurat. The log fold change and adjusted p-value for each DEG were calculated using the non-parametric two-sided Wilcoxon rank-sum test. Only DEGs with an absolute average log fold change greater than 0.25 and an adjusted p value less than 0.05 were considered significant.

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[0250] Gene Ontology ( GO) Analysis and KEGG pathway analysis

[0251] GO and KEGG pathway analysis of DEGs was performed by EnrichR (version 3.2) and visualized with the ggplot2 R package. Representative terms selected from the top 20 ranked GO terms or KEGG pathways (p < 0.01) were displayed.

[0252] Pseudotime analysis

[0253] Epidermal cell developmental trajectories were computed using the STREAM algorithm. Gene filtering was performed using the filter_genes function with the parameter min_num_cell set to 5. Variable gene selection was then conducted with the select_variable_genes function, using a loess_frac parameter of 0.01. To initialize the tree structure, the parameter n_cluster was set to 10. The elastic principal graph was obtained with the following parameters: epg_alpha set to 0.015, epg_mu set to 0.2, and epg_lambda set to 0.02.

[0254] Gene set scoring

[0255] Individual pathways were collected from the GenAge and KEGG database and scored in single cells Seurat AddModuleScore function and visualized in ggpubr R package (version 0.5.0). Individual pathways were tested for significance using a Wilcoxon rank-sum test and Bonferroni corrected through the ‘rstatix’ R package (version 0.7.2).

[0256] Perturbation plot and pathway enrichment analysis

[0257] Pathway activity was determined using the ‘clusterProfiler’ R package (version 3.0.4) using the enrichGO function with default parameters. Enrichment plots were generated using the ‘enrichplot’ (version 3.1.8)

[0034] . Differentially expressed genes were determined by the Seurat function FindMarkers with default parameters. The overall ‘perturbation score’ for each cell state was calculated by summing all log2 fold changes for DEGs in each cell state.

[0258] Gene modules

[0259] Gene modules were clustered by performing a hierarchical cluster analysis using a set of dissimilarities for the gene expressions. Initially, each object is assigned to its cluster and then the algorithm proceeds iteratively, at each stage joining the two most similar clusters, continuing until there is just a single cluster. At each stage, distances between clusters are recomputed by the Lance— Williams dissimilarity update formula according to the particular clustering method

[0260] 44

[0261] #14392914vl Attorney Docket No. H0498.70834WO00 being used. Gene-gene interactions within a module were identified by mapping the genes to the STRING network.

[0262] Cell-cell communication analysis

[0263] To assess cell-cell communication molecules between different cell types, we used CellPhoneDB software (version 1.1.0) to infer the intercellular communication network from single-cell transcriptome data. Only receptors and ligands expressed in more than 10% of the cells in the specific cell types were considered in the analysis. First, by randomly permuting the cluster labels of all cells 1,000 times, we determined the mean of the average receptor expression level of a cluster and the average ligand expression level of the interacting cluster. Then, we performed pairwise comparisons between all cell types and obtained a likelihood of p-value to filter the false-positive interaction. Only interactions with p < 0.05 were considered to be significant.

[0264] ATF3 immunohistochemistry staining

[0265] Immunohistochemistry staining was performed according to previously developed protocols. Paraffin-embedded sections were deparaffinized with three washes of 100% xylene and rehydrated through a series of graded alcohols (100%, 100%, 100%, 95%, and 80%), followed by a brief wash in distilled water. Antigen retrieval was carried out using heat-mediated treatment in sodium citrate buffer (pH 6.0) for 20 minutes. Sections were then treated with 3% H2O2 to quench endogenous peroxidase activity, followed by a 1-hour incubation in blocking buffer at room temperature. Primary ATF3 antibody incubation was performed overnight at 4°C. Following PBS washes, HRP-conjugated secondary antibodies were applied, and detection was conducted using DAB. The sections were counterstained with eosin and dehydrated through a series of graded alcohols (80%, 95%, 95%, 95%, 100%, 100%, and 100%) and 100% xylene before cover-slipping with a resinous mounting medium.

