AXL signaling pathway activator for wound healing

Abstract

Methods of promoting healing of a wound in a dermal location of a subject are provided. Aspects of the methods include administering an effective amount of an AXL signaling pathway activator, e.g., a GAS6 protein active agent, to the wound to promote healing of the wound, e.g., to promote regenerative healing of the wound with reduced scarring. Also provided are kits including an amount of an AXL signaling pathway activator composition.

Description

[0001] AXL SIGNALING PATHWAY ACTIVATOR FOR WOUND HEALING

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0003] This invention was made with Government support under contracts DE026602, DE027346, DE029463, DE029987, GM116892, and GM136659 awarded by the National Institutes of Health. The Government has certain rights in the invention.

[0004] CROSS-REFERENCE

[0005] Pursuant to 35 U.S.C. § 1 19 (e), this application claims priority to the filing date of U.S. Provisional Application No. 63 / 469,645, filed on May 30, 2023, the disclosure of which application is incorporated herein by reference in its entirety.

[0006] INCORPORATION BY REFERENCE OF SEQUENCE LISTING XML FILE

[0007] A Sequence Listing is provided herewith as a Sequence Listing XML, “STAN- 2111 WO_S23-199_S EQ_L I ST”, created on March 15, 2024 and having a size of 3,940 bytes. The contents of the Sequence Listing XML are incorporated herein by reference in their entirety.

[0008] INTRODUCTION

[0009] The skin is the largest organ in the body consisting of several layers and plays an important role in biologic homeostasis. The skin has multiple functions, including thermal regulation, metabolic function (vitamin D metabolism), and immune functions. Mammalian skin includes two main layers, the epidermis and the dermis. The epidermis is outermost layer of skin and serves as a protective barrier to the environment. The dermis is the layer of skin beneath the epidermis and serves a location for the appendages of skin including, e.g., hair follicles, sweat glands, sebaceous glands, apocrine glands, lymphatic vessels and blood vessels. The dermis provides strength and elasticity to the skin through an extracellular matrix or connective tissue made of structural proteins (collagen and elastin), specialized proteins (fibrillin, fibronectin, and laminin), and proteoglycans. The epidermis and dermis are separated by the basement membrane, a thin, fibrous extracellular matrix. Wound healing or tissue healing is a biological process that involves the replacement of damaged or destroyed tissue with living tissue. When the skin barrier is broken, a regulated sequence of biochemical events is activated to repair the damage. The process is regulated by numerous biological components including, e.g., growth factors, cytokines, and chemokines, and employs several components including, e.g., soluble mediators, blood cells, extracellular matrix components, and parenchymal cells. Wound healing generally proceeds through several stages. The process is divided into several phases including hemostasis, inflammation, proliferation, and remodeling. The end point of wound healing may include the formation of a scar. Skin wounds involving the dermis invariably heal by developing fibrotic scar tissue, which can result in disfigurement, growth restriction, and permanent functional loss. Various types of scars may form after skin tissue repair including, e.g., a “normal” fine line and abnormal scars including widespread scars, atrophic scars, scar contractures, hypertrophic scars, and keloid scars.

[0010] SUMMARY

[0011] Following injury, human skin repairs itself through a predetermined wound healing process, resulting in the formation of fibrotic scar tissue. Skin scarring produces significant functional and esthetic burdens for patients. However, no current therapeutic strategies exist for successfully preventing or reversing the fibrotic process that leads to scarring. Attempts at reducing scarring often entail ablation of cell populations known to be fibrogenic, but this approach could impair or delay wound repair by nonspecifically eliminating cells that are needed for proper healing. Skin regeneration - as defined by recovery of three features of normal skin: 1 ) secondary elements (e.g., dermal appendages), 2) ECM structure, and 3) mechanical strength - has not been achieved. Furthermore, limited understanding of the molecular mechanisms underpinning scar formation, and importantly, the mechanisms that govern repair without scarring have restricted the development of effective scarring treatments.

[0012] The oral mucosa repairs wounds more rapidly and efficiently than any other barrier site in the adult mammalian body, with scarless healing in just 1 -3 days. Oral wounds heal at nearly three times the rate of matched dermal wounds (e.g., dermal facial wounds), exhibiting a much lower inflammatory response and reduced or no scarring. Despite the remarkable regenerative capacity of the oral mucosa, the tissue-specific programs that regulate this rapid regeneration are relatively unexplored.

[0013] The present inventors demonstrate the use of single-cell RNA sequencing (scRNA-seq) of paired buccal mucosal and facial skin wounds in mice to characterize their molecular profiles. Oral and facial wounds across the time course of wound healing were examined and key differences between the fibrotic and regenerative response in the facial skin and oral mucosa were found. The mechanisms underlying scarless wound healing in the mouth are identified and it is demonstrated that by upregulating oral wound healing programs in the skin, facial skin healing with a regenerative phenotype can be produced.

[0014] Methods of promoting healing of a wound in a dermal location of a subject are provided. Aspects of the methods include administering an effective amount of an AXL signaling pathway activator, e.g., a GAS6 protein active agent, to the wound to promote healing of the wound, e.g., to promote regenerative healing of the wound with reduced scarring. Also provided are kits including an amount of an AXL signaling pathway activator.

[0015] BRIEF DESCRIPTION OF THE FIGURES

[0016] Figs. 1A-1G. Single cell RNA-sequencing reveals distinct mouse fibroblast subpopulations following oral and facial injury. (Fig. 1 A) Schematic depicting the timeline of wounding prior to harvest for single cell RNA-sequencing. (Fig. 1 B) H&E histology of oral and dermal tissue at unwounded (UW), post-operative day (POD) 1 , 2, 4, and 7; dashed black and white lines indicate the wound bed. (Fig. 1 C) Immunofluorescent staining of Collagen I (top) and Collagen III (bottom) of facial and oral POD4 wounds. (Fig. I D) Dendrogram of bulk RNA- sequencing of oral and facial unwounded and POD7 fibroblasts (Fig. 1 E) UMAP plots visualizing all cells captured in both tissues across all timepoints, the fibroblasts (outlined) were selected based on differential gene expression and isolated in silico to generate (Fig. 1 F) a UMAP plot showing the 9 types of fibroblasts identified, with their key genes and functions noted in the corresponding table. (Fig. 1 G) Ratio plots showing the ratio of each fibroblast cluster at each time point in facial and oral wounds. Scale bar; (Fig. 1 B, top) 250 p.m, (Fig. 1 B, bottom) 50 |im (Fig. 1 C) 25 |j.m. (n= 5 mice per group, unless otherwise stated).

[0017] Figs. 2A-2E. Axl signaling is upregulated in the oral mucosa fibroblasts upon wound repair. (Fig. 2A) Heatmap showing the differential number of interactions between each cell type, with red representing an increased number of interactions in the OM compared to FS, and blue indicating a lower number of interactions. Each row and column represent a cell type, labeled by color, with the populations with the largest increase in OM signaling outlined in black. (Fig. 2B) Heatmap of the Gas6-Axl signaling network in facial vs oral non-immune cells. (Fig. 2C) Violin plot of Axl expression levels in fibroblasts within the scRNA-seq dataset, split by FSF (black) and OMF (red). (Fig. 2D) RNAscope in situ hybridization for Axl transcripts (red) + IF staining for Col1 a1 (green) + DAPI (blue) for facial (left) and oral (right) samples at all time points, dashed white lines indicate the wound bed. (Fig. 2E) Quantification of FACS experiment showing the percentage of AXL and pAXL positive fibroblasts in the wound bed of Facial (black) and Oral (red) tissue at each time point. Scale bar; (Fig. 2A) 25 pm.

[0018] Figs. 3A-3F. Spatial Analysis of facial and oral wounds by VISIUM and CODEX analysis. (Fig. 3A) Schematic of the CODEX experiment and Visium spatial plots of unwounded oral (I), unwounded facial (II), POD4 oral wounds (III) and POD4 facial wounds (IV) by UMAP (left) and scRNA-seq annotations (right). (Fig. 3B) Spatial plots of Axl and Ptk2 expression in facial (top) and oral wounds (bottom). (Fig. 3C) Differential interaction maps in Facial Unwounded vs. Oral unwounded- (top) and Facial POD4 wounds vs. Oral POD4 wounds (right). (Fig. 3D) UMAP of CODEX defined clusters. (Fig. 3E) Bar graph quantifying Fibroblast 2 and Fibroblast 4 proportions in facial and oral wounds. (Fig. 3F) Histograms of Axl expression in CODEX defined fibroblast clusters. (n= 3, unless otherwise stated). (n= 3 mice per group, unless otherwise stated).

[0019] Figs. 4A-4H. FAK expression following mechanical stimulation suppresses AXL expression in vitro. (Fig. 4A) Schematic depicting the workflow of in vitro stretching experiments with FAK inhibitor. Facial and oral fibroblasts were isolated and plated on a 3D hydrogel stretching device. Gels were either unstretched, stretched, or treated with inhibitor during stretching. (Fig. 4B) Schematic depicting the hypothesized inhibition of AXL by FAK, and the predicted outcome of application of FAKi. (Fig. 4C) IF staining of unstretched (top), stretched (middle) and stretched + FAKi (bottom) gels plated with FSFs (left) and OMFs (right). Gels were stained for FAK (green), GAS6 (red), and AXL (yellow) along with DAPI (blue). (Fig. 4D) Quantification of FAK, GAS6, and AXL staining from in vitro stretching experiments in (Fig. 4C). (Fig. 4E) Schematic depicting the work flow of in vitro stretching experiments with AXLi. (Fig. 4F) Schematic depicting the hypothesized inhibition of FAK by AXL, and the predicted outcome of application of AXLi. (Fig. 4G) IF staining of unstretched (top), stretched (middle) and stretched + AXLi (bottom) gels plated with FSFs (left) and OMFs (right). Gels were stained for FAK (green), GAS6 (red), and AXL (yellow) along with DAPI (blue). (Fig. 4H) Quantification of FAK, GAS6, and AXL staining from in vitro stretching experiments in (Fig. 4G). Scale bar; (Figs. 4C, 4G) 100 pm. (n= 6 gels per group, unless otherwise stated).

[0020] Figs. 5A-5E. AXL signaling upregulation can revert facial wounds to heal like oral wounds. (Fig. 5A) Schematic of the experiment workflow for AXLi and exogenous GAS6 experiments and (Fig. 5B) schematic depicting the predicted outcome of these experiments. (Fig. 5C) H&E staining of unwounded, POD4 wounded control, POD4 +GAS6 protein, and POD4 +AXLi facial and oral tissues, with dermal thickness quantified. (Fig. 5D) IF staining of the controls, +GAS6 protein and + AXLi tissues. Merged FAK (green), AXL (red), and DAPI (blue) images (top) and FAK (middle) and AXL (bottom) channels. (Fig.5E) Quantification of AXL staining (left) and FAK staining (right). Scale bar: (Figs. 5C, 5D) 250 |im. (n= 3 mice per group, unless otherwise stated).

[0021] Figs. 6A-6F. Human and Mouse Oral Scars Lose Gas6 / Axl Expression. (Fig. 6A) Schematic depicting the timeline for obtaining human samples. (Fig. 6B) H&E staining of healthy (left) and reinjured (right) facial and oral tissues. (Fig. 6C) IF staining for DAPI (blue), COL1 (red), FAK (white) and AXL (green) in human facial (left) and oral (right) reinjured tissue. (Fig. 6D) Schematic showing the timeline of mouse repetitive injury model and predicted outcomes of untreated vs. GAS6 treated injuries (Fig. 6E) H&E staining of POD14 and POD30 facial (top) and oral (bottom) reinjured tissue vs reinjured tissue + GAS6 protein. (Fig. 6F) IF staining as in (Fig. 6C), with POD14 + Gas6 samples from facial (top) and oral tissue(bottom). Scale bar: (Fig. 6A) 250 pm, (Fig. 6B left) 250 p.m, (Fig. 6B right) 50 pm, (Fig. 6E) 150 p.m, (Fig. 6F) 150 |im. (n= 3 human samples per group and n=3 mice per group, unless otherwise stated).

[0022] Figs. 7A-7D. (Fig. 7A) Schematic and H&E staining showing the gross histology of the oral mucosa and facial skin. (Fig. 7B) Masson’s Trichrome staining of facial (top) and oral (bottom) tissue at unwounded, POD1 , POD2, POD4, and POD7. (Fig. 7C) Picrosirius red staining of both tissues showing ECM fiber ultrastructure at all time points. (Fig. 7D) GO terms derived from bulk RNA-sequencing of FSFs and OMFs at unwounded and POD7, depicting the top GO terms from multiple databases. Scale bar; (Fig. 7A) 150 |j.m, (Fig. 7B) 100 pm, (Fig. 7C) 25 |j.m. (n= 5 mice per group, unless otherwise stated).

[0023] Figs. 8A-8E. (Fig. 8A) Violin plots showing key genes used to determine the broad cell type of each identified cluster. (Fig. 8B) Bar plots depicting ratios of immune, endothelial and epithelial cell clusters within the scRNA-seq dataset, split by tissue and timepoint. (Fig. 8C) UMAP plot of all fibroblasts, split by tissue, with the bar chart depicting the ratio of each tissue per cluster. (Fig. 8D) Pie charts depicting the composition of facial vs oral fibroblast populations. (Fig. 8E) GO term plots showing the top 9 GO terms for Fibroblast 1 and Fibroblast 2.

[0024] Figs. 9A-9D. (Fig. 9A) Heat map of the top 5 genes for each fibroblast cluster. (Fig. 9B) Immunofluorescent staining of Fibroblast 1 , Fibroblast 2, and Fibroblast 4 markers in facial (top) and oral (bottom) POD4 wounds. (Fig. 9C) Trajectory plots generated using scVelo in Python for facial and oral fibroblasts. (Fig. 9D) Violin plot of Seal expression in fibroblasts. Scale bar; (Fig. 9B) 25 jam. Figs. 10A-10F. (Fig. 10A) Relative information flow for each significant pathway identified via CellChat. X-axis indicates the percentage of interactions per pathway identified in the dataset, split by tissue, with OM in red and FS in black. Arrows demarcate Gas6 and Prosl, two ligands of Axl that are upregulated in the OM. (Fig. 10B) Top GO terms for Fibroblast 4 derived from the scRNA-seq dataset. (Fig. 10C) Violin plot of Gas6 expression levels in fibroblasts. (Fig. 10D) Violin plot of Axl and Gas6 expression within the immune subsets within the scRNA-seq dataset, split by FSF (black) and OMF (red). (Fig. 10E) Violin plot of Axl and Gas6 expression within the endothelial subsets within the scRNA-seq dataset, split by FSF (black) and OMF (red). (Fig. 10F) Ratio of spliced to unspliced transcripts of Axl and Gas6 in the facial mesenchyme (fibroblasts + endothelium) and oral mesenchyme, respectively. Purple dashed line represents the expected ratio if there is no activation of the pathway in the tissue.

[0025] Figs. 11A-11 F. (Fig. 11A) IF staining for AXL (white), GAS6 (red), FAK (green) and DAPI (blue) across timepoints. (Fig. 11 B) Quantification of FACS experiment showing the percentage of FAK and pFAK positive fibroblasts in the wound bed of FS (black) and OM (red) at each time point. (Fig. 11 C) IF of AXL staining in immune cells. (Fig. 11 D) Schematic of FAK KO experiment. (Fig. 11 E) H&E staining of facial (top) and oral (bottom) tissue: WT control (Unwounded; left), Col1GreERT;R26mTmG(POD4; middle), and Col1GreERT;R26mTmG;Fak,l / +(POD4; right). (Fig. 11 F) IF staining for AXL (red), FAK (green), and DAPI (blue). Scale bar; (Figs. 11A, 11 E, 11F) 150 p.m. (n= 3 mice per group, unless otherwise stated).

[0026] Figs. 12A-12B. (Fig. 12A) Picrosirius red staining (PSR) of both tissues showing extracellular matrix fiber components in WT control (Unwounded; left), Col1CreERT;R26mTmG(POD4; middle), and Col1CreERT;R26mTmG;Fakfl / +(POD4; right). (Fig. 12B) UMAP of PSR parameters. Scale bar; (Fig. 12A) 25 |im. n= 3 mice per group, unless otherwise stated).

[0027] Figs. 13A-13D. (Fig. 13A) Visium spatial plots of Col1A1, Krt14, and Ptprc (Fak) in oral mucosa and facial skin unwounded. (Fig. 13B) Visium spatial plots of scRNA-seq fibroblast annotated subpopulations onto oral and facial unwounded and POD4 samples. (Fig. 13C) Visium spatial plots of Col1A 1, Krt14, and Ptprc (Fak) in oral and facial unwounded. (Fig. 13D) Differential interaction maps in Facial Unwounded vs. Facial POD4 wounds (left) and Oral unwounded vs Oral POD4 wounds (right). (n= 3 mice per group, unless otherwise stated).