[0266] Validation of ATF 3 Expression with Published Data

[0267] To validate ATF3 expression variation between young and old samples, we manually curated sequencing data from four research papers (PMID: 33238152, 35069694, 34031030, 32327715), selecting samples from healthy individuals. These samples were stratified into young (<30 years) and old (>60 years) groups according to the criteria set in the original publications. The curated data were integrated using Scanpy version 1.9.2 and annotated based on either the 45

[0268] #14392914vl Attorney Docket No. H0498.70834WO00 original publications or in-house developed models. ATF3 expression levels were compared by averaging the raw counts for keratinocytes in each sample. Statistical analysis was performed using a student’s t-test. mRNA synthesis

[0269] Double-stranded DNA (dsDNA) template vectors of ATF3 (data not shown) were PCR amplified from the donor plasmid (pUC-GW-Kan, Genewiz) using primers. The resulting dsDNA template vectors were purified using the QIAquick PCR Purification Kit (Qiagen). Gene block inserts (IDT) were cloned into the dsDNA template vector using Gibson Assembly (New England Biolabs). The resulting circular dsDNA plasmid was then transformed into TOP 10 competent cells (Thermo Fisher Scientific). The cells were cultured for 12 hours at 37°C. A single bacterial colony was picked and cultured for 12 hours in LB at 37°C. The next day, the plasmids were extracted and purified using the QIAprep Spin Miniprep kit (Qiagen). The resulting plasmids were digested at 37° C for 30 minutes using BamHI-HF (New England Biolabs) and subsequently purified using the QIAquick PCR Purification Kit. Linearized dsDNAs from subsequent steps were used to synthesize the mRNAs. mRNAs were in vitro transcribed for 12 hours at 37 °C using a MEGAscript T7 transcription kit (Thermo Fisher Scientific). The transcription mixture contained 1 pg of template dsDNA, 5 mM of ATP nucleotide-triphosphate, 5 mM of CTP nucleotide-triphosphate, 3 mM of N1 -methyl pseudouridine-5 '-triphosphate (TriLink Biotechnologies), 4 mM of CleanCap reagent AG (TriLink Biotechnologies), 0.1 U Inorganic pyrophosphatase (Thermo Fisher Scientific). Transcribed mRNAs were purified using LiCl precipitation following the MEGAscript T7 transcription kit instruction.

[0270] Cell culture

[0271] Human primary keratinocytes were isolated from healthy skin samples obtained during surgical procedures, which were generated as part of the surgical procedure. Subcutaneous adipose tissue was excised from the human skin specimens and the tissue pieces were incubated in dispase solution (Stemcell, 07913) at 4°C overnight. Following digestion, the epidermis was separated from the dermis, chopped into small pieces, and incubated in 0.05% Trypsin-EDTA (Thermo Fisher Scientific, 25300054) for 10 minutes. The human primary keratinocytes were collected through a 70 mm cell strainer, seeded in a precoated dish, and cultured in supplemented Keratinocyte SFM (K-SFM, Thermo Fisher Scientific, 17005042), which included 0.2 ng / mL 46

[0272] #14392914vl Attorney Docket No. H0498.70834WO00 human recombinant EGF, 30 pg / mE BPE, and 1% antibio tic / antimycotic. The cells were cultured at 37 °C under an atmosphere of 5% CO2 in the air. Cells were cultured in K-SFM supplemented with 30ug / ml bovine pituitary extract (BPE) and 0.3ng / ml recombinant epidermal growth factor (rEGF) with 5% CO2 at 37 °C. Human dermal Fibroblasts were obtained from the Aging Cell Culture Repository (NIA, Coriell Institute for Medical Studies) and were cultured in DMEM medium containing 10% FBS at 5% CO2 at 37°C. siRNA Treatment

[0273] Primary human basal stem cell keratinocytes were plated the day before treatment in a 6- well plate to achieve 70-80% confluency at the time of treatment. 150 pmol of Dharmacon ON- TARGETplus siRNA (Non-targeting: D-001810-01-05; ATF3: J-008663-05-0002) was complexed with 7.5 pL of Lipofectamine RNAiMax (Invitrogen, 13778075) according to the manufacturer’s instructions and then added to the cells. The keratinocytes were incubated with the siRNA complexes for six hours, then the medium was changed. Cell samples were collected at 48 hours post-transfection for RT-qPCR to assess target knock-down, and SASP RT-qPCR was performed on samples collected at 72 hours post-transfection. qPCR

[0274] Total RNA was extracted using the RNeasy Plus mini kit (Qiagen, 74104) following the manufacturer’s protocol. cDNA synthesis was performed using the SuperScript III First-Strand Synthesis System (Invitrogen, 18080051), and qPCR was performed in technical triplicate using the KAPA SYBR FAST Universal qPCR kit (KK4602) on a Roche EightCycler 96 instrument. Relative quantitation was determined using the AACt method with GAPDH as an endogenous normalization control.

[0275] Senescence-associated, fi-galactosidase staining

[0276] Senescence-associated P-galactosidase (SA-P-gal) staining was performed using the Senescence P-Galactosidase Staining Kit (Cell Signaling Technology, 9860). Briefly, cells were rinsed with PBS and fixed with lx fixative solution for 15 minutes. Fresh X-gal stock solution was prepared by adding 1 ml of DMSO to 20 mg of X-gal. The P-Galactosidase staining solution was then prepared according to the manufacturer's instructions. The fixed cells were rinsed with PBS twice, and a total volume of 1 ml of the staining solution was added to each well. The plate was sealed and incubated in a 37 °C dry incubator for 18 hours. The staining results were 47

[0277] #14392914vl Attorney Docket No. H0498.70834WO00 observed using a microscope, and P-galactosidase-positive cells were considered senescent cells, which were counted in 3 randomly chosen fields. The SA-P-gal staining was further quantified by flow cytometry.

[0278] C12FDG staining for SA-P-gal was performed by initially incubating cells with 100 nM Bafilomycin Al (VWR, 102513) for 1 hour. Subsequently, cells were incubated for an additional hour with 100 nM Bafilomycin Al and 33 nM 5-Dodecanoylaminofluorescein Di-P-D- Galactopyranoside (C12FDG, ThermoFisher, D2893). Then, flow cytometry was conducted using a BD® LSR II Flow Cytometer, and data were analyzed using FlowJo software (Version 10.10.0).

[0279] Flow Cytometry

[0280] Cells were initially stained with Hoechst 33342 (Dojindo) for 30 minutes to label the nuclei before dissociated into a single-cell suspension. We used the eBioscience™ Intracellular Fixation & Permeabilization Buffer Set (Invitrogen) for fixation and permeabilization. A total of 1 mL of the fixation working solution was added to each sample, which was then incubated for 30 minutes at 4°C. After incubation, the samples were washed twice with 2 mL of IX Permeabilization Buffer. Post-washing, the cell pellet was resuspended in 100 pL of IX Permeabilization Buffer and incubated with 1 pg of Alexa Fluor® 488 anti-human Ki-67 Antibody (BioLegend, 350532) for at least 30 minutes at room temperature. Following this incubation, the samples were washed twice with 2 mL of IX Permeabilization Buffer. The final stained cells were resuspended in 150 pL of Cell Staining Buffer (BioLegend) and analyzed using a BD® LSR II Flow Cytometer.