[0028] Figs. 14A-14E. (Fig. 14A) Representative images of identified UMAP CODEX clusters showing their spatial distribution in facial and oral samples. (Fig. 14B) Representative images showing the distribution of the CODEX defined clusters. (Fig. 14C) Representative images of distribution of CODEX annotated cell populations on sample types. (Fig. 14D) Bar graph quantifying fibroblast proportions in facial and oral wounds. (Fig. 14E) Bar graph quantifying cell interactions in facial and oral wounds. Scale bar; (Fig. 14C) 150 pm. (n= 3 mice per group, unless otherwise stated).

[0029] Figs. 15A-15C. (Fig. 15A) Anchor transfer of CODEX defined fibroblast clusters onto murine scRNA-seq fibroblast clusters. (Fig. 15B) Histograms of Fak+ and Axl+Gas6+ expression in CODEX defined fibroblast clusters. (Fig. 15C) Differential interaction maps in Facial Unwounded vs. Facial POD-4 wounds (top left), Oral unwounded vs Oral POD-4 wounds (top right), Facial Unwounded vs Oral Unwounded (bottom left), and Facial POD-4 versus Oral POD- 4 (bottom left).

[0030] Figs. 16A-16F. (Figs. 16A, 16C, 16D) IF staining of unstretched (top), stretched (middle) and stretched + FAKi (bottom) gels plated with FSFs (left) and OMFs (right). (Fig. 16B) Quantification of aSMA and COL1 A1 staining of gels. (Fig. 16E) FACS analysis of FAK and AXL positive cells in gels at 48 hours. (Fig. 16F) COL1A1 secretion of fibroblasts in gels by ELISA. Scale bar; (Figs. 16A, 16C, 16D) 100 pm. (n= 6 gels per group, unless otherwise stated).

[0031] Figs. 17A-17R (Figs. 17A, 17C, 17D) IF staining of unstretched (top), stretched (middle) and stretched + AXLi (bottom) gels plated with FSFs (left) and OMFs (right). (Fig. 17B) Quantification of aSMA and COL1 A1 staining of gels. (Fig. 17E) FACS analysis of FAK and AXL positive cells in gels at 48 hours. (Fig. 17F) COL1A1 secretion of fibroblasts in gels by ELISA. Scale bar; (Figs. 17A, 17C, 17D) 100 pm. (n= 6 gels per group, unless otherwise stated).

[0032] Figs. 18A-18C. (Figs. 18A-18C) IF staining of unstretched (top), stretched (middle) and stretched + GAS6 protein (bottom) gels plated with FSFs (left) and OMFs (right). (Fig. 18A) Quantification of GAS6, AXL (top) and COL1A1 , aSMA staining of gels (bottom). Scale bar; (Figs. 18A-18C) 100 pm. (n= 6 gels per group, unless otherwise stated).

[0033] Figs. 19A-19C. (Fig. 19A) Schematic of Ax / shRNA gel experiment. (Figs. 19B, 19C) IF staining of unstretched scramble (first panel), unstretched Ax / shRNA (second panel), stretched scrambled (third panel) and stretched Ax / shRNA (fourth panel). Scale bar; (Figs. 19A, 19B) 100 |im. (n= 6 gels per group, unless otherwise stated).

[0034] Figs. 20A-20C. (Fig. 20A) Masson’s Trichrome of unwounded, untreated POD4 control, POD4 + Gas6 protein, and POD4 +AXLi facial and oral tissues, with blue pixel density quantified. (Fig. 20B) IF staining of Fibroblast 1 and Fibroblast 2 markers in facial and oral treated samples with DAPI (blue), ENPP2 (green), LUMICAN (red). (Fig. 20C) Picrosirius red staining of AXLi / GAS6 experiments with PCA of ECM pseudocolored by sample type (left) with quantification (right). Scale bar; (Fig. 20A) 250 p.m, (Fig. 20B) 50 p.m, (Fig. 20C) 25 |im. (n= 3 mice per group, unless otherwise stated).

[0035] Figs. 21A-21 D. (Fig. 21 A) Quantification of pAXL (left) and pFAK staining of Axli / GAS6 experiments. (Figs. 21 B, 21 C) IF staining of Axli / GAS6 experiments. (Fig. 21 D) Quantification of COL1A1 IF staining in ofAxI inhibitor / activator experiments. Scale bar; (Figs. 21 B, 21C) 150 p.m. (n= 3 mice per group, unless otherwise stated).

[0036] Figs. 22A-22C. (Fig. 22A) Schematic depicting the experiment workflow for Axl KO experiments. (Fig. 22B) H&E staining of unwounded and POD4 oral and facial tissue in AxH (top), Axl+ / _(middle), and Axl+ / +(bottom) mice. (Fig. 22C) Masson's Trichrome staining of the same samples as above. Scale bar; (Fig. 22B) 250 .m, (Fig. 22C) 250 p.m. (n= 3 mice per group, unless otherwise stated).

[0037] Figs. 23A-23B. (Fig. 23A) Picrosirius red staining (PSR) of Axl KO experiments. (Fig. 23B) PCA of PSR analysis pseudocolored by sample type (left) with quantification (right). Scale bar; (Fig. 23A) 25 pm. (n= 3 mice per group, unless otherwise stated).

[0038] Fig. 24. IF staining of AxI KO experiments of facial and oral samples in Axl+ / +(top), Axl+A(middle), and AxlA(bottom) mice. Scale bar; 150 p.m. (n= 3 mice per group, unless otherwise stated).

[0039] Figs. 25A-25B. (Fig. 25A) Masson Trichome staining of human facial and oral samples. (Fig. 25B) RNAscope in situ hybridization for Axl transcripts (red) + DAPI (blue) for human oral and facial samples. Scale bar; (Figs. 25A, 25B) 250 p.m (n= 3 human samples per group, unless otherwise stated).

[0040] Figs. 26A-26C. (Fig. 26A) Masson’s Trichrome of mouse POD14 facial (top) and oral (bottom) reinjury (right) vs reinjury + Gas6 protein (left). (Fig. 26B) Picrosirius red staining (PSR) of reinjury experiments in oral (left) and facial (right) tissue. (Fig. 26C) Picrosirius Red PCA analysis pseudocolored by sample type. Scale bar; (Fig. 26A) 250 p.m (Fig. 26B) 25 |im. (n= 3 mice per group, unless otherwise stated).

[0041] DEFI ITIONS

[0042] As used herein in its conventional sense, the term “fibroblast” refers to a cell responsible for synthesizing and organizing extracellular matrix. Two dermal fibroblast lineages include Engrailed-1 En1) positive fibroblasts and Paired Related Homeobox 1 (Prrx ) and Ectonucleotide Pyrophosphatase / Phosphodiesterase (Enpp2) positive fibroblasts. Oral mucosal fibroblast (OMF) lineages include Lumican (Lum) and Adipocyte enhancer-binding protein 1 (Aebpl) positive fibroblasts.

[0043] As used herein, the term “AXL signaling pathway” is used interchangeably with “GAS6- AXL signaling pathway” and refers to one or more attributes of a biological cell, population of cells, or a component of a cell that are modulated by activation of the AXL receptor, e.g., by binding of a GAS6 ligand to the AXL receptor. In some cases, the one or more attributes include, e.g., an amount, activity, modification (e.g., post-translational modification), or expression level (DNA or RNA expression levels) of a component of a cell (e.g., a protein, nucleic acid, etc.).

[0044] As used herein, the term “modulating” means increasing, reducing or inhibiting an attribute of a biological cell, population of cells, or a component of a cell (e.g., a protein, nucleic acid, etc.). In some cases, the attribute includes, e.g., activation of a signaling pathway. In some cases, the attribute includes an amount and / or activity of one or more cells. In some cases, the attribute includes, e.g., an amount, activity, or expression level (DNA or RNA expression levels) of a component of a cell (e.g., a protein, nucleic acid, etc.). In some cases, "modulate" or "modulating" or “modulation” may be measured using an appropriate in vitro assay, cellular assay or in vivo assay. In some cases, the increase or decrease is 10% or more relative to a reference, e.g., 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 97% or more, 98% or more, up to 100% relative to a reference. For example, the increase or decrease may be 2 or more times, 3 times or more, 4 times or more, 5 times or more, 6 times or more, 7 times or more, 8 times or more, 9 times or more, 10 times or more, 50 times or more, or 100 times or more relative to a reference.

[0045] The term "fibrosis" as used herein in its conventional sense refers to the formation or development of excess fibrous connective tissue in an organ or tissue as a result of injury or inflammation of a part or interference with its blood supply. It can be a consequence of the normal healing response that leads to a scar, an abnormal reactive process or no known or understood cause.

[0046] As used herein in its conventional sense, the term “scarring” refers to a condition in which fibrous tissue replaces normal tissue destroyed by injury or disease. The term “scarring” further refers to abnormality in one or more of color, contour (bulging / indentation), rugosity (roughness / smoothness), strength (skin strength is reduced / scars are weaker than skin), overall appearance, e.g., due to lack of hair (which does not regrow in scars), and texture (softness / hardness), arising during the skin healing process. The expression “preventing” or ’’prevent” used herein in the context of scarring refers to an adjustment to the extent of development of scarring, whereby one or more of the color, contour, rugosity and texture of the healed skin surface approximates on ordinary visual inspection to that of the subject’s normal skin. The expression “reducing” or “reduce” or “ameliorating” or “ameliorate” used herein in the context of scarring refers to an adjustment to the extent of development of scarring, whereby one or more of the color, contour, rugosity and texture of the healed skin surface approaches measurably closer to that of the patient's normal skin.

[0047] As used herein in its conventional sense, the term "scar" refers to a fibrous tissue that replaces normal tissue destroyed by injury or disease. Damage to the outer layer of skin (the epidermis) is healed by rebuilding the tissue, and in these instances, scarring is slight or absent. When the thick layer of tissue beneath the skin’s outer surface (i.e., the dermis) is damaged, however, rebuilding is more complicated. The body lays down collagen fibers (a protein which is naturally produced by the body) in a composition that is different from that found in uninjured skin, and this usually results in a noticeable scar. After the wound has healed, the scar continues to alter as new collagen is formed, existing collagen is enzymatically remodeled, and the blood vessels return to normal, allowing most scars to fade and improve in appearance over the two years following an injury. However, there permanently remains some visible evidence of the injury, and hair follicles and sweat and oil glands do not grow back. As used herein, the term "scar area" refers to the area of normal tissue that is destroyed by injury or disease and replaced by fibrous tissue.

[0048] Scars differ from normal skin in three key ways: (1) they are devoid of any dermal appendages (hair follicles, sweat glands, etc.); (2) their collagen structure is fundamentally different, with dense, parallel fibers rather than the “basketweave” pattern that lends normal skin its flexibility and strength; and (3) as a result of their inferior matrix structure, they are weaker than skin.

[0049] The term "scar-related gene" as used herein refers to a nucleic acid encoding a protein that is activated in response to scarring as part of the normal wound healing process. The term "scar-related gene product" as used herein refers to the protein that is expressed in response to scarring as part of the normal wound healing process.

[0050] The term “regenerative OMF-like gene expression” as used herein refers to relative expression of genes or gene products that are indicative of oral mucosal fibroblast-like wound healing compared to dermal fibroblast-like (e.g., facial skin fibroblast-like) wound healing. For example, regenerative OMF-like gene expression relative to a control comprises down-regulation of genes that have lower expression in oral mucosal fibroblasts during wound healing compared to dermal fibroblasts during wound healing. Additionally, regenerative OMF-like gene expression relative to a control comprises up-regulation of genes that have higher expression in oral mucosal fibroblasts during wound healing compared to dermal fibroblasts during wound healing.

[0051] Scar tissue consists mainly of disorganized collagenous extracellular matrix. This is produced by myofibroblasts, which differentiate from dermal fibroblasts in response to wounding, which causes a rise in the local concentration of Transforming Growth Factor-p, a secreted protein that exists in at least three isoforms called TGF-pi, TGF- 2 and TGF-[33 (referred to collectively as TGF-p). TGF-p is an important cytokine associated with fibrosis in many tissue types (Beanes, S. et al, Expert Reviews in Molecular Medicine, vol. 5, no. 8, pp. 1 - 22 (2003)). Types of scars are further described in, e.g., PCT Application No. WO 2014 / 040074, the disclosure of which is incorporated herein by reference in its entirety.

[0052] The term “skin” used herein in its conventional sense includes all surface tissues of the body and sub-surface structure thereat including, e.g., mucosal membranes and eye tissue as well as ordinary skin. The expression “skin” may include a wound zone itself. The reapproximation of skin over the surface of a wound has long been a primary sign of the completion of a significant portion of wound healing. This reclosure of the defect restores the protective function of the skin, which includes protection from bacteria, toxins, and mechanical forces, as well as providing the barrier to retain essential body fluids. The epidermis, which is composed of several layers beginning with the stratum corneum, is the outermost layer of the skin. The innermost skin layer is the deep dermis.

[0053] As used herein in its conventional sense, the term “dermal appendages” includes hair follicles, sebaceous and sweat glands, fingernails, and toenails.

[0054] As used herein, the term “dermal location” refers to a region of a skin of a subject having any size and area. The dermal location may encompass a portion of skin of a subject such as, e.g., the scalp. The dermal location may include one or more layers of skin including, e.g., the epidermis and the dermis. In some cases, the dermal location includes a wound.

[0055] As used herein in its conventional sense, a “photosensitizer” or “photoreactive agent” or “photosensitizing agent” is a light-activated drug or compound. A photosensitizer may be defined as a substance that absorbs electromagnetic radiation, most commonly in the visible spectrum, and releases it as another form of energy, most commonly as reactive oxygen species and / or as thermal energy. In some cases, a photosensitizing agent is useful in photodynamic therapy. Such agents may be capable of absorbing electromagnetic radiation and emitting energy sufficient to exert a therapeutic effect, e.g., the impairment or destruction of unwanted cells or tissue, or sufficient to be detected in diagnostic applications. For example, the photosensitizer can be any chemical compound that collects in one or more types of selected target tissues and, when exposed to light of a particular wavelength, absorbs the light and induces impairment or destruction of the target tissues. Virtually any chemical compound that homes to a selected target and absorbs light may be used. The photosensitizer may be nontoxic to a subject to which it is administered and is capable of being formulated in a nontoxic composition. The photosensitizer may also be nontoxic in its photodegraded form. In some cases, the photosensitizers are characterized by a lack of toxicity to cells in the absence of the photochemical effect and are readily cleared from non-target tissues.

[0056] As used herein in its conventional sense, the term “wound” includes any disruption and / or loss of normal tissue continuity in an internal or external body surface of a human or non-human animal body, e.g., resulting from a non-physiological process such as surgery or physical injury. The expression “wound” or “wound environment” used herein refers to any skin lesion capable of triggering a healing process which may potentially lead to scarring, and includes wounds created by injury, wounds created by burning, wounds created by disease and wounds created by surgical procedures. The wound may be present on any external or internal body surface and may be penetrating or non-penetrating. The methods herein described may be beneficial in treating problematic wounds on the skin's surface. Examples of wounds which may be treated in accordance with the method of the invention include both superficial and non-superficial wounds, e.g., abrasions, lacerations, wounds arising from thermal injuries (e.g., burns and those arising from any cryo-based treatment), and any wound resulting from surgery.

[0057] The term "wound healing" as used herein in its conventional sense refers to a regenerative process with the induction of a temporal and spatial healing program, including, but not limited to, the processes of inflammation, granulation, neovascularization, migration of fibroblast, endothelial and epithelial cells, extracellular matrix deposition, re-epithelialization, and remodeling.