[0281] Ki67 Immunohistochemistry Staining

[0282] For Ki67 immunohistochemistry, cells were stained with Hoechst 33342 (Dojindo) for 30 minutes before fixation in a 24-well plate. After three washes with PBS, cells were fixed with 4% paraformaldehyde (PFA) at room temperature for 20 minutes. Following fixation, the supernatant was removed, and the cells were permeabilized with 0.1% Triton-X at room temperature for 20 minutes. The cells were then washed three additional times with PBS. A 25 pL aliquot of a 1:200 dilution of Alexa Fluor® 647 anti-human Ki-67 Antibody (Abeam, ab281928) was added to each well. Cells were stained overnight at 4°C and subsequently inspected using a FLoid™ Cell Imaging Station.

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[0285] Immunohistochemistry Staining for the Target Gene

[0286] 5 p m-thick sections of formalin-fixed paraffin-embedded (FFPE) human skin specimens from young (20-30 years old) and aged (> 80 years) patients were deparaffinized and rehydrated using a graded ethanol series. After quenching endogenous peroxidase activity (BLOXALL Blocking Solution, Vector Laboratories), heat-induced epitope retrieval was performed using citric acid-based antigen unmasking solution (Vector Laboratories) at 110°C for 15 minutes in a programmable antigen retrieval chamber (ARC, Biocare Medical). Subsequently, tissue sections were blocked with 2.5% normal goat serum (Vector Laboratories) at room temperature for 20 minutes followed by incubation at 4°C overnight with mouse anti-ATF3 (1:100, Abeam ab 191513) in a humidified chamber. The next day, after washings with phosphate-buffered saline, tissue sections were incubated with ImmPRESS HRP Goat Anti-Mouse IgG Polymer Reagent (Vector Laboratories) for 30 minutes in a humidified chamber according to the manufacturer’s recommendation. Chromogenic detection was performed using ImmPACT DAB Peroxidase (Vector Laboratories) prepared according to the manufacturer’s instructions and applied to the tissue sections for a total of 7 minutes, followed by counterstaining with Hematoxylin QS (Vector Laboratories), dehydration in 100% isopropanol, and coverslipping with toluene-based mounting medium (VectaMount Express, Vector Laboratories). The following semi-quantitative scoring rubric was employed to evaluate staining: 3+: >50% of cells had strong nuclear / nucleolar staining, 2+: <50% of cells had variable nuclear / nucleolar staining, and 1+: <50% of cells had weak primarily nucleolar staining. mRNA treatment

[0287] Old (Female 62 years old) primary human epidermal keratinocytes were cultured in the K-SFM, supplemented with 30 ug / ml bovine pituitary extract (BPE, Gibco) and 0.3 ng / ml epidermal growth factor (EGF, Gibco) at 37 °C and 5% CO2 atmosphere, splitting the cells every 2-4 days to maintain monolayer coverage. Two days before transfection, cells were seeded in 6- well plates at a density of 200,000 cells per well.

[0288] For transfection, the purified mRNA was prepared at a concentration of 1 p g / p 1 in UltraPure Distilled Water (Invitrogen). For each treated well, the mRNA was diluted in 125 pl of Opti-MEM Reduced Serum Medium (Thermo Fisher Scientific) at the desired concentration. In a separate tube, MessengerMAX reagent was diluted in 125 pl of Opti-MEM at a ratio of 2 pl of MessengerMAX per 1 pg of mRNA. The diluted mRNA and MessengerMAX reagent were

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[0290] #14392914vl Attorney Docket No. H0498.70834WO00 combined, gently mixed, and incubated at room temperature for 10 minutes to allow complex formation.

[0291] After the incubation period, 250 pl of mRNA-MessengerMAX complexes were added dropwise to each well of the cell culture, with gentle swirling to ensure uniform distribution. After transfection, the plate was incubated at 37 °C in a CO2 incubator to promote optimal cellular uptake. After 30 minutes, the transfection medium was removed, and cells were washed twice with PBS. Media was replaced with fresh K-SFM and cells were returned to 37°C incubator.