[0058] As used herein in its conventional sense, the term “microneedling” refers to the use of microneedles on an area of the body. An individual microneedle is designed to puncture the skin up to a predetermined distance, which may be greater than the nominal thickness of the stratum corneum layer of skin (the very outer layer of the skin out-covering the epidermis). Using such microneedles may overcome the barrier properties of the skin. At the same time, the microneedles are relatively painless and bloodless if they are made to not penetrate through the epidermis, which is approximately less than 2.0-2.5 mm beneath the outer surface of the skin. Microneedles may require a direct pushing motion against the skin of sufficient force to penetrate completely through the stratum corneum. In general, microneedle stimulation systems are well known for their use in skin care treatment of various conditions such as wrinkles, acne scarring, stretch marks, skin whitening and facial rejuvenation. In certain embodiments of microneedling, a method of piercing holes in the skin and applying drugs or cosmetics to the skin provides a way to rapidly and sufficiently permeate the skin. In some cases, using microneedles is sufficient to injure the skin just enough to begin natural healing processes and stimulate collagen and elastin production, and the like, to heal the skin. In these methods, hundreds to thousands of tiny holes or microconduits are created in the skin with the microneedling device without damaging the deeper layers of the skin. This injury to the skin begins a natural healing process that leads to the release of natural stimulants and growth factors which stimulates the formation of new natural collagen and elastin in the papillary dermis to produce new, healthy skin tissue. Also, new capillaries are formed. This neovascularisation and neocollagenesis associated with the wound healing process leads to the formation of younger looking skin, reduction of skin pathologies and improvement of scars. Generally called percutaneous collagen induction therapy, microneedling has also been used in the treatment of photo ageing. Furthermore, medical substances may be applied to the site where the holes are created and the substances are supposed to permeate into the skin through the tiny holes. Microneedling is generally applied to the face, neck, scalp, and just about anywhere on the body where a particular condition warrants without removing or permanently damaging the skin. A predetermined number of needles are inserted into the skin to the desired depth. As a reaction to the minor injury, the skin tissue begins a natural wound-healing cascade. This natural process forms new healthy dermal tissue that helps smooth scars, remove wrinkles and improve pigmentation, and yields a younger, healthier and a cleaner-looking skin.

[0059] As used herein in its conventional sense, the term “fractional laser resurfacing treatment” or “fractional laser resurfacing” or “fractional resurfacing” refers to the use of electromagnetic radiation to improve skin defects by inducing a thermal injury to the skin, which results in a complex wound healing response of the skin. This leads to a biological repair of the injured skin. Various techniques providing this objective have been introduced. The different techniques can be generally categorized in two groups of treatment modalities: ablative laser skin resurfacing (“LSR”) and non-ablative collagen remodeling (“NCR”). The first group of treatment modalities, i.e., LSR, includes causing thermal damage to the epidermis and / or dermis, while the second group, i.e., NCR, is designed to spare thermal damage of the epidermis. LSR with pulsed CC^ or Er:YAG lasers, which may be referred to in the art as laser resurfacing or ablative resurfacing, is considered to be an effective treatment option for signs of photo aged skin, chronically aged skin, scars, superficial pigmented lesions, stretch marks, and superficial skin lesions. NCR techniques are variously referred to in the art as nonablative resurfacing, non-ablative subsurfacing, or non-ablative skin remodeling. NCR techniques generally utilize non-ablative lasers, flashlamps, or radio frequency current to damage dermal tissue while sparing damage to the epidermal tissue. The concept behind NCR techniques is that the thermal damage of only the dermal tissues is thought to induce wound healing which results in a biological repair and a formation of new dermal collagen. This type of wound healing can result in a decrease of photoaging related structural damage. Avoiding epidermal damage in NCR techniques decreases the severity and duration of treatment related side effects. In particular, post procedural oozing, crusting, pigmentary changes and incidence of infections due to prolonged loss of the epidermal barrier function can usually be avoided by using the NCR techniques. Additional methods and devices for practicing fractional laser resurfacing are described in, e.g., PCT Application No. WO 2005 / 007003; U.S. Application No. 20160324578; and Beasley et al. (2013) Current Dermatology Reports. 2:135-143, the disclosures of which are incorporated herein by reference in their entireties.

[0060] As used herein, the term “administering” includes in vivo administration as well as direct administration to tissues ex vivo. Generally, administration is, for example, oral, buccal, parenteral (e.g., intravenous, intraarterial, subcutaneous), intraperitoneal (i.e., into the body cavity), topically, e.g., by inhalation or aeration (i.e., through the mouth or nose), or rectally systemic (i.e., affecting the entire body). A composition may be administered in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants, and vehicles as desired. The term “topically” may include injection, insertion, implantation, topical application, or parenteral application.

[0061] DETAILED DESCRIPTION

[0062] Methods of promoting healing of a wound in a dermal location of a subject are provided. Aspects of the methods include administering an effective amount of an AXL signaling pathway activator, e.g., a GAS6 protein active agent, to the wound to promote regenerative healing of the wound with reduced scarring. Also provided are kits including an amount of an AXL signaling pathway activator composition.

[0063] Before the present invention is described in greater detail, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0064] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0065] Certain ranges are presented herein with numerical values being preceded by the term "about." The term "about" is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes. In determining whether a number is near to or approximately a specifically recited number, the near or approximating unrecited number may be a number which, in the context in which it is presented, provides the substantial equivalent of the specifically recited number.

[0066] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, representative illustrative methods and materials are now described.

[0067] All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

[0068] It is noted that, as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation.

[0069] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present invention. Any recited method can be carried out in the order of events recited or in any other order which is logically possible.

[0070] While the apparatus and method has or will be described for the sake of grammatical fluidity with functional explanations, it is to be expressly understood that the claims, unless expressly formulated under 35 ll.S.C. §112, are not to be construed as necessarily limited in any way by the construction of "means" or "steps" limitations, but are to be accorded the full scope of the meaning and equivalents of the definition provided by the claims under the judicial doctrine of equivalents, and in the case where the claims are expressly formulated under 35 U.S.C. §112 are to be accorded full statutory equivalents under 35 U.S.C. §112.

[0071] In further describing various aspects of the invention, the methods are reviewed first in greater detail, followed by a review of kits. Applications in which the methods and kits find use are also provided in greater detail below.

[0072] METHODS

[0073] As summarized above, aspects of the invention include methods of promoting healing of a wound in a dermal location of a subject. In some cases, the methods prevent scarring during healing of a wound in a subject. In certain embodiments, aspects of the methods include administering an effective amount of an AXL signaling pathway activator to a wound to promote healing of the wound. The methods may be applied to any cell or population of cells as described herein.

[0074] As summarized above, aspects of the methods may include administering an effective amount of an AXL signaling pathway activator to a wound. AXL is a member of the Tyro3-Axl- MerTK (TAM) family of receptor tyrosine kinases. The AXL receptor is composed of an extracellular domain composed of two immunoglobulin-like (IgL) domains and two fibronectin III (FNIII) domains, a transmembrane domain, and an intracellular tyrosine kinase domain. TAM receptor ligands include Growth Arrest Specific 6 (GAS6), and Protein S (PROS1 ) proteins. GAS6 and PR0S1 are members of the vitamin K-dependent protein family that resemble blood coagulation factors rather than typical growth factors. GAS6 has high structural homology with PROS1 , sharing the same modular composition and having 40% sequence identity. PROS1 can only activate TYRO3 and MERTK receptors, while GAS6 can activate all members of the TAM receptor family. The AXL receptor is specifically activated by GAS6 which has highest affinity for the AXL receptor and can activate AXL receptors in a concentration dependent manner. Upon binding of GAS6 to the AXL receptor, the GAS6-AXL complex dimerizes with another GAS6-AXL complex to form a 2:2 homodimerized complex. Human GAS6 is a 678 amino acid protein that consists of a y-carboxyglutamic acid (Gla)-rich domain, four epidermal growth factor (EGF)-like domains, and two C-terminal laminin G-like (LG) domains. The y-carboxyglutamic acid residues of the Gla-rich domain are formed by vitamin K-dependent carboxylation and mediate binding to phosphatidylserine on plasma membranes in a calcium-dependent manner. The first LG domain (LG-1 ) of GAS6 binds directly to the extracellular lgL-1 and lgL-2 domains of AXL and binding of a GAS6 fragment composed of the two GAS6 LG domains (i.e., LG-1 and LG-2 domains) alone is sufficient for activation of the AXL receptor.

[0075] A variety of different AXL signaling pathway activators may be used in various embodiments of the invention. In general, an AXL signaling pathway activator can belong to any of a number of compound classes such as peptides, polypeptides, peptidomimetics, antibodies, nucleic acids, and small molecules. In certain embodiments, the AXL signaling pathway activator is an AXL receptor agonist. By AXL receptor agonist, it is meant that the AXL signaling pathway activator binds to the AXL receptor and modulates the AXL receptor activity, e.g., by initiating a physiological response mediated by the AXL receptor.

[0076] In some instances, the AXL signaling pathway activator is a protein or fragment thereof. In some cases, the AXL signaling pathway activator is an antibody binding agent or derivative thereof. In some cases, the antibody binding agent or derivative thereof binds the extracellular domain of an AXL receptor and cross-links and activates the AXL receptor in an AXL receptordimer. The term "antibody binding agent" as used herein includes polyclonal or monoclonal antibodies or fragments that are sufficient to bind to an analyte of interest. The antibody fragments can be, for example, monomeric Fab fragments, monomeric Fab' fragments, or dimeric F(ab)'2 fragments. Also within the scope of the term "antibody binding agent" are molecules produced by antibody engineering, such as single-chain antibody molecules (scFv) or humanized or chimeric antibodies produced from monoclonal antibodies by replacement of the constant regions of the heavy and light chains to produce chimeric antibodies or replacement of both the constant regions and the framework portions of the variable regions to produce humanized antibodies. Antibody binding agent activators of the AXL signaling pathway are known in the art and described for example in WO 2009 / 062690 A1 and WO 2015 / 193428 A1 , the disclosures of which are herein incorporated by reference.

[0077] In some instances, the AXL signaling pathway activator is a GAS6 protein or a fragment thereof. Unless otherwise specified, the term “Growth Arrest Specific 6” or “GAS6” as used herein refers to both serum-derived and recombinant GAS6. As discussed above, the GAS6 protein is encoded by the Growth Arrest Specific 6 gene (Example gene, transcript, and protein sequences of GAS6 may be accessed at Genbank at NG_029703.1 , NM 000820.4, and NP 000811.1 , respectively). As used in the present invention, GAS6 encompasses any natural variants, alternative sequences, isoforms, or mutant proteins that can be found in a serum sample, for example a human serum sample. Examples of GAS6 variants found in the human population include, without limitation: F41 L; S231Y; V347M; G500R; S580L; E612K; and R616Q. Additional examples of GAS6 variants include GAS6 variants which are not y-carboxylated or are substantially less y-carboxylated than serum-derived GAS6. As will be appreciated, the GAS6 used in the methods and compositions described herein may be serum-derived or recombinant and may further comprise one or more different variants (including full length and truncated forms). In some instances, the truncated forms of GAS6 may comprise the two C-terminal laminin G-like domains (i.e., LG-1 and LG-2 domains). Methods of making and / or purifying serum-derived or recombinant GAS6 protein variants are known in the art and described for example in WO 1996 / 028548 A1 , WO 2004 / 029209 A2, WO 2004 / 108748 A2, and WO 2016 / 063522 A1 , the disclosures of which are herein incorporated by reference.

[0078] In some cases, the AXL signaling pathway activator comprises an amino acid sequence having 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% or greater amino acid sequence identity to the following GAS6 amino acid sequence: MAPSLSPGPAALRRAPQLLLLLLAAECALAALLPAREATQFLRPRQRRAFQVFEEAKQGHLERECVEELC SREEAREVFENDPETDYFYPRYLDCINKYGSPYTKNSGFATCVQNLPDQCTPNPCDRKGTQACQDLMGNF FCLCKAGWGGRLCDKDVNECSQENGGCLQICHNKPGSFHCSCHSGFELSSDGRTCQDIDECADSEACGEA RCKNLPGSYSCLCDEGFAYSSQEKACRDVDECLQGRCEQVCVNSPGSYTCHCDGRGGLKLSQDMDTCEDI LPCVPFSVAKSVKSLYLGRMFSGTPVIRLRFKRLQPTRLVAEFDFRTFDPEGILLFAGGHQDSTWIVLAL RAGRLELQLRYNGVGRVTSSGPVINHGMWQTI SVEELARNLVIKVNRDAVMKIAVAGDLFQPERGLYHLN LTVGGIPFHEKDLVQP INPRLDGCMRSWNWLNGEDTTIQETVKVNTRMQCFSVTERGSFYPGSGFAFYSL DYMRTPLDVGTESTWEVEVVAHIRPAADTGVLFALWAPDLRAVPLSVALVDYHSTKKLKKQLVVLAVEHT ALALMEIKVCDGQEHWTVSLRDGEATLEVDGTRGQSEVSAAQLQERLAVLERHLRSPVLTFAGGLPDVP VTSAPVTAFYRGCMTLEVNRRLLDLDEAAYKHSDITAHSCPPVEPAAA (SEQ ID N0:1 ).

[0079] In some cases, the AXL signaling pathway activator comprises an amino acid sequence having 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% or greater amino acid sequence identity to the following GAS6 LG domain amino acid sequence: HCDGRGGLKLSQDMDTCEDILPCVPFSVAKSVKSLYLGRMFSGTPVIRLRFKRLQPTRLVAEFDFRTFDP EGILLFAGGHQDSTWIVLALRAGRLELQLRYNGVGRVTSSGPVINHGMWQTISVEELARNLVIKVNRDAV MKIAVAGDLFQPERGLYHLNLTVGGIPFHEKDLVQPINPRLDGCMRSWNWLNGEDTTIQETVKVNTRMQC FSVTERGSFYPGSGFAFYSLDYMRTPLDVGTESTWEVEVVAHIRPAADTGVLFALWAPDLRAVPLSVALV DYHSTKKLKKQLVVLAVEHTALALMEIKVCDGQEHVVTVSLRDGEATLEVDGTRGQSEVSAAQLQERLAV LERHLRSPVLTFAGGLPDVPVTSAPVTAFYRGCMTLEVNRRLLDLDEAAYKHSDITAHSCPPVEPAAA (SEQ ID NO:2).

[0080] In some instances, a nucleic acid coding sequence is administered to the subject under conditions sufficient for the coding sequence to be expressed in the subject. Depending on the desired polypeptide, the nucleic acid coding sequence may vary. Nucleic acids of interest include those encoding the AXL signaling pathway activators provided above. By nucleic acid composition, it is meant a composition comprising a sequence of DNA or RNA having an open reading frame that encodes a polypeptide of interest, i.e. , a polypeptide coding sequence, and is capable, under appropriate conditions, of being expressed as a polypeptide of interest, e.g., a GAS6 protein or active fragment thereof. Also encompassed in this term are nucleic acids that are homologous, substantially similar or identical to the specific nucleic acids described above. In addition to the above-described specific nucleic acid compositions, also of interest are homologues of the above sequences. In certain embodiments, sequence similarity between homologues is 20% or higher, such as 25 % or higher, and including 30 %, 35%, 40%, 50%, 60%, 70% or higher, including 75%, 80%, 85%, 90% and 95% or higher. Sequence similarity is calculated based on a reference sequence, which may be a subset of a larger sequence, such as a conserved motif, coding region, flanking region, etc. A reference sequence may be 18 nt long or longer, such as 30 nt long, and may extend to the complete sequence that is being compared. Algorithms for sequence analysis are known in the art, such as BLAST, described in Altschul et al. (1990), J. Mol. Biol. 215:403-10 (using default settings, i.e., parameters w=4 and T=17). Of particular interest in certain embodiments are nucleic acids of substantially the same length as nucleic acids mentioned above, where by substantially the same length is meant that any difference in length does not exceed about 20 number %, usually does not exceed about 10 number % and more usually does not exceed about 5 number %; and have sequence identity to any of these sequences of at 90% or greater, such as 95% or greater and including 99% or greater over the entire length of the nucleic acid. In some embodiments, the nucleic acids have a sequence that is substantially similar or identical to the above specific sequences. By substantially similar is meant that sequence identity is 60% or greater, such as 75% or greater and including 80, 85, 90, or even 95% or greater. Nucleic acids of interest also include nucleic acids that encode the proteins encoded by the above described nucleic acids, but differ in sequence from the above described nucleic acids due to the degeneracy of the genetic code.

[0081] Nucleic acids as described herein may be present in a vector. Various vectors (e.g., viral vectors, bacterial vectors, or vectors capable of replication in eukaryotic and prokaryotic hosts) can be used in accordance with the present invention. Numerous vectors which can replicate in eukaryotic and prokaryotic hosts are known in the art and are commercially available. In some instances, such vectors used in accordance with the invention are composed of a bacterial origin of replication and a eukaryotic promoter operably linked to a DNA of interest.

[0082] Viral vectors used in accordance with the invention may be composed of a viral particle derived from a naturally-occurring virus which has been genetically altered to render the virus replication-defective and to express a recombinant gene of interest in accordance with the invention. Once the virus delivers its genetic material to a cell, it does not generate additional infectious virus but does introduce exogenous recombinant genes into the cell, preferably into the genome of the cell. Numerous viral vectors are well known in the art, including, for example, retrovirus, adenovirus, adeno-associated virus, herpes simplex virus (HSV), cytomegalovirus (CMV), vaccinia and poliovirus vectors.

[0083] The nucleic acid of interest may be administered using a non-viral vector, for example, as a DNA- or RNA-liposome complex formulation. Such complexes comprise a mixture of lipids which bind to genetic material (DNA or RNA), providing a hydrophobic coat which allows the genetic material to be delivered into cells. Liposomes which can be used in accordance with the invention include DOPE (dioleyl phosphatidyl ethanol amine), CUDMEDA (N-(5-cholestrum-3-p- ol 3-urethanyl)-N',N'-dimethylethylene diamine). When the nucleic acid of interest is introduced using a liposome, in some instances one first determines in vitro the optimal values for the nucleic acid: lipid ratios and the absolute concentrations of nucleic acid and lipid as a function of cell death and transformation efficiency for the particular type of cell to be transformed. These values can then be used in or extrapolated for use in in vivo transformation. The in vitro determinations of these values can be readily carried out using techniques which are well known in the art. Other non-viral vectors may also be used in accordance with the present invention. These include chemical formulations of DNA or RNA coupled to a carrier molecule (e.g., an antibody or a receptor ligand) which facilitates delivery to host cells for the purpose of altering the biological properties of the host cells. By the term "chemical formulations" is meant modifications of nucleic acids to allow coupling of the nucleic acid compounds to a carrier molecule such as a protein or lipid, or derivative thereof. Exemplary protein carrier molecules include antibodies specific to the cells of a targeted secretory gland or receptor ligands, i.e., molecules capable of interacting with receptors associated with a cell of a targeted secretory gland.

[0084] DNA constructs may include a promoter to facilitate expression of the DNA of interest within a target cell, such as a strong, eukaryotic promoter. Exemplary eukaryotic promoters include promoters from cytomegalovirus (CMV), mouse mammary tumor virus (MMTV), Rous sarcoma virus (RSV), and adenovirus. More specifically, exemplary promoters include the promoter from the immediate early gene of human CMV (Boshart et al., Cell 41 :521 -530, 1985) and the promoter from the long terminal repeat (LTR) of RSV (Gorman et al., Proc. Natl. Acad. Sci. USA 79:6777-6781 , 1982).

[0085] Instead of administration of a polypeptide of interest, e.g., GAS6, such as described above, the level of systemic polypeptide active agent in the subject may be enhanced by stimulating endogenous production and / or release of the polypeptide in vivo. In some instances, an agent may be administered that stimulates production and / or release of GAS6 in vivo. Agents that stimulate production and / or release of GAS6 in vivo include, but are not limited to, proteins (e.g., antibodies), peptides, small molecules and nucleic acid molecules. In some instances, the agent that stimulates production and / or release of GAS6 in vivo is vitamin K (e.g., vitamin K1 ). (See, for example, al., Nutrients 13:4101 , 2021 ). In some instances, the agent that stimulates production and / or release of GAS6 in vivo is a pro-inflammatory molecule. (See, for example, Ekman, et al., Crit Care. 14:R158, 2010; Hurtado, et al., Crit Care. 14:1003, 2010). By pro-inflammatory molecule is meant a molecule that increases inflammation. Pro-inflammatory molecules, including pro-inflammatory cytokines, are well known in the art and described for example in Chen, et aL, Oncotarget. 9:7204-7218, 2018.

[0086] In some instances, the AXL signaling pathway activator is a peptidomimetic that exhibits the desired activity. A peptidomimetic is a protein-like or peptide-like chain that mimics a peptide. In some cases, the peptidomimetic is an AXL receptor agonist. Peptidomimetics of interest include, without limitation: peptidomimetics incorporating D-amino acids, that may, for example, improve enzymatic stability; peptidomimetics incorporating unnatural amino acids that may, for example, support bio-orthogonal chemistries (e.g., to enable selective cross-linking); p-turn mimetics; cyclic peptides that may, for example, improve biological activity; or any combination thereof.

[0087] In some instances, the AXL signaling pathway activator is a small molecule that exhibits the desired activity. In some cases, the small molecule is an AXL receptor agonist. Naturally occurring or synthetic small molecule compounds of interest include numerous chemical classes, such as organic molecules, e.g., small organic compounds having a molecular weight of more than 50 and less than about 2,500 Daltons. Candidate agents comprise functional groups for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical groups. The candidate agents may include cyclical carbon or heterocyclic structures and / or aromatic or polyaromatic structures substituted with one or more of the above functional groups. Candidate agents are also found among biomolecules including peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof. Such molecules may be identified, among other ways, by employing screening protocols. Methods of screening for small molecule AXL signaling pathway activators are known in the art and described for example in WO 2009 / 005813 A1 , the disclosure of which is herein incorporated by reference. Methods of screening for TAM receptor agonists are known in the art and described for example in Zhong, Fang, et al. "TYRO3 agonist as therapy for glomerular disease." JCI Insight 8.1 (2023), the disclosure of which is herein incorporated by reference.

[0088] In some instances, the AXL signaling pathway activator may be a small molecule that is not an AXL receptor agonist. In some cases, the small molecule is a small molecule that comprises one or more unpaired electrons. Small molecules comprising one or more unpaired electrons include, but are not limited to, reactive oxygen species (ROS). In some embodiments, the AXL signaling pathway activator may be an ROS, including, but not limited to, hydrogen peroxide. (See, for example, Konishi, et al., J Biol Chem. 279:28766-28770, 2004).

[0089] As used herein, “an effective amount of an AXL signaling pathway activator” refers to an amount of an AXL signaling pathway activator suitable to promote healing of a wound. In some cases, an effective amount of an AXL signaling pathway activator includes one or more unit doses of the AXL signaling pathway activator such as, e.g., one or more doses, two or more doses, three or more doses, four or more doses, five or more doses, six or more doses, seven or more doses, eight or more doses, nine or more doses, or ten or more doses. In some cases, an effective amount of an AXL signaling pathway activator composition includes a single dose, e.g., a single injection, of the AXL signaling pathway activator. The AXL signaling pathway activator composition may include any suitable amount of an AXL signaling pathway activator such as, e.g., an effective amount of an AXL signaling pathway activator. In some cases, the effective amount of an AXL signaling pathway activator does not delay wound closure or the wound closure rate.

[0090] In some cases, the AXL signaling pathway activator includes an effective amount of an AXL signaling pathway activator ranging from, e.g., 0.1 mg / ml to 2 mg / ml, 0.5 mg / ml to 2 mg / ml, 1 mg / ml to 2 mg / ml, 0.1 mg / ml to 1 mg / ml, 0.5 mg / ml to 1 mg / ml, 1 mg / ml to 5 mg / ml, or 5mg / ml to 10mg / ml. The effective amount of the AXL signaling pathway activator may be administered, e.g., after wound formation, over any suitable period of time including, e.g., one day or more, 2 days or more, 3 days or more, 4 days or more, 5 days or more, 7 days or more, 8 days or more, 9 days or more, 10 days or more, 11 days or more, 12 days or more, 13 days or more, 14 days or more, 21 days or more, 30 days or more, 60 days or more, or 90 days or more. The effective amount of the AXL signaling pathway activator may be administered, e.g., before wound formation, over any suitable period of time including, e.g., one day or more, or 2 days or more before wound formation.

[0091] In some embodiments, the AXL signaling pathway activator is administered as a pharmaceutically acceptable composition in which one or more AXL signaling pathway activators may be mixed with one or more carriers, thickeners, diluents, buffers, preservatives, surface active agents, excipients and the like. Pharmaceutical compositions may also include one or more additional active ingredients such as antimicrobial agents, anti-inflammatory agents, anesthetics, and the like in addition to the one or more AXL signaling pathway activators. In some cases, the AXL signaling pathway activator composition includes, e.g., a derivative of AXL signaling pathway activator. “Derivatives” include pharmaceutically acceptable salts and chemically modified agents.

[0092] The pharmaceutical compositions of the present invention may be administered by any route commonly used to administer pharmaceutical compositions. For example, administration may be done topically (including ophthalmically, vaginally, rectally, intranasally), orally, by inhalation, or parenterally, for example by intravenous drip or subcutaneous, intraperitoneal or intramuscular injection.

[0093] Pharmaceutical compositions formulated for topical administration may include ointments, lotions, creams, gels, drops, sprays, liquids, salves, sticks, soaps, aerosols, and powders. Any conventional pharmaceutical excipient, such as carriers, aqueous, powder or oily bases, thickeners and the like may be used. Ointments and creams may, for example, be formulated with an aqueous or oily base with the addition of suitable thickening and / or gelling agents. Lotions may be formulated with an aqueous or oily base and will, in general, also contain one or more emulsifying, dispersing, suspending, thickening or coloring agents. Powders may be formed with the aid of any suitable powder base. Drops may be formulated with an aqueous or non-aqueous base also comprising one or more dispersing, solubilizing or suspending agents. Aerosol sprays are conveniently delivered from pressurized packs, with the use of a suitable propellant.

[0094] The AXL signaling pathway activator composition may be stored at any suitable temperature. In some cases, the AXL signaling pathway activator composition is stored at temperatures ranging from 1eC to 30fiC, from 2sC to 27eC, or from 5BC to 25eC. The AXL signaling pathway activator composition may be stored in any suitable container, as described in detail below.

[0095] The AXL signaling pathway activator composition may be administered to a wound in a dermal location a subject. In some cases, the AXL signaling pathway activator composition is administered to a dermal location surrounding a wound in a subject. The administration can be by any suitable route, including, e.g., topical, intravenous, intradermal, subcutaneous, and intramuscular. In some cases, the administering comprises injecting the composition below a topical dermal location of the subject. The injecting may be performed with any suitable device such as, e.g., a needle. Other delivery means include coated microneedles, i.e., microneedles having a AXL signaling pathway activator composition deposited thereon, as well as microneedles that include internal reservoirs that are configured to receive a AXL signaling pathway activator composition therein and disperse the AXL signaling pathway activator composition therefrom. In some cases, the administering comprises delivering the composition to a topical dermal location. The delivering may be performed with any suitable device or composition such as, e.g., a transdermal patches, gels, creams, ointments, sprays, lotions, salves, sticks, soaps, powders, pessaries, aerosols, drops, solutions and any other convenient pharmaceutical forms.

[0096] The AXL signaling pathway activator composition may be administered at any suitable time. In some cases, the AXL signaling pathway activator composition is administered to a wound immediately after formation of the wound in a subject. In some cases, the AXL signaling pathway activator composition is administered to a wound any suitable amount of time before formation of the wound such as, e.g., 1 minute, 2 minutes, 5 minutes, 10 minutes, 30 minutes, an hour, 1 day, or 2 days before formation of the wound. In some cases, the AXL signaling pathway activator composition is administered to a wound after any suitable amount of time after formation of the wound such as, e.g., 1 minute, 2 minutes, 5 minutes, 10 minutes, 30 minutes, or an hour after formation of the wound. In some cases, the AXL signaling pathway activator composition is administered to a closed wound after any suitable amount of time after formation of the wound such as, e.g., 7 days or longer, 14 days or longer, 30 days or longer, 60 days or longer, 120 days or longer, 150 days or longer, 180 days or longer, 210 days or longer, 240 days or longer, 270 days or longer, 300 days or longer, 330 days or longer, 360 days or longer, 1 year or longer, 1.5 years or longer, 2 years or longer, 5 years or longer, etc.

[0097] In certain embodiments, the methods as provided herein promote healing of a wound. In some cases, the healing includes a regenerative response from one or more cells. In some cases, the methods do not compromise healing of a wound, e.g., wound closure and repair. For example, in some cases, the methods do not delay wound closure or the wound closure rate. In some cases, the healing of the wound is completed in an amount of time substantially equal to an amount of time for healing of a wound not treated with the AXL signaling pathway activator composition. In some cases, the healing of the wound is completed in an amount of time that is less than an amount of time for healing of a wound not treated in accordance with the invention. In certain embodiments, the methods reduce, prevent, or reverse scarring during healing of a wound in a subject, as described in detail below.

[0098] In some cases, the healing of the wound includes regeneration of dermal appendages. In some cases, the dermal appendages include hair follicles, sweat glands, and sebaceous glands. In some cases, the healing of the wound produces a healed wound with reduced levels of collagen hyperproliferation compared to levels of collagen hyperproliferation in a healed wound not treated with the AXL signaling pathway activator composition. In some cases, the healing of the wound produces a healed wound comprising improved connective tissue architecture compared to the connective tissue architecture in a healed wound not treated with the AXL signaling pathway activator composition. In certain embodiments, the healing includes recovery or regrowth of one or more of dermal appendages, ultrastructure (i.e., matrix structure), and mechanical strength (e.g., wound breaking strength) that is, e.g., comparable to that of normal skin or unwounded skin.

[0099] In certain embodiments, the methods further include forming a wound in a dermal location of a subject. In some cases, the wound is formed to perform a procedure, e.g., a surgical procedure. In some cases, the wound is formed to improve tissue quality. For example, the methods may include forming microscopic injuries to induce tissue regeneration. In some cases, the wound is formed to disrupt an outer dermal layer, e.g., stratum corneum, to increase penetration and absorption of one or more substances or compositions, e.g., a therapeutic composition, through the skin of a subject. In some cases, the methods include forming one or more wounds at a plurality of dermal locations. In some cases, the methods include forming one or more wounds across a dermal location. The nature and size of the wound may vary. In certain embodiments, the wound is a microscopic wound. The microscopic wound may be formed by any suitable means as described in detail below such as, e.g., a laser, microneedle, etc. In certain embodiments, the wound is a partially healed wound.

[0100] The wound may be formed by any suitable means, e.g., mechanical, physical or chemical injury of the skin. In some cases, the wound results from non-physiological processes, e.g., a surgical wound or a wound resulting from physical injury, abrasions, lacerations, thermal injuries (e.g., a burn or a wound arising from a cryo-based treatment). In some cases, the wound is formed by the application of one or more of, e.g., ultrasound, radio frequency (RF), laser (e.g., fraxel), ultraviolet energy, infrared energy, or mechanical disruption. In some cases, the wound is formed by, e.g., microdermabrasion (e.g., with an adapted skin preparation pad, sandpaper), microneedling, tape-stripping, pan-scrubber, exfoliating scrub, compress rubbing, non-ablative lasers at a low-energy delivery. Additional mechanical treatments include, e.g., curettage or dermoabrasion (e.g., with an adapted sandpaper or micro-needling (or micro-perforation)). In certain aspects, wounding is accomplished using chemical treatments (e.g., a caustic agent, etc.), or mechanical or electromagnetic or physical treatments including but not limited to dermabrasion (DA), particle-mediated dermabrasion (PMDA), microdermabrasion, microneedles, laser (e.g., a laser that delivers ablative, non-ablative, fractional, non-fractional, superficial, or deep treatment, and / or that is CO2-based, or erbium-YAG-based, erbium-glass based (e.g. Sciton Laser), neodymiurmyttrium aluminum garnet (Nd:YAG) laser, etc.), a low-level (low- intensity) laser therapy treatment (e.g., HairMax® Laser comb), laser abrasion, irradiation, radio frequency (RF) ablation, dermatome planing (e.g. dermaplaning), a coring needle, a puncture device, a punch tool or other surgical tool, suction tool or instrument, electrology, electromagnetic disruption, electroporation, sonoporation, low voltage electric current, intense pulsed light, or surgical treatments (e.g., skin graft, hair transplantation, strip harvesting, scalp reduction, hair transplant, follicular unit extraction (FUE), robotic FUE, etc.), or supersonically accelerated saline. In some cases, the wound is formed by a tissue disrupting device, as described in detail below.

[0101] Embodiments of the methods of the present invention can be practiced on any suitable subject. A subject of the present invention may be a “mammal” or “mammalian”, where these terms are used broadly to describe organisms which are within the class mammalia, including the orders carnivore {e.g., dogs and cats), rodentia e.g., mice, guinea pigs, and rats), and primates {e.g., humans, chimpanzees, and monkeys). In some instances, the subjects are humans. The methods may be applied to human subjects of both genders and at any stage of development (i.e., neonates, infant, juvenile, adolescent, adult), where in certain embodiments the human subject is a juvenile, adolescent or adult. While the present invention may be applied to samples from a human subject, it is to be understood that the methods may also be carried-out on samples from other animal subjects (that is, in “non-human subjects”) such as, but not limited to, birds, mice, rats, dogs, cats, livestock and horses.

[0102] SCAR REDUCTION

[0103] In certain embodiments, the methods provided herein reduce, reverse, or prevent scarring during healing of a wound in a subject. In certain embodiments, the methods include forming a wound in a dermal location of a subject, e.g., according to any of the embodiments described herein, and administering an effective amount of an AXL signaling pathway activator composition to the wound to promote healing of the wound, e.g., according to any of the embodiments described herein. In certain embodiments, the methods include forming a wound in a dermal location of a subject, e.g., according to any of the embodiments described herein, and administering an effective amount of an AXL signaling pathway activator composition to the wound to prevent scarring of the wound, e.g., according to any of the embodiments described herein.