[0292] Crosstalk

[0293] Primary human epidermal keratinocytes from a 27 -year-old donor (PromoCell, C- 12003) and 56-year donor (Lifeline Cell Technology, C-0025) were cultured in Keratinocyte Growth Medium (Lifeline Cell Technology, LL-0007), which was devoid of Gentamicin and Amphotericin B but supplemented with lOOU / ml Penicillin-Streptomycin. At 80% confluence, the cell was washed twice with Hanks' Balanced Salt solution (Sigma Aldrich, H6648) and cultured in fresh low-glucose Dulbecco's modified Eagle's medium (Gibco, 11885-084) for an additional 48 hours. The culture media were then collected and employed as conditioned media for experiments on the same day. A comparative negative control was established by incubating low-glucose Dulbecco's modified Eagle's medium at 37°C for the equivalent duration.

[0294] Human dermal fibroblasts were sourced from 28-year-old female from the Aging Cell Culture Repository (NIA, Coriell Institute for Medical Studies) and were maintained as monolayer cultures in low-glucose Dulbecco's modified Eagle's medium (DMEM, Gibco, 11885- 084) supplemented with 10% fetal bovine serum (Gibco, A3160501) and 100 U / ml Penicillin- Streptomycin until they reached 75% confluence. The fibroblasts were then seeded at IxlO5cells / ml and incubated overnight to allow cell attachment. Afterward, the cells underwent two washes with PBS and were subjected to a 24-hour serum starvation period in low-glucose DMEM without fetal bovine serum. This was followed by a 24-hour incubation with a mixture consisting of 50% low-glucose DMEM and 50% keratinocyte-conditioned media. The cell count in each well was then normalized with the Cell Count Normalization kit (Dojindo, C544), and tested for SA-P-gal activity.

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[0297] RNA-isolation and real-time reverse transcription-quantitative polymerase chain reaction (qPCR)

[0298] Total RNA was extracted using the RNeasy Plus mini kit (Qiagen, 74104) following the manufacturer’s protocol. cDNA synthesis was performed using the SuperScript III First-Strand Synthesis System (Invitrogen, 18080051), and qPCR was performed in technical triplicate using the KAPA SYBR FAST Universal qPCR kit (KK4602) on a Roche LightCycler 96 instrument. Relative quantitation was determined using the AACt method with GAPDH as an endogenous normalization control. qPCR assays were performed using a minimum of three independent samples. Group differences were evaluated using an independent samples t-test. Results are reported as mean ± SEM, p-value < 0.05 is considered statistically significant.

[0299] ELISA

[0300] To quantify the total amount of Collagen Type I in the culture, both the cells and the supernatant were collected separately post-treatment. The cells were lysed on ice using RIPA lysis buffer for 20 minutes. Collagen Type I concentrations in both the cell lysate and supernatant were quantified using the Human Collagen Type I ELISA Kit (Abeam, ab285250). The assay was performed by following the manufacturer’s protocol to ensure accuracy and consistency in measurements.

[0301] All references, patents and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which in some cases may encompass the entirety of the document.

[0302] The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.”

[0303] It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

[0304] In the claims, as well as in the specification above, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” “composed of,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases “consisting of’ and “consisting essentially of’ shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03.

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[0307] The terms “about” and “substantially” preceding a numerical value mean ±10% of the recited numerical value.

[0308] Where a range of values is provided, each value between and including the upper and lower ends of the range are specifically contemplated and described herein.

[0309] #14392914vl

Claims

Attorney Docket No. H0498.70834WO00CLAIMSWhat is claimed is:

1. A method of inhibiting cellular senescence, the method comprising expressing Activating Transcription Factor 3 (ATF3) in a cell at a level sufficient to inhibit senescence of the cell.

2. A method of inhibiting cellular senescence, the method comprising: delivering a messenger ribonucleic acid (mRNA) comprising an open reading frame encoding Activating Transcription Factor 3 (ATF3) to a skin cell in an amount effective to inhibit senescence of the skin cell.