[0104] The level or amount of scarring may be assessed and measured according to any convenient metric. For example, the Vancouver Scar Scale (VSS), Visual Analog Scale (VAS), Patient and Observer Scar Assessment Scale (POSAS), Manchester Scar Scale (MSS), the Stony Brook Scar Evaluation Scale (SBSES), or combinations thereof may be used to assess and measure scarring. Scarring-related metrics that may be assessed and / or measured include, but are not limited to, color, thickness, relief, pliability, surface area, pain, pruritus, functional outcomes, psychosocial outcomes, clinician assessment and / or measurement, patient assessment and / or measurement, and combinations thereof. The levels of scarring, e.g., in a wound treated with an AXL signaling pathway activator composition during healing or a healed wound treated with an AXL signaling pathway activator composition, may be assessed relative to a control, e.g., a wound or healed wound not treated with an AXL signaling pathway activator composition. In some cases, the level of scarring is assessed by measuring a physical property of a healed wound such as, e.g., extent of re-epithelialization, scar thickness, etc. In some cases, the level of scarring is assessed by detecting the presence of or quantitating the amount of one or more dermal appendages including, e.g., hair follicles, sweat glands, and sebaceous glands, in a dermal location. In some cases, the level of scarring is assessed by detecting and / or characterizing the formation of connective tissue or an ECM matrix in a dermal location. In certain embodiments, the level of scarring is assessed by detecting and / or quantitating the amount of cells, e.g., types or subpopulations of cells, in a dermal location. In some cases, the level of scarring is assessed by measuring and / or quantitating the expression and / or activity or one or more scar-related genes and / or scar-related gene products. In some cases, levels of scarring are assessed by one or more of the following: visual examination, histology, immunohistochemical analysis, immunofluorescence, and machine learning. In some cases, the level of scarring is assessed with a machine learning algorithm for quantitatively assessing connective tissue and fibrosis based on histological stains. In some embodiments, evaluated metrics include, e.g., ECM fiber length and width, packing and alignment of groups of ECM fibers, and ECM fiber branching. Various scar assessment scales are provided, e.g., in PCT Application No. WO 2014 / 040074, the disclosure of which is incorporated herein by reference in its entirety. According to some embodiments, the methods reduce scarring compared to a control as measured by visual analog scale (VAS) score, color matching (CM), matte / shiny (M / S) assessment, contour (C) assessment, distortion (D) assessment, texture (T) assessment, or a combination thereof. While the magnitude of scarring reduction may vary, in some instances the magnitude ranges from 10% to 98%, such as, 10% to 95%, 20% to 95%, 30% to 95%, 40% to 95%, 50% to 95%, 60% to 95%, 70% to 95%, 80% to 95%, or 90% to 95%.

[0105] The levels of reduction of scarring during the healing process may vary. In certain embodiments, the methods are effective to reduce the occurrence, severity, or both of scars. In some cases, the method produces a healed wound with reduced levels of scarring compared to levels of scarring in a healed wound not treated with the AXL signaling pathway activator composition. In certain embodiments, the method produces a scarless healed wound. In some cases, the methods produce a healed wound comprising improved connective tissue architecture compared to the connective tissue architecture in a healed wound not treated with the AXL signaling pathway activator composition. In some cases, the methods produce a healed wound with reduced levels of collagen hyperproliferation compared to levels of collagen hyperproliferation in a healed wound not treated with the AXL signaling pathway activator composition. In some embodiments, the methods improve the alignment of collagen fibers in the wound. In some embodiments, the methods reduce collagen formation in the wound. In some cases, the methods produce a healed wound with increased growth of dermal appendages. In certain embodiments, the methods reduce the wound size. In some case, a dermal location having a healed wound treated with a AXL signaling pathway activator composition according to the methods provided herein is indistinguishable in appearance (e.g., pigmentation, texture) from normal skin or unwounded skin. In some cases, a dermal location having a healed wound treated with an AXL signaling pathway activator composition according to the methods provided herein has physical properties (e.g., tensile strength) indistinguishable from normal skin or unwounded skin. In some cases, a dermal location having a healed wound treated with an AXL signaling pathway activator composition according to the methods provided herein has growth and generation of dermal appendages that are indistinguishable from normal skin or unwounded skin. In some cases, a dermal location having a healed wound treated with an AXL signaling pathway activator composition according to the methods provided herein has a connective tissue architecture, e.g., ECM matrix, that is indistinguishable from normal skin or unwounded skin. In certain embodiments, the methods do not impair normal wound healing or delay the wound closure rate compared to a control. In certain embodiments, the methods increase wound healing, e.g., the wound closure rate compared to a control. In some cases, one or more of the produced effects of the methods as described herein indicate a reduction of scarring or the prevention of scarring.

[0106] According to some embodiments, the methods decrease scar area compared to a control. According to some embodiments, the methods decrease scar area compared to a control by 1% or more, 2% or more, 3% or more, 4% or more, 5% or more, 6% or more, 7% or more, 8% or more, 9% or more, 10% or more, 11% or more, 12% or more, 13% or more, 14% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more. According to some embodiments, the methods decrease scar area compared to a control within one day or more, 2 days or more, 3 days or more, 4 days or more, 5 days or more, 7 days or more, 8 days or more, 9 days or more, 10 days or more, 11 days or more, 12 days or more, 13 days or more, 14 days or more, 21 days or more, 30 days or more, 60 days or more, or 90 days or more of the administration, e.g., of an AXL signaling pathway activator composition.

[0107] According to some embodiments, the methods decrease scar thickness compared to a control. According to some embodiments, the methods decrease scar thickness compared to a control by 1% or more, 2% or more, 3% or more, 4% or more, 5% or more, 6% or more, 7% or more, 8% or more, 9% or more, 10% or more, 11% or more, 12% or more, 13% or more, 14% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more. According to some embodiments, the methods decrease scar thickness compared to a control within one day or more, 2 days or more, 3 days or more, 4 days or more, 5 days or more, 7 days or more, 8 days or more, 9 days or more, 10 days or more, 11 days or more, 12 days or more, 13 days or more, 14 days or more, 21 days or more, 30 days or more, 60 days or more, or 90 days or more of the administration, e.g., of an AXL signaling pathway activator composition.

[0108] According to some embodiments, the methods decrease fibrosis at a dermal location compared to a control. In some cases, the methods decrease fibrosis at a dermal location compared to a control by 1 % or more, 2% or more, 3% or more, 4% or more, 5% or more, 6% or more, 7% or more, 8% or more, 9% or more, 10% or more, 11% or more, 12% or more, 13% or more, 14% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more. According to some embodiments, the methods decrease fibrosis at a dermal location compared to a control within 1 day or more, 2 days or more, 3 days or more, 4 days or more, 5 days or more, 7 days or more, 8 days or more, 9 days or more, 10 days or more, 11 days or more, 12 days or more, 13 days or more, 14 days or more, 21 days or more, 30 days or more, 60 days or more, or 90 days or more of the administration, e.g., of an AXL signaling pathway activator composition.

[0109] According to some embodiments, the methods produce a wound or healed wound with increased tensile strength, e.g., as measured by wound breaking force and Young’s modulus, compared to a control. According to some embodiments, the methods increase tensile strength compared to a control within one day or more, 2 days or more, 3 days or more, 4 days or more, 5 days or more, 7 days or more, 8 days or more, 9 days or more, 10 days or more, 11 days or more, 12 days or more, 13 days or more, 14 days or more, 21 days or more, 30 days or more, 60 days or more, or 90 days or more of the administration. According to some embodiments, the methods increase tensile strength compared to a control by 1% or more, 2% or more, 3% or more, 4% or more, 5% or more, 6% or more, 7% or more, 8% or more, 9% or more, 10% or more, 11% or more, 12% or more, 13% or more, 14% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more.

[0110] According to some embodiments, the methods produce detectible levels of dermal appendages such as hair follicles, sweat glands, and / or sebaceous glands, or any combination thereof, at a dermal location compared to a control. According to some embodiments, the methods increase the number of dermal appendages such as hair follicles, sweat glands, and / or sebaceous glands, or any combination thereof, at a dermal location compared to a control. In some cases, the methods increase the number of dermal appendages such as hair follicles, sweat glands, and / or sebaceous glands, or any combination thereof, at a dermal location compared to a control by 1 % or more, 2% or more, 3% or more, 4% or more, 5% or more, 6% or more, 7% or more, 8% or more, 9% or more, 10% or more, 11% or more, 12% or more, 13% or more, 14% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more. According to some embodiments, the methods produce detectible levels of or increase the number of dermal appendages such as hair follicles, sweat glands, and / or sebaceous glands, or any combination thereof, at a dermal location compared to a control within 1 day or more, 2 days or more, 3 days or more, 4 days or more, 5 days or more, 7 days or more, 8 days or more, 9 days or more, 10 days or more, 11 days or more, 12 days or more, 13 days or more, 14 days or more, 21 days or more, 30 days or more, 60 days or more, or 90 days or more of the administration.

[0111] In some embodiments, the methods may modulate the expression and / or activity of scar- related genes or the production of scar-related gene products. In some cases, the level of scarring may be assessed by measuring the expression and / or activity of scar-related genes. In some cases, the level of scarring may be assessed by measuring the amount and / or activity of scar- related gene products. According to another embodiment, an effective amount of an AXL signaling pathway activator composition is effective to modulate messenger RNA (mRNA) levels expressed from scar-related genes. According to another embodiment, an effective amount of an AXL signaling pathway activator composition is effective to modulate the level of scar-related gene product expressed from the scar related gene. According to some embodiments, the scar-related gene and / or product is transforming growth factor-|31 (TGF-pi), tumor necrosis factor-a (TNF-a), collagen, interleukin-6 (IL-6), chemokine (CC motif) Ligand 2 (CCL2) (or monocyte chemotactic protein-1 (MCP-1 )), chemokine (CC motif) receptor 2 (CCR2), EGF-like module-containing mucinlike hormone receptor-like 1 (EMR1 ), CD26, YAP, fibronectin, or one or more of the sma I mad- related proteins (SMAD). According to some embodiments, the methods modulate, e.g., decrease, the expression and / activity of one or more of collagen type 1 , CD26, and YAP in a wound, e.g., in cells present in a wound, compared to a control. According to some embodiments, the methods modulate, e.g., increase, the expression and / activity of fibronectin in a wound, e.g., in cells present in a wound, compared to a control. According to some embodiments, the methods produce detectible levels of markers of hair follicle and sebaceous or sweat gland identity such as, e.g., cytokeratin 14 and / or cytokeratin 19, respectively, at a dermal location compared to a control. In some cases, the methods increase the levels of markers of hair follicle and sebaceous or sweat gland identity, e.g., cytokeratin 14 and / or cytokeratin 19, at a dermal location compared to a control.

[0112] In certain embodiments, the methods decrease or increase the expression and / activity of one or more scar-related genes or scar-related gene products by 1% or more, 2% or more, 3% or more, 4% or more, 5% or more, 6% or more, 7% or more, 8% or more, 9% or more, 10% or more, 11 % or more, 12% or more, 13% or more, 14% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more. According to some embodiments, the methods decrease or increase the expression and / or activity of one or more scar-related genes or scar-related gene products compared to a control within one day or more, 2 days or more, 3 days or more, 4 days or more, 5 days or more, 7 days or more, 8 days or more, 9 days or more, 10 days or more, 11 days or more, 12 days or more, 13 days or more, 14 days or more, 21 days or more, 30 days or more, 60 days or more, or 90 days or more of the administration, e.g., of an AXL signaling pathway activator.

[0113] In some embodiments, the methods may modulate regenerative OMF-like gene expression. In some cases, wound healing may be assessed by measuring OMF-like gene expression. According to another embodiment, an effective amount of an AXL signaling pathway activator composition is effective to induce regenerative OMF-like gene expression. According to some embodiments, OMF-like gene expression comprises down-regulation of focal adhesion kinase (FAK) expression, down-regulation of collagen type 1 expression, down-regulation of ectonucleotide pyrophosphatase / phosphodiesterase 2 (ENPP2) expression, and up-regulation of lumican (LUM) expression. According to some embodiments, the methods modulate OMF-like gene expression in a wound, e.g., in cells present in a wound, compared to a control. In some cases, the methods modulate OMF-like gene expression at a dermal location compared to a control.

[0114] In certain embodiments, the methods decrease or increase regenerative OMF-like gene expression by 1% or more, 2% or more, 3% or more, 4% or more, 5% or more, 6% or more, 7% or more, 8% or more, 9% or more, 10% or more, 11% or more, 12% or more, 13% or more, 14% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more. According to some embodiments, the methods decrease or increase regenerative OMF-like gene expression compared to a control within one day or more, 2 days or more, 3 days or more, 4 days or more, 5 days or more, 7 days or more, 8 days or more, 9 days or more, 10 days or more, 11 days or more, 12 days or more, 13 days or more, 14 days or more, 21 days or more, 30 days or more, 60 days or more, or 90 days or more of the administration, e.g., of an AXL signaling pathway activator.

[0115] In some instances, the methods reverse existing scarring in healed wounds / existing scars. In such embodiments, one or more metrics of scars, e.g., as described above, may be improved as described above. The existing scar may be scar that is has existed for a period of time, e.g., 7 days or longer, 14 days or longer, 30 days or longer, 60 days or longer, 120 days or longer, 150 days or longer, 180 days or longer, 210 days or longer, 240 days or longer, 270 days or longer, 300 days or longer, 330 days or longer, 360 days or longer, 1 year or longer, 1 .5 years or longer, 2 years or longer, 5 years or longer, etc.

[0116] COMBINATION THERAPY

[0117] For use in the subject methods, the AXL signaling pathway activator(s), such as described above, may be administered in combination with other pharmaceutically active agents, including other agents that treat the underlying condition or a symptom of the condition, e.g., scarring. "In combination with" as used herein refers to uses where, for example, the first compound is administered during the entire course of administration of the second compound; where the first compound is administered for a period of time that is overlapping with the administration of the second compound, e.g. where administration of the first compound begins before the administration of the second compound and the administration of the first compound ends before the administration of the second compound ends; where the administration of the second compound begins before the administration of the first compound and the administration of the second compound ends before the administration of the first compound ends; where the administration of the first compound begins before administration of the second compound begins and the administration of the second compound ends before the administration of the first compound ends; where the administration of the second compound begins before administration of the first compound begins and the administration of the first compound ends before the administration of the second compound ends. As such, "in combination" can also refer to regimen involving administration of two or more compounds. "In combination with" as used herein also refers to administration of two or more compounds which may be administered in the same or different formulations, by the same of different routes, and in the same or different dosage form type.

[0118] Examples of other agents for use in combination therapy in embodiments of methods of the invention include, but are not limited to, YAP inhibitors. In some instances, the YAP inhibitor is a small molecule agent that exhibits the desired activity, e.g., inhibiting YAP expression and / or activity. Naturally occurring or synthetic small molecule compounds of interest include numerous chemical classes, such as organic molecules, e.g., small organic compounds having a molecular weight of more than 50 and less than about 2,500 Daltons. Candidate agents comprise functional groups for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical groups. The candidate agents may include cyclical carbon or heterocyclic structures and / or aromatic or polyaromatic structures substituted with one or more of the above functional groups. Candidate agents are also found among biomolecules including peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof. Such molecules may be identified, among other ways, by employing the screening protocols.

[0119] In some cases, the YAP inhibitor is a photosensitizing agent. In some cases, the Yap inhibitor is a benzoporphyrin derivative (BPD). The benzoporphyrin derivative may be any convenient benzoporphyrin derivative such as, e.g., those described in U.S. Patent No. 5,880,145; U.S. Patent No. 6,878,253; U.S. Patent No. 10,272,261 ; and U.S. Application No. 2009 / 0304803, the disclosures of which are incorporated herein by reference in their entireties. In some cases, the benzoporphyrin derivative is a photosensitizing agent. In some cases, the YAP inhibitor is verteporfin (benzoporphyrin derivative monoacid ring A, BPD-MA; tradename: Visudyne®).

[0120] Further details regarding YAP inhibitors and methods of using the same are provided in United States Patent Application Serial No. 17 / 626,699 published as US 2022-0313658 A1 ; the disclosure of which is herein incorporated by reference.

[0121] In some instances, aspects of the methods may include administering an effective amount of an AXL signaling pathway activator in combination with a Piezo inhibitor. In certain embodiments, the Piezo inhibitor includes a Piezol and / or Piezo2 inhibitor. In some cases, the Piezo inhibitor is a Piezol inhibitor. In some cases, the Piezo inhibitor is a Piezo2 inhibitor. In some cases, both a Piezol inhibitor and Piezo2 inhibitor are administered to a subject. In some cases, the method consists essentially of administering a Piezo inhibitor. As used herein, a “Piezo inhibitor” refers to a molecule that may inhibit Piezo protein function and signaling. In some cases, the Piezo inhibitor inhibits cellular mechanical signaling. In some cases, the Piezo inhibitor reduces or inhibits Piezo protein expression (DNA or RNA expression) or activity (e.g., nuclear translocation). In some cases, the Piezo inhibitor reduces or inhibits the interaction of a Piezo protein with other signaling molecules. In certain embodiments, administering the Piezo inhibitor reduces mechanical activation of one or more cells, e.g., adipocytes, in a wound, wherein, e.g., the level of mechanical activation of the one or more cells, e.g., adipocytes, in a wound is reduced compared to a suitable control. Further details regarding Piezo inhibitors and methods of using the same are provided in PCT Application Serial No. PCT / US2023 / 018997 published as WO 2023 / 211728; the disclosure of which is herein incorporated by reference.

[0122] In some instances, aspects of the methods may include administering an effective amount of an AXL signaling pathway activator in combination with a complement inhibitor. As used herein, a “complement inhibitor” refers to any of a number of compound classes such as peptides, polypeptides, antibodies, small molecules, and nucleic acids (e.g., aptamers, RNAi agents such as short interfering RNAs) that may inhibit at least the classical pathway of complement activation, at least the alternative pathway of complement activation, or both. In some cases, the complement inhibitor is a Factor H polypeptide or nucleic acid coding sequence therefor. Further details regarding complement inhibitors and methods of using the same are provided in PCT Application Serial No: PCT / US2023 / 020698 published as WO 2023 / 215294; the disclosure of which is herein incorporated by reference.

[0123] In the context of a combination therapy, combination therapy compounds may be administered by the same or different route of administration (e.g., intrapulmonary, oral, enteral, etc.) that the AXL signaling pathway activator is administered. The AXL signaling pathway activator and the combination therapy compound may be administered in any combination of administration routes described herein. In some embodiments, the AXL signaling pathway activator and the combination therapy compound are administered via the same administration route (e.g., the AXL signaling pathway activator and the combination therapy compound are both injected below a topical dermal location in a subject). In some embodiments, the AXL signaling pathway activator and the combination therapy compound are administered via different administration routes (e.g., the AXL signaling pathway activator is injected below a topical dermal location in a subject and the combination therapy compound is administered orally, the AXL signaling pathway activator is injected below a topical dermal location in a subject and the combination therapy compound is administered intravenously, etc.). The AXL signaling pathway activator and the combination therapy compound may be administered according to a variety of protocols. For example, the AXL signaling pathway activator and the combination therapy compound may be administered at the same time or sequentially, where when the two agents are administered sequentially, the AXL signaling pathway activator may be administered before or after the combination therapy compound. As such, the AXL signaling pathway activator may be administered before, substantially at the same time, at the same time, or after the combination therapy compound.

[0124] Where one agent is administered before the other agent, the first agent may be administered before from 14 days before, up to 0.5 days before, the second agent. In some instances, the AXL signaling pathway activator is administered before the combination therapy compound, e.g., where the AXL signaling pathway activator is administered 0.5 to 14 days before, such as 1 to 7 days before, 0.5 to 7 days before, 0.5 to 5 days before, 0.5 to 1 .5 days before, including 14 days before, 7 days before, and 1 day before of the combination therapy compound. In some instances, the combination therapy compound is administered before the AXL signaling pathway activator, e.g., where the combination therapy compound is administered 0.5 to 14 days before, such as 1 to 7 days before, 0.5 to 7 days before, 0.5 to 5 days before, 0.5 to 1.5 days before, including 14 days before, 7 days before, and 1 day before the AXL signaling pathway activator.

[0125] In some embodiments, the AXL signaling pathway activator is administered at substantially the same time as the combination therapy compound, e.g., where the AXL signaling pathway activator is administered within 0.5 days (i.e. , within 12 hours), including within 10 hours, within 8 hours, within 6 hours, within 4 hours, within 2 hours, within 1 hour (i.e., within 60 minutes), within 30 minutes, within 15 minutes, within 10 minutes, within 5 minutes and within 1 minute of the combination therapy compound. For example, co-administration of the AXL signaling pathway activator and the combination therapy compound may be injected below a topical dermal location in a subject on the same day at the same time. Where the two agents are administered simultaneously, they may be administered as separate formulations or as a co-formulation, as desired.

[0126] KITS

[0127] Aspects of the present disclosure also include kits. The kits are suitable for practicing embodiments of the methods described herein. The kits may include, e.g., an amount of an AXL signaling pathway activator composition. In some instances, the kits may further include a tissue disrupting device. In some cases, the kits are suitable for practicing embodiments of the methods for promoting wound healing. In some cases, the kits are suitable for practicing embodiments of the methods for ameliorating scar formation.

[0128] The AXL signaling pathway activator composition may be present in any suitable amount. In some cases, the kit includes an effective amount of an AXL signaling pathway activator composition, e.g., according to the embodiments described above. The AXL signaling pathway activator composition may be present in any suitable container that is compatible with the AXL signaling pathway activator composition. By “compatible” it is meant that the container is substantially inert (e.g., does not significantly react with) the liquid and / or reagent(s) of the AXL signaling pathway activator in contact with a surface of the container. Containers of interest may vary and may include but are not limited to a test tube, centrifuge tube, culture tube, falcon tube, microtube, Eppendorf tube, specimen collection container, specimen transport container, and syringe.

[0129] The container for holding the AXL signaling pathway activator composition may be configured to hold any suitable volume of the AXL signaling pathway activator composition. In some cases, the size of the container may depend on the volume of AXL signaling pathway activator composition to be held in the container. In certain embodiments, the container may be configured to hold an amount of AXL signaling pathway activator composition ranging from 0.1 mg to 1000 mg, such as from 0.1 mg to 900 mg, such as from 0.1 mg to 800 mg, such as from 0.1 mg to 700 mg, such as from 0.1 mg to 600 mg, such as from 0.1 mg to 500 mg, such as from 0.1 mg to 400 mg, or 0.1 mg to 300 mg, or 0.1 mg to 200 mg, or 0.1 mg to 100 mg, 0.1 mg to 90 mg, or 0.1 mg to 80 mg, or 0.1 mg to 70 mg, or 0.1 mg to 60 mg, or 0.1 mg to 50 mg, or 0.1 mg to 40 mg, or 0.1 mg to 30 mg, or 0.1 mg to 25 mg, or 0.1 mg to 20 mg, or 0.1 mg to 15 mg, or 0.1 mg to 10 mg, or 0.1 mg to 5 mg, or 0.1 mg to 1 mg, or 0.1 mg to 0.5 mg. In certain embodiments, the container is configured to hold an amount of AXL signaling pathway activator composition ranging from 0.1 g to 10 g, or 0.1 g to 5 g, or 0.1 g to 1 g, or 0.1 g to 0.5 g. In certain instances, the container is configured to hold a volume (e.g., a volume of a liquid AXL signaling pathway activator composition) ranging from 0.1 ml to 200 ml. For instance, the container may be configured to hold a volume (e.g., a volume of a liquid) ranging from 0.1 ml to 1000 ml, such as from 0.1 ml to 900 ml, or 0.1 ml to 800 ml, or 0.1 ml to 700 ml, or 0.1 ml to 600 ml, or 0.1 ml to 500 ml, or 0.1 ml to 400 ml, or 0.1 ml to 300 ml, or 0.1 ml to 200 ml, or 0.1 ml to 100 ml, or 0.1 ml to 50 ml, or 0.1 ml to 25 ml, or 0.1 ml to 10 ml, or 0.1 ml to 5 ml, or 0.1 ml to 1 ml, or 0.1 ml to 0.5 ml. In certain instances, the container is configured to hold a volume (e.g., a volume of a liquid AXL signaling pathway activator composition) ranging from 0.1 ml to 200 ml.

[0130] The shape of the container may also vary. In certain cases, the container may be configured in a shape that is compatible with the assay and / or the method or other devices used to perform the assay. For instance, the container may be configured in a shape of typical laboratory equipment used to perform the assay or in a shape that is compatible with other devices used to perform the assay. In some embodiments, the liquid container may be a vial or a test tube. In certain cases, the liquid container is a vial. In certain cases, the liquid container is a test tube.

[0131] As described above, embodiments of the container can be compatible with the AXL signaling pathway activator composition in contact with the reagent device. Examples of suitable materials for the containers include, but are not limited to, glass and plastic. For example, the container may be composed of glass, such as, but not limited to, silicate glass, borosilicate glass, sodium borosilicate glass (e.g., PYREX™), fused quartz glass, fused silica glass, and the like. Other examples of suitable materials for the containers include plastics, such as, but not limited to, polypropylene, polymethylpentene, polytetrafluoroethylene (PTFE), perfluoroethers (PFE), fluorinated ethylene propylene (FEP), perfluoroalkoxy alkanes (PFA), polyethylene terephthalate (PET), polyethylene (PE), polyetheretherketone (PEEK), and the like.

[0132] In some embodiments, the container may be sealed. That is, the container may include a seal that substantially prevents the contents of the container from exiting the container. The seal of the container may also substantially prevent other substances from entering the container. For example, the seal may be a water-tight seal that substantially prevents liquids from entering or exiting the container, or may be an air-tight seal that substantially prevents gases from entering or exiting the container. In some instances, the seal is a removable or breakable seal, such that the contents of the container may be exposed to the surrounding environment when so desired, e.g., if it is desired to remove a portion of the contents of the container. In some instances, the seal is made of a resilient material to provide a barrier (e.g., a water-tight and / or air-tight seal) for retaining a sample in the container. Particular types of seals include, but are not limited to, films, such as polymer films, caps, etc., depending on the type of container. Suitable materials for the seal include, for example, rubber or polymer seals, such as, but not limited to, silicone rubber, natural rubber, styrene butadiene rubber, ethylene-propylene copolymers, polychloroprene, polyacrylate, polybutadiene, polyurethane, styrene butadiene, and the like, and combinations thereof. For example, in certain embodiments, the seal is a septum pierceable by a needle, syringe, or cannula. The seal may also provide convenient access to a sample in the container, as well as a protective barrier that overlies the opening of the container. In some instances, the seal is a removable seal, such as a threaded or snap-on cap or other suitable sealing element that can be applied to the opening of the container. For instance, a threaded cap can be screwed over the opening before or after a sample has been added to the container.

[0133] As used herein, a “tissue disrupting device” is a device that causes cellular damage or injury. The tissue disrupting device may be configured to form a wound in a dermal location of a subject, e.g., according to any of the methods described herein. In some cases, the device may apply to a dermal location one or more of, e.g., ultrasound, radio frequency (RF), laser, ultraviolet energy, infrared energy, or mechanical disruption. Suitable tissue disrupting devices include, but are not limited to, surgical instruments (e.g., scalpels, lancets, etc.), needles, microneedles (e.g., a Dermaroller®), lasers, etc. In certain embodiments, the devices include 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 skin-penetrating component(s) (e.g., a needle, a drill, a microauger, a tube comprising cutting teeth, a spoon bit, a wire, a fiber, a blade, a high-pressure fluid jet, a cryoprobe, a cryoneedle, an ultrasound needle, a multi-hole needle including one or more chemical agents, a microelectrode, and / or a vacuum, or any other component described herein) that can penetrate the skin simultaneously. In some cases, the tissue disrupting device is configured to administer or deliver an effective amount of an AXL signaling pathway activator composition to a wound, e.g., a wound formed by the tissue disrupting device. In certain embodiments, the tissue disrupting device is configured to administer, e.g., inject, the AXL signaling pathway activator composition to a topical dermal location or below a topical dermal location of the subject. The administration may be performed with any suitable mechanism or medium according to any of the embodiments described above such as, e.g., a needle, microneedle, gel, etc. In some cases, one or more portions of the tissue disrupting device contains an effective amount of an AXL signaling pathway activator composition. In some cases, the tissue disrupting device includes one or more microneedles. In some cases, the tissue disrupting device includes an array of microneedles. In certain embodiments, the tissue disrupting device is a microneedling device including, e.g., the Dermaroller® or Dermapen®. In some cases, the tissue disrupting device is a laser, e.g., for practicing fractional laser resurfacing.

[0134] UTILITY The subject methods find use in applications involving wound healing including, e.g., clinical and research applications. In certain embodiments, the methods find use in postnatal wound healing or wound healing in adults. The methods also find use in reversing existing scarring, e.g., of healed wounds / existing scars. The methods may find use in any applications where a wound is intentionally, e.g., via surgery, or unintentionally created. Methods of embodiments of the invention also find use in ameliorating, e.g., reducing or inhibiting, organ fibrosis, e.g., liver fibrosis, heart fibrosis, inflammatory bowel fibrosis, muscle fibrosis, kidney fibrosis, etc., in a subject.

[0135] In certain embodiments, the subject methods find use in applications where it is desirable to reduce or prevent scarring, or reverse existing scarring. The subject methods may be applied to the treatment of all types of skin, including wound zones and eyes, where scarring is a possibility. In certain embodiments, the methods may be used to treat or prevent scarring of human skin resulting from burns, scalds, grazes, abrasions, cuts and other incisional wounds, surgery and pathological skin scarring conditions such as, e.g., Dupuytren's disease, and the conditions of fibrotic dermal scarring, hypertrophic scarring, keloid scarring and corneal and other ocular tissue scarring.

[0136] The following example(s) is / are offered by way of illustration and not by way of limitation.

[0137] EXPERIMENTAL

[0138] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.

[0139] General methods in molecular and cellular biochemistry can be found in such standard textbooks as Molecular Cloning: A Laboratory Manual, 3rd Ed. (Sambrook et aL, HaRBor Laboratory Press 2001); Short Protocols in Molecular Biology, 4th Ed. (Ausubel et al. eds., John Wiley & Sons 1999); Protein Methods (Bollag et al., John Wiley & Sons 1996); Nonviral Vectors for Gene Therapy (Wagner et al. eds., Academic Press 1999); Viral Vectors (Kaplift & Loewy eds., Academic Press 1995); Immunology Methods Manual (I. Lefkovits ed., Academic Press 1997); and Cell and Tissue Culture: Laboratory Procedures in Biotechnology (Doyle & Griffiths, John Wiley & Sons 1998), the disclosures of which are incorporated herein by reference. Reagents, cloning vectors, cells, and kits for methods referred to in, or related to, this disclosure are available from commercial vendors such as BioRad, Agilent Technologies, Thermo Fisher Scientific, Sigma- Aldrich, New England Biolabs (NEB), Takara Bio USA, Inc., and the like, as well as repositories such as e.g., Addgene, Inc., American Type Culture Collection (ATCC), and the like.

[0140] Example 1 - Oral wounds heal with regeneration while facial skin wound repair results in fibrosis

[0141] Histologically, the oral mucosa is similar to the skin; both tissues are comprised of a squamous stratified epithelium, with an underlying lamina propria or mesenchymal layer, subtended by a third layer composed of muscle and fatty tissue (Fig. 7A). To compare oral mucosa and facial skin wound repair, a mouse model was developed that allows for parallel assessment of healing in both tissues (Fig. 1A); the oral mucosa was compared with the facial skin as both tissues are neural crest derived tissues. 2.5 mm dermal wounds were created in both the oral mucosa and facial skin within the same animal and wounds were harvested at postoperative day (POD) 1 , 2, 4, and 7 to evaluate repair following dermal injury.

[0142] H&E staining demonstrated that by POD4, oral mucosal wounds had repaired, such that the tissue architecture resembled that of unwounded oral tissue. In contrast, at POD4 facial skin wounds appeared distinct from the control, and they healed with a scar by POD7 (Fig. 1 B). Masson Trichome staining revealed greater collagen deposition in facial wounds compared to oral wounds by POD7, and Picrosirius Red Staining indicated that the extracellular matrix (ECM) of POD4 oral mucosal wounds resembled unwounded mucosa; this contrasted with facial wounds, which showed disorganized ECM structure at POD4 and POD7, indicative of fibrosis (Figs. 7B- 7C). Collagen type I and III immunostaining was also greater in the facial wounds at POD4 compared to oral wounds, indicative of a more fibrotic phenotype (Fig. 1C).

[0143] These initial results confirmed that the oral mucosa and skin healed with regenerative and fibrotic outcomes, respectively. Next, it was sought to identify the cellular subpopulations responsible for such disparate outcomes. Because fibroblasts are primarily responsible for laying down collagen following an injury and determine the degree of fibrosis, fibroblasts isolated from facial and oral wounds at POD7 were compared by bulk RNA-sequencing. Fibroblast subpopulations differed between the oral mucosa and facial skin upon wound repair, and gene ontology (GO) pathway analysis showed that pathways associated with skin fibrosis were upregulated in FSFs, including collagen synthesis and ECM organization (Fig. 1 D and Fig. 7D).