3. The method of claim 1 or 2, wherein the cell is a dermal cell or an epidermal cell.

4. The method of claim 3, wherein the cell is a keratinocyte.

5. The method of claim 1 or 2, wherein the cell is a hair follicle stem cell.

6. The method of any preceding claim, wherein ATF3 is expressed in the cell at a level sufficient to reduce expression of one or more senescence-associated secretory phenotype (SASP) factors.

7. The method of claim 6, wherein the SASP factors are selected from cytokines, chemokines, growth factors, proteases, lipid metabolites and extracellular vesicles.

8. The method of claim 6, wherein the SASP factors are selected from interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin- 1 beta (IL-ip), interferon beta (IFN-P), tumor necrosis factor alpha (TNF-a), cyclin-dependent kinase inhibitor 2A (CDKN2A), CXCL family members, C-X- C motif chemokine ligand 2 (CXCL2), matrix metalloproteinases (MMPs), serine proteases, cathepsin, cyclooxygenase, prostaglandin E2 (PGE2), and leukotriene D.

9. The method of any preceding claim, wherein ATF3 is expressed in the cell at a level sufficient to reduce expression of senescence-associated P-galactosidase.53#14392914vlAttorney Docket No. H0498.70834WO0010. The method of any preceding claim, wherein ATF3 is expressed in the cell at a level sufficient to increase the rate of proliferation of the cell.

11. The method of any of claims 2-10, wherein the open reading frame encodes a wild-type ATF3 protein.

12. The method of any of claims 2-11, wherein the open reading frame comprises a nucleic acid sequence that is at least 90%, 95%, 97%, or 100% identical to SEQ ID NO: 1.

13. The method of any of claims 2-12, wherein the ATF3 has an amino acid sequence that is at least 90%, 95%, 97%, or 100% identical to SEQ ID NO: 2.

14. The method of any of claims 2-13, wherein the mRNA comprises at least one uridine nucleoside comprising N1 -methylpseudouridine triphosphate.

15. The method of any of claims 2-14, wherein the mRNA comprises a 5' human alpha- globulin- 1 (HBA1) untranslated region (UTR) and / or a 3' HBA1 UTR.

16. The method of any of claims 2-15, wherein the mRNA comprises a nucleic acid sequence that is at least 90%, 95%, 97%, or 100% identical to any one of SEQ ID NOs: 3-8.

17. The method of any of claims 2-16, wherein the mRNA is delivered using a viral vector, transfection reagent, lipid nanoparticle (LNP), microneedle, and / or electroporation.

18. The method of claim 17, wherein the viral vector is an adeno-associated viral, adenoviral, or lentiviral vector.

19. The method of claim 17, wherein the transfection reagent is a cationic lipid, a polymer- based reagent, a calcium phosphate-based reagent, or a dendrimer-based reagent.

20. The method of claim 17, wherein the lipid nanoparticle comprises ionizable lipids and / or polyethylene glycol (PEG)-lipid conjugates.54#14392914vlAttorney Docket No. H0498.70834WO0021. A method of inhibiting cellular senescence, the method comprising: delivering a messenger ribonucleic acid (mRNA) comprising an open reading frame encoding Activating Transcription Factor 3 (ATF3) to a subject in an amount effective to inhibit cellular senescence in the subject.

22. A method of increasing cellular collagen and / or elastin production, the method comprising: delivering a messenger ribonucleic acid (mRNA) comprising an open reading frame encoding Activating Transcription Factor 3 (ATF3) to a subject in an amount effective to increasing cellular collagen and / or elastin production in the subject.

23. The method of 22, wherein the increased cellular collagen and / or elastin production is produced by dermal fibroblasts.