[0144] Example 2 - Single cell RNA-sequencing reveals distinct fibroblast subpopulations in oral mucosal and facial skin wounds

[0145] To characterize the fibroblast lineages residing in the wound environment in vivo, fluorescence-activated cell sorting (FACS) was utilized to isolate fibroblasts from oral mucosa and facial skin wounds using a lineage negative sorting strategy at POD1 , 2, 4, and 7, with unwounded tissue as a control. Lineage positive cells were also captured for comparison at each timepoint. scRNA-seq was performed, wherein all sequenced cells from facial and oral wounds were pooled for downstream analysis, including epithelial, endothelial, smooth muscle, and immune cells (Fig. 1 E). Cell types were identified based on expression of hallmark genes for each cell subtype (Fig. 8A). An increase in immune cells was observed upon wounding, with both tissues having a peak immune response at POD1 and higher numbers of immune cells in the facial wounds than oral wounds. Facial wounds were biased towards inflammatory immune subsets, such as Macrophage 1 and Neutrophils, while oral wounds were biased towards anti-inflammatory immune subsets, such as Macrophage 2. Higher numbers of oral endothelial cells were recovered. Recovery of endothelial cell numbers occurred by POD4 in the oral mucosa, while facial skin still showed low numbers of endothelial cells at POD7. Overall, when examining captured cell composition at each time point, the oral mucosa returned to steady state by POD7, while facial skin was still in the process of wound healing (Fig. 8B). These transcriptional data paralleled histological observations showing that by POD7 the oral mucosa had regenerated and returned to the homeostatic state, while the facial skin was still undergoing scar formation (Fig. 1 B).

[0146] When fibroblasts from all timepoints in both tissues were analyzed together, Louvainbased (Seurat) clustering identified nine transcriptionally distinct fibroblast clusters (Fig. 1 F and Fig. 9A). Putative functions were assigned to each cluster based on differential gene expression and identified markers for each population (Fig. 1 F). Certain fibroblast populations were predominantly composed of cells from one tissue, while others showed tissue-specific expression, such as Fibroblast 6 (Fig. 8C). The study was focused on the most abundant fibroblast populations, Fibroblast 1 and Fibroblast 2. Fibroblast 1 is marked by expression of Paired Related Homeobox 1 (Prrx ) and Ectonucleotide Pyrophosphatase / Phosphodiesterase (Enpp2). Prrx1+fibroblasts contribute to ventral skin scarring, but also are a crucial fibroblast progenitor population in the hard palate mucosa that are necessary for scarless regeneration. This population constituted 33% of all captured fibroblasts in the facial skin, and only 20% of all captured fibroblasts in the oral mucosa (Fig. 8D). Additionally, timepoint analysis in the facial skin showed that Fibroblast 1 is a highly wound responsive population and accounted for 55% of all FSFs at POD1 and 46% at POD2, suggesting that this population plays a key role in the fibroblast wound healing response in the face. In contrast, the predominant fibroblast population in the oral mucosa was Fibroblast 2, which accounted for 29% of all OMFs captured. This cluster was marked by expression of Lumican (Lum) and Adipocyte enhancer-binding protein 1 (Aebpl). GO term analysis revealed key differences between pathways in these fibroblast clusters. Fibroblast 1 was enriched in pathways involved in epithelial to mesenchymal transition, focal adhesion, cancer and inflammation, all of which are associated with fibrosis. In contrast, Fibroblast 2 was enriched in pathways related to ECM degradation, matrix metalloproteinases, inhibition of mechanotransduction and anti-inflammatory pathways, all of which point toward a regenerative phenotype (Fig. 8E).

[0147] Examining the several timepoints during the wound healing process revealed that Fibroblast 1 peaks after wounding at POD1 in the facial skin, while Fibroblast 2 maintains low numbers throughout wound healing (Fig. 1G). In contrast in oral mucosa, Fibroblast 2 remains high throughout wound healing (Fig. 1G). Markers for all Fibroblasts were determined by differential gene expression (Fig. 9A). Immunostaining for Fibroblast 1 and Fibroblast 2 showed high expression of Fibroblast 1 markers (Ennp2, Prrxl) in facial wounds, while oral wounds showed higher expression of Fibroblast 2 markers (Lum, Aebpl) compared to facial wounds (Fig. 9B).

[0148] Next, scVelo was used to assess predicted cell trajectories based on expression of unspliced (immature) and spliced (mature) mRNA variants. Oral fibroblast velocity showed a convergence at Fibroblast 2, indicating that this population is an important end-state for oral fibroblasts after wounding. Both skin and oral velocity showed a trajectory from Fibroblast 1 into Fibroblast 2, suggesting that the Prrx1+Fibroblast 1 could be a progenitor population in both tissues with differing fates; in the skin, this cluster transitioned into Fibroblast 5 and 8 (more fibrotic populations) while in the oral mucosa it transitioned into Fibroblast 2 (regenerative population) (Fig. 9C). These differences in cell fate could account for the more fibrotic versus more regenerative wound healing response observed in the skin and oral mucosa, respectively. Recent work has indicated that Prrx1+fibroblasts in the hard palate differentiate into Sca1+fibroblasts after wounding, and that these cells are crucial for a regenerative response. In accordance with this finding, Fibroblast 2 and Fibroblast 4 are Sca1+(Fig. 9D). Together, these data highlight the regenerative phenotype of the oral fibroblasts following injury.

[0149] Example 3 - Gas6-Axl signaling is upregulated in oral fibroblasts following dermal injury

[0150] To understand cell-cell interactions in the oral and facial wounds, CellChat analysis was performed based on known receptor-ligand interactions. Analysis of all cells from the scRNA-seq highlighted differences in the fibroblasts’ interactions in the two tissues during wound repair. Notably, “cell-cell interactions” involving Fibroblast 2, 4 and, 9 were up-regulated in the oral mucosa compared to facial skin (Fig. 2A). Fibroblast 2 was predicted to interact with nearly all cell types present in the dataset but was specifically enriched for interactions with other fibroblasts and endothelial cells, and these interactions were much higher in the oral mucosa than in the facial skin (Fig. 2A). Several upregulated pathways were identified in the oral mucosa. A pathway of interest was the signaling between cell surface receptor-tyrosine kinase Axl and its ligand Gas6, which were specifically enriched in all three of these populations (Fig. 2B and Fig. 10A).

[0151] AXL belongs to a subfamily of receptor tyrosine kinases that mediates homeostasis as well as several physiological responses. AXL is activated by a single ligand, GAS6, a widely expressed vitamin K dependent protein. The GAS6-AXL pathway has been associated with fibrotic resolution in many body sites, including lungs, kidney, brain, central nervous system, intestines, liver, and tumor progression. AXL transduces signals from the ECM to the cytoplasm, and the GAS6-AXL pathway regulates numerous cellular activities such as proliferation, survival, adhesion, and migration. Furthermore, AXL is an important driver of epithelial cell sternness and plasticity, supporter of vascular remodeling and regeneration, and promoter of Wnt-mediated regenerative response. AXL signaling is often associated with macrophages, as well as pericytes and cancer-associated fibroblasts, but it has not been investigated in the context of fibroblasts and oral wound healing. The dataset showed Gas6 signaling upregulated in the following populations: Endothelial 1 , Endothelial 4, Fibroblast 1 , Fibroblast 4, and Fibroblast 9 (Fig. 2A). This analysis indicated that Fibroblast 2 and 4 are both critical senders and receivers of Gas6 / Axl signaling, raising the question of whether activation of Axl in these populations is critical to the regenerative process of the oral mucosa. Fibroblast 4, shared between tissues, was marked by C-x-c motif chemokine ligand 5 (Cxcl5) and Decorin (Den), and associated GO terms were heavily biased towards inflammatory and other immune signaling (Fig. 9B and Fig. 10B).

[0152] Sequencing data confirmed high Axl expression in oral fibroblasts across the timepoints investigated, while the expression in immune and endothelial subsets remained low (Fig. 2C and Figs. 10D-10E). Axl expression did increase in skin fibroblasts upon wounding, in accordance with recent data published by other groups, but oral mucosal levels were higher throughout the course of wound healing. Axl, and its ligand Gas6 were investigated specifically to target the fibroblast-specific role in utilizing this pathway. RNAscope single molecule in situ hybridization confirmed high Axl mRNA expression in oral samples at all time points when compared to facial wounds, and immunohistochemistry for AXL and GAS6 with Collagen I confirmed expression in fibroblasts (Fig. 2D). Additionally, scVelo-driven comparison of spliced / unspliced mRNA to generate phase portraits of Axl and Gas6 expression showed Gas6-Axl activation in the oral mucosa, limited Axl activation in the facial skin, and no transcriptional dynamic change in Gas6 (Fig. 10F). It was confirmed that oral fibroblasts expressed greater AXL and pAXL following wounding across all timepoints compared to FSFs by immunostaining and FACS (Fig. 2E and Fig. 11A).

[0153] Example 4 - AXL and FAK expression during wound repair in vivo demonstrate an inverse relationship

[0154] Next, potential downstream targets of AXL signaling were probed in the facial and oral fibroblasts. Fibroblasts are activated by mechanical forces, driving scarring and fibrosis following skin injury. Mechanotransduction is initiated at the interface between the cell membrane and the ECM, where mechanical signals are transformed into chemical signals. Different tissue types have varying mechanical properties, and the oral mucosa has low tensile properties compared to the skin. The focal adhesion kinase (FAK) mechanotransduction pathway has been implicated in fibroblast induced fibrosis of the skin. Inhibition of FAK reduces skin scarring in small and large animal models. FAK localizes in focal adhesion complexes on the cytoplasmic side of the cell and is activated by a mechanical stimulus. Mechanical activation of fibroblasts through FAK drives a more fibrotic molecular trajectory, and previous work in pericytes has demonstrated a reduction of FAK expression with increased GAS6-AXL signaling. A similar link between FAK and AXL in fibroblasts was investigated following oral and skin dermal injury.

[0155] Given the lower tensile properties in the oral mucosa, it is possible that the oral mucosa may have lower levels of FAK than the facial skin. Using immunostaining and FACS, it was confirmed that FSFs showed greater FAK and pFAK following wounding compared to OMFs at all timepoints (Figs. 11 A-11 B). Immunostaining further illustrated that the enhanced GAS6 and AXL expression following wound injury in the oral mucosa was specific to fibroblasts and not observed in the immune cells (Fig. 11 C). To further evaluate the relationship between FAK and AXL in vivo, Col1Cre EF!T;R26,FAK,l / +mice were bred (Fig. 11 D). A recombination was induced with tamoxifen and the mice were wounded with the same oral and facial wounding as described above. H&E analysis demonstrated reduced scarring in the facial wounds in Col1Cre'ERT;R26,FAKfl / +mice at POD4 compared to WT mice (Fig. 11 E). Immunostaining and FACS confirmed decreased FAK expression in oral and facial wounds in the Col1Cre'ERT;R26,FAKfl / +mice (Fig. 11 F). The ECM ultrastructure analysis also demonstrated that, following FAK deletion in FSFs upon wounding, the ECM resembled that of regenerative oral wounds, illustrating that FAK inhibition led facial wounds to heal like oral wounds (Figs. 12A-12B). Importantly, immunostaining following FAK11^ revealed that AXL was upregulated in the OMF and FSF wounds at POD4 (Fig. 11 F). Collectively, these data suggest that that the AXL-FAK inverse relationship observed in vitro was maintained in vivo and that facial wounds can heal with reduced scar formation in FAK ' fibroblasts.

[0156] Example 5 - Spatial transcriptomics defines Gas6-Axl sensitive fibroblasts in oral wound healing

[0157] To further explore the significance of fibroblasts in oral and facial wound repair, the 10x Genomics Visium platform was applied to analyze spatial gene expression in wounds at POD2, 4, and 7 (Fig. 3A, left). The epidermal, dermal, and hypodermal layers were identified both histologically and according to their gene expression profiles (Fig. 13A).

[0158] Anchor-based integration was performed to assess membership of each of the nine scRNA-seq defined fibroblast clusters in the Visium-defined clusters (Fig. 3AI-3AIV). Following overlay of the scRNA-seq fibroblasts onto the spatial data, the fibroblast cluster distributions were largely congruent (Fig. 3AI-3AIV). scRNA-seq Fibroblast 1 was enriched in facial wounds compared to oral wounds (Fig. 13B). In contrast, scRNA-seq Fibroblast 2 was enriched in oral wounds compared to facial wounds (Fig. 13B). In support of the scRNA-seq findings, Ax / spatial expression was higher in oral compared to facial wounds at POD4 (Fig. 3B). Furthermore, Fak was higher in facial compared to oral wounds (Fig. 3B and Fig. 13C).

[0159] To assess communication networks among cell types in oral and facial wounds at POD4, differential interaction maps were generated comparing the groups. Oral wounds showed greater interactions between cell groups compared to facial wounds (Fig. 3C and Fig. 13D). Collectively, these data suggested that oral wounds may express Gas6-Axl spatially through similar fibroblast subclusters. Furthermore, the spatial organization of OMF and FSF subpopulations are distinct in wounds with regenerative and scarring phenotypes. Example 6 - Spatial phenetyping by CODEX demenstrates that fibroblast crosstalk differs in healing of oral and facial wounds

[0160] Building upon the spatial transcriptomic analyses described above, oral and facial wounds were examined at POD4 using multiplexed protein staining. CO-Detection by indEXing (CODEX), an assay in which a panel of 41 individual protein markers are sequentially labeled with, and iteratively imaged via cyclic additions and washouts of dye-labeled oligonucleotide-conjugated antibodies, was utilized (Fig. 3A, right). Across all treatment groups, 20 cell clusters were based on CODEX protein expression signatures (Fig. 3D and Figs. 14A-14C).

[0161] Six fibroblast CODEX defined clusters were identified. Anchor based transfer of the CODEX defined fibroblasts onto the scRNA-seq defined fibroblasts indicated that CODEX defined Fibroblast Cluster 4 was very similar to the molecular profile of scRNA-seq defined Fibroblast 2, and CODEX defined Fibroblast Cluster 2 was very similar to the molecular profile of scRNA-seq defined Fibroblast 1 (Fig. 15A). Interestingly, CODEX defined Fibroblast 2 was greater in facial POD4 group compared to the oral POD4 group, while CODEX defined Fibroblast 4 was greater in oral POD4 compared to facial POD4, in accordance with the scRNA-seq data (Fig. 3E and Fig. 14D). Consistent with the scRNA-seq findings, Fibroblast Cluster 4 had high AXL protein expression compared to other clusters (Fig. 3F and Fig. 15B).

[0162] Differential interaction maps were then used to visualize the strength of spatial cell-cell interactions based on K-nearest-neighbor localization, which can be uniquely assessed using CODEX and other spatial phenotyping technologies. These spatial relationships were assessed in POD4 oral versus facial wounds (Fig. 15C). Interestingly, CODEX defined Fibroblast 4 showed strong interactions in oral POD4 wounds with epithelial and endothelial cells, indicative of more regenerative cell-cell communications (Fig. 14E). In contrast, CODEX defined Fibroblast 2 showed strong interactions with smooth muscle cells suggestive of more fibrotic cell-cell communications (Fig. 14E).

[0163] Collectively, these data indicated that oral wounds expressed higher levels of GAS6-AXL pathway members at a protein level in a spatial environment. Furthermore, the spatial cell proteomic expression of oral and facial wounds is distinct.

[0164] Example 7 -Axl signaling suppresses FAK expression in fibroblasts upon mechanical stimulation in vitro

[0165] To further interrogate the role of FAK-associated mechanical forces and GAS6-AXL signaling in regenerative healing of the oral mucosa, a 3D culture system was developed to perform controlled stretching on fibroblasts, permitting precise mechanomodulation. It was investigated if FAK inhibition could promote AXL upregulation and lead to antifibrotic effects in fibroblasts. FSFs and OMFs (106cells) were seeded in culture media mixed with 2 mg / mL collagen to create collagen gels. At day 0, gels were subjected to 1 of 3 conditions: ( 1) no strain; (2) 10% equibiaxial strain (mimicking in vivo scar conditions); or (3) strain with FAK inhibitor (FAKi). For condition (3), media included 5 pM FAKi PF-573228 at initiation of strain. Following stretching, gels were incubated for an additional 48 hours (Fig. 4A). Given the previous results, it was expected that following the application of FAKi to stretched fibroblasts, AXL expression would be increased (Fig. 4B). Gels fixed with 4% paraformaldehyde underwent immunostaining for FAK, GAS6, and AXL. In the absence of strain, OMFs showed high levels of AXL and GAS6, unlike FSFs which showed minimal protein staining (Figs. 4B-4D). When strain was applied, increased staining for FAK was observed in FSFs (Figs. 4B-4D). In contrast, little staining for FAK was observed with OMFs, which retained higher levels of AXL and GAS6 with stretching (Figs. 4B- 4D). Interestingly, FAKi increased AXL and GAS6 staining in FSFs, more closely resembling immunostaining observed with OMFs (Figs. 4B-4D and Figs. 16A-16C). These findings were corroborated by FACS analysis (Fig. 16E). Collagen type I secretion was also greater in FSFs with strain compared to OMFs, which was reduced with FAK inhibition, pointing to a more fibrotic phenotype in the FSFs upon stretching (Fig. 16F). Together, these data suggested that OMFs are less sensitive to FAK-mediated mechanical stress than FSFs, and that FAK inhibition in FSFs may promote transition of FSFs to a more regenerative OMF-like phenotype.