24. The method of any of claims 21-23, wherein the open reading frame encodes a wild-type ATF3 protein.

25. The method of any of claims 21-24, wherein the open reading frame comprises a nucleic acid sequence that is at least 90%, 95%, 97%, or 100% identical to SEQ ID NO: 1.

26. The method of any of claims 21-25, wherein the ATF3 has an amino acid sequence that is at least 90%, 95%, 97%, or 100% identical to SEQ ID NO: 2.

27. The method of any of claims 21-26, wherein the mRNA comprises at least one uridine nucleoside comprising N1 -methylpseudouridine triphosphate.

28. The method of any of claims 21-27, wherein the mRNA comprises a 5' human alpha- globulin- 1 (HBA1) untranslated region (UTR) and / or a 3' HBA1 UTR.

29. The method of any of claims 21-28, wherein the mRNA comprises a nucleic acid sequence that is at least 90%, 95%, 97%, or 100% identical to any one of SEQ ID NOs: 3-8.55#14392914vlAttorney Docket No. H0498.70834WO0030. The method of any of claims 21-29, wherein the mRNA is delivered using topical, intravenous, intramuscular, intradermal, transdermal, oral, or subcutaneous administration.

31. The method of any one of claims 21-30, wherein the mRNA is delivered to the subject using a viral vector, lipid nanoparticle (LNP), or microneedle.

32. The method of claim 31, wherein the viral vector is an adeno-associated viral, adenoviral, or lentiviral vector.

33. The method of claim 31, wherein the lipid nanoparticle comprises ionizable lipids and / or polyethylene glycol (PEG)-lipid conjugates.

34. The method of any one of claims 21-33, wherein the subject has a skin disorder and / or an autoimmune disorder.

35. The method of any one of claims 21-34, wherein the subject has psoriasis, atopic dermatitis, lupus, lichen planus, epidermolysis bullosa, scleroderma, alopecia, acne vulgaris, actinic keratosis, bruises, bums, dermatitis herpetiformis, hidradenitis suppurativa, hypertrophic scars, keloids, prurigo, pyoderma gangrenosum, or vitiligo.

36. The method of claim 35, wherein the subject has alopecia, optionally selected from androgenetic alopecia, alopecia areata, telogen effluvium, traction alopecia, cicatricial alopecia, alopecia barbae, ophiasis alopecia, alopecia universalis, and pseudopelade.

37. The method of any one of claims 21-33, wherein the subject has impaired wound healing, hyperpigmentation disorders, or a chronic inflammatory condition.

38. A method of treating a skin disorder in a subject, the method comprising: delivering a messenger ribonucleic acid (mRNA) comprising an open reading frame encoding Activating Transcription Factor 3 (ATF3) to a subject in an amount effective to treat the skin disorder.

39. The method of claim 38, wherein the skin disorder is psoriasis, atopic dermatitis, lupus, lichen planus, epidermolysis bullosa, scleroderma, alopecia, acne vulgaris, actinic keratosis,56#14392914vlAttorney Docket No. H0498.70834WO00 bruises, burns, dermatitis herpetiformis, hidradenitis suppurativa, hypertrophic scars, keloids, prurigo, pyoderma gangrenosum, or vitiligo.

40. The method of claim 39, wherein the skin disorder is alopecia, optionally selected from androgenetic alopecia, alopecia areata, telogen effluvium, traction alopecia, cicatricial alopecia, alopecia barbae, ophiasis alopecia, alopecia universalis, and pseudopelade.

41. The method of claim 38 or 39, wherein the subject has impaired wound healing, hyperpigmentation disorders, or a chronic inflammatory condition.

42. A skin cell comprising an engineered messenger ribonucleic acid (mRNA) comprising an open reading frame encoding Activating Transcription Factor 3 (ATF3), wherein the ATF3 is expressed by the cell at a level sufficient to inhibit senescence of the skin cell.

43. The skin cell of claim 42, wherein the skin cell is a dermal cell or an epidermal cell, optionally a keratinocyte.57#14392914vl

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