[0166] Next, it was investigated whether AXL inhibition allowed for FAK upregulation and fibrotic effects using the same 3D culture system. At day 0, gels were subjected to 1 of 3 conditions: ( 1) no strain; (2) 10% equibiaxial strain; or (3) strain with AXL inhibitor (AXLi, Bemcentinib). For condition (3), media included 0.1 pM AXLi at initiation of strain (Fig. 4E). Given the previous results, it was expected that following the application of AXLi to stretched fibroblasts, FAK expression would be increased (Fig. 4F). Following stretching, gels were incubated for an additional 48 hours (Fig. 4E). When strain was applied with AXLi, the OMFs expressed high levels of FAK and low levels of AXL and GAS6, resembling FSFs with stretch. In contrast, the FSFs expressed high levels of FAK following AXLi with low levels of AXL and GAS6, similar to FSFs with stretch alone (Figs. 4G-4H and Figs. 17A-17D). FACS analysis also confirmed these results (Fig. 17E). Collagen I secretion was also greater in OMFs after AXLi, suggesting that the oral regenerative phenotype can be modulated through mechanical signals (Fig. 17F). To further evaluate the association between AXL and FAK, the AXL ligand, GAS6, was applied to the gels. FSFs showed a reduction of FAK with strain and GAS6, to similar levels as OMFs (Figs. 18A-18C). To confirm the AXLi results, a short hairpin RNA (shRNA) was utilized to knockdown Axl (Fig. 19A). Similar findings were observed as with the AXLi gels, with increased FAK and Collagen type I expression following Axl shRNA knockdown in both OMFs and FSFs with stretch (Figs. 19B-19C).

[0167] Overall, these data indicated that FAK and AXL expression in fibroblasts have an inverse relationship in vitro, such that OMFs downregulate FAK through AXL expression upon mechanical stimulation, leading to a regenerative phenotype. In contrast, FSFs upregulate FAK and repress AXL expression following mechanical cues, leading to a fibrotic phenotype. Thus, mechanotransduction is a key driver of skin fibrosis, and inhibiting it results in reduced skin fibrosis.

[0168] Example 8 - Modulation of FSF behavior in vivo reverts facial wounds to an oral like regenerative phenotype

[0169] Following the discovery of the close relationship between AXL and FAK in vitro, it was investigated whether this relationship could be exploited to improve wound healing outcomes of facial wounds in vivo (Fig. 5A). As AXL signaling modulated FAK expression in vitro, it was possible that activation of GAS6-AXL signaling could revert facial wounds to heal like oral wounds, and that inhibition of GAS6-AXL signaling may allow oral wounds to repair like facial wounds (Fig. 5B). The activation of GAS6-AXL signaling was evaluated by wounding with the application of recombinant GAS6 protein (50 ng protein per wound) (Fig. 5A). Oral and facial wounds were created in 60-day-old C57BL / 6 mice and wounds were evaluated at POD4. Administration of recombinant GAS6 was performed two days prior, one day prior, and on the day of wounding to oral mucosa and facial skin. H&E analysis revealed that the facial wounds had decreased dermal thickness and reduced scar formation following GAS6 treatment compared to untreated facial wounds, suggesting that upregulation of GAS6-AXL could rescue the facial scarring phenotype (Fig. 5C). Oral wounds showed a similar dermal thickness following GAS6 treatment to untreated wounds (Fig. 5C). Furthermore, Masson Trichome staining showed reduced collagen deposition in facial wounds with GAS6 compared to untreated facial wounds, revealing that facial wounds could revert to an oral-like regenerative phenotype (Fig. 20A). Staining for Fibroblast 2 makers, including Lum, was also upregulated in the wounds following GAS6 application in the facial wounds suggested that GAS6 could activate regenerative fibroblasts in the facial skin (Fig. 20B). As AXL signaling modulated FAK expression in vitro, it was investigated whether the inhibition of GAS6-AXL signaling could revert oral wounds to heal like facial wounds (Fig. 5B). To evaluate the effects of downregulating the GAS6-AXL pathway during wounding, oral and facial wounds were treated with AXLi (10 ul of 100uM Bemcentinib) two days prior, one day prior, and on the day of wounding and analyzed POD4 wounds (Fig. 5A). H&E analysis revealed an increased dermal thickness in oral wounds with AXLi compared to untreated oral wounds, illustrating that AXLi drives oral wounds to a more fibrotic phenotype (Figs. 5B-5C). Facial wounds also showed a similar thickened dermis following injury with AXLi to facial untreated wounds. Masson Trichome staining further showed increased collagen deposition in oral wounds treated with AXLi compared to oral wounds alone, revealing that oral wounds could revert to a facial like phenotype (Fig. 20A). Staining for Fibroblast 1 markers, including Enpp2, was also upregulated in the wounds following AXLi in the facial wounds, suggesting that AXLi could activate fibrotic fibroblasts in the oral mucosa (Fig. 20B). Additionally, ECM ultrastructure Picrosirius Red analysis of wounds treated with GAS6 protein or AXLi demonstrated that oral wounds resembled facial wounds following AXLi and facial wounds mimicked oral wounds after Gas6 treatment (Fig. 20C).

[0170] The interaction of FAK and AXL was evaluated following the activation and inhibition of the Gas6-AXL signaling. Immunostaining of wounds treated with Gas6 protein and AXLi demonstrated an inverse relationship between AXL and FAK (Figs. 5D-5E and Figs. 21 A-21 D). Oral wounds treated with AXLi showed an upregulation of FAK compared to oral untreated wounds. In contrast, facial wounds treated with GAS6 protein, demonstrated an increase in AXL and downregulation of FAK (Figs. 5D-5E and Figs. 21 A-21 D).

[0171] To corroborate the effects of AXLi in vivo using genetic approaches, AXL knockout (KO; AXltm1D9en / J) mice, including homozygous KO (AXL_ / ) and heterozygous (AXL+ / ) mice, were utilized and their wound healing outcomes were compared to wild type mice (WT) (AXL+ / +). The same oral and facial wound model was performed and wounds were evaluated at POD 1 , 4, and 7 (Fig. 22A). Oral wounds in AXL+ / _or AXL / _showed a reduced regenerative phenotype following dermal injury, with increased dermal thickness compared to Wild Type (WT) mice at POD 4 and 7 (Fig. 22B). Collagen deposition was also greater in the AXL+or AXLZoral wounds compared to WT mice at POD4 and 7 (Fig. 22C). ECM Picrosirius Red analysis demonstrated that AXL / _oral wounds mimicked facial wounds compared to oral wounds alone (Figs. 23A-23B). Immunostaining revealed that AXL / _mice showed an upregulation of FAK following oral and facial wounding compared to WT wounds (Fig. 24). Taken together, these findings indicate that upregulating AXL signaling can promote FSFs to adopt a more oral-like regenerative phenotype and facilitate scarless repair of facial wounds. In contrast, the inhibition of AXL in oral wounds results in fibrosis. A key finding from this study was the use of GAS6 in driving regenerative wound repair in facial skin.

[0172] Example 9 -AXL-FAK signaling during murine wound repair is conserved in human facial and oral scars

[0173] To determine if the observations in mice were conserved in humans, human facial and oral scars were obtained and analyzed. While acute oral injuries heal without a scar, repetitive injuries cause the oral mucosa to heal with fibrosis, similar to the facial skin. As biopsies from acute facial and oral injuries (single injury) are ethically difficult to obtain, mature facial and oral scars from three individuals were analyzed. Following informed consent, three adult patients with oral scars from repetitive injuries (oral scar) were compared to three facial scars resulting from a previous incision (facial scar) (Figs. 6A-6C and Fig. 25A). Immunostaining and RNA scope confirmed that Gas6 and Axl mRNA and protein expression was low in oral scar and facial scars, with high expression of FAK in both tissues (Fig. 6C and Fig. 25B). Given these findings, it was hypothesized that in cases of aberrant wound healing, such as after repetitive injury, the fibrotic program “overcomes” the anti-fibrotic potential of GAS6-AXL, resulting in decreased expression and utilization of this pathway, increased expression of FAK, and scarring in the typically nonscarring tissue.

[0174] To test this hypothesis, a repetitive injury model in mice was used that mimicked the human samples. The facial skin and oral mucosa of C57BL / 6 mice were injured three times with a seven-day rest period in between each injury (Fig. 6D). Wounds were harvested at POD14 and POD30. Following repetitive injury, oral and facial wounds formed a scar at POD14, unlike oral wounds following a single injury, as shown by H&E analysis (Fig. 6E, left). Masson Trichome staining also showed a greater deposition of collagen in oral repetitive injuries compared to single injuries, confirming scar formation (Fig. 26A, left). Immunostaining further showed that AXL and GAS6 were downregulated and FAK was upregulated in mouse oral scars compared to single injuries at POD14, similar to the observations in human samples (Fig. 6F, top). These data confirm what was observed in the human oral scars: following repetitive injury oral scars in both systems had decreased expression of AXL and GAS6, and an increase in FAK. This led to investigating if activating the AXL pathway throughout the repetitive wounding process could rescue the regenerative phenotype of the oral mucosa. To evaluate if repetitively oral and facial injuries could be rescued by GAS6 treatment, GAS6 was applied intradermally at each repeat injury (Fig. 6D). This activation of GAS6-AXL signaling yielded oral and facial healing with reduced dermal thickening and collagen deposition (Fig. 6E, right and Fig. 26A, right). Furthermore, ECM Picrosirius Red analysis showed that reinjured oral and facial wounds treated with GAS6 showed a similar architecture to non-scarring single oral injuries (Figs. 26B-26C). Immunostaining further showed that AXL and GAS6 were upregulated and FAK was downregulated in oral and facial reinjuries (Fig. 6F, bottom). Taken together these data suggest that in oral wounds high activation of the AXL pathway drives regenerative wound healing in the oral mucosa, and loss of this pathway upon repetitive injury results in a fibrotic phenotype. Remarkably, activation of AXL via GAS6 application can rescue the regenerative phenotype of repetitively injured oral mucosa, as well as induce reduced scarring in the skin. Thus, the GAS6-AXL pathway is a potential therapeutic target for prevention of scarring, reduction of scaring, and scarless healing in both the oral mucosa and skin.

[0175] Materials and methods

[0176] Mice

[0177] Transgenic mouse strains (acquired from Jackson Laboratories): B6.Cg-Tg(Col1 a2- cre / ERT,-ALPP)7Cpd / 2J (Stock: 029567), mTmG (B6.129(Cg)-Gt(ROSA)26Sortm4(AC7B- tdTomato,-EGFP)Luo / J Stock: 007676), B6 (C57BL / 6J, Stock: 000664). Mice were housed at the Stanford University Comparative Medicine Pavilion per Stanford APLAC guidelines, under the supervision of the Veterinary Service Center (VSC). All animals were genotyped under Jackson Laboratory instructions, using Transnetyx's Automated Genotyping PCR services.

[0178] Facial and Oral Wounding

[0179] Induction:

[0180] Intraperitoneal tamoxifen injections (90% corn oil / ethanol v / v; 200 mg / kg body weight) were used to induce Wnt1tCre / ERT2;ROSA26mTmGmice (n = 6 mice per experimental group) every day for 5 consecutive days prior to surgery.

[0181] Oral and Skin Wounding:

[0182] For facial and oral wounding, anesthesia was induced and maintained with 1-3% isoflurane at a flow rate of 2L / min. Adequate anesthesia was confirmed with the loss of hind-limb reflex to nociceptive stimuli. Facial skin was sterilized with betadine Surgical Scrub Veterinary (Avrio Health L.P.™, Stamford, CT) followed by sterile alcohol prep pads (FisherScientific™, Pittsburgh, PA). Next, 2.5 mm full-thickness circular wounds were made with punch biopsies. Wounds were dressed using 3M Tegaderm Transparent Film 1626w dressings (3M™, Cat:1626W). For oral wounds, 2.5mm punch biopsies were made in the buccal mucosa. Oral mucosa wounds were not dressed. For the Axl inhibition in vivo, at -2, -1 and at the day of surgery 10ul of (10nm) was injected in the oral and skin sites prior to wounding. To upregulate Axl signaling mouse Gas6 recombinant protein (10mg / ml) was applied at -2, -1 and at the day of surgery onto the wound sites.

[0183] Harvest.

[0184] Skin and oral wounds were then collected at different timepoints including POD 1 , 2, 4, and 7, at which time the wounds were harvested with dissecting scissors. Surrounding unwounded skin was taken as a control and Dissection followed fascial planes. Surrounding skin was also harvested for use as unwounded controls. For oral wounds the uninjured side was taken as a control. Mice were euthanized by CO2 narcosis and cervical dislocation. Harvested skin and mucosa for Fluorescence-activated cell sorting (FACS) was mechanically digested using dissecting scissors to finely mince each specimen. Harvested skin and mucosa for use in histology and immunofluorescent (IF) staining was placed in tissue embedding cassettes.

[0185] Harvesting Cells for Fluorescence-activated cell sorting (FACS):

[0186] Following CO2 euthanasia, facial and oral wounds were dissected and washed once in PBS. The wounded tissue was then diced into a fine consistency using sharp surgical scissors. Minced skin tissue was then enzymatically digested using a 1 :1 ratio of Collagenase Type IV (ThermoFisher, Cat:17104019) and Collagenase Type II (ThermoFisher, Cat:17101015) at a concentration of 1500U / ml in DMEM (ThermoFisher, Cat:10569010) for 90 minutes at 37°C. Tissue was then agitated at 150rpm during enzymatic digestion. The collagenase activity was stopped using FACS buffer, and digested tissue was strained through 70um cell strainers. Cells were pelleted at 1500rpm for 5 minutes at 4C and then resuspended in 150uL of FACS buffer for primary antibody staining. For lineage negative (Lin-) FACS analysis, the following primary antibodies were used: Tie2 (ThermoFisher, Cat:13598782), CD45 (ThermoFisher, Cat:48045182 or ThermoFisher, Cat:13045181 ), CD31 (ThermoFisher, Cat:RM52280 or ThermoFisher 13031181), CD324 (ThermoFisher, Cat: 13324982), CD326 (ThermoFisher, Cat:45592185), Ter119 (ThermoFisher, Cat:14592182). Following primary antibody staining, samples were washed with 500uL of FACS buffer, spun at 1500rpm for 5 minutes (4C), and resuspended in 150uL of FACS buffer for secondary antibody staining using either Streptavidin-eFluor450 (ThermoFisher, Cat: 48431782) or Streptavidin-Alexa Fluor 647 (S21374). Cells were stained with secondary for 20 minutes on ice. Cells were washed again with 500uL of FACS buffer, and DAPI (4',6-diamidino-2-phenylindole) (Biolegend, Cat: 422801) was added to label dead cells. A BD II FACS Aria machine was used for FACS sorting and analysis.

[0187] Histology and Immunofluorescent Staining:

[0188] Fixation-.

[0189] Both oral and skin tissue in transgenic mice were fixed in 4% paraformaldehyde (PFA) solution in PBS (Electron Microscopy Sciences, Cat: 15710) for 24h at 4°C. All other skin and oral samples were fixed in 10% neutral buffered formalin (NBF; ThermoFisher Scientific™, Waltham, MA) for 24 hours at 4°C.

[0190] Cryosectioning-.

[0191] Following fixation oral and skin samples were soaked in 30% sucrose dissolved in PBS at 4°C. Following one week, samples were removed from the sucrose solution and embedded as tissue blocks using Tissue Tek O.C.T. (Sakura Finetek, Torrance, CA). Frozen blocks were mounted on a Thermo Scientific CryoStar NX70 cryostat, and 8 pm-thick sections were transferred to Superfrost / Plus adhesive slides (ThermoFisher Scientific™, Waltham, MA).

[0192] Paraffin Sectioning-.

[0193] An Automated Tissue Processor (ThermoFisher Scientific™, Waltham, MA) was used to dehydrate oral and skin samples in a gradient of alcohols. ThermoFisher Histostar Tissue Embedding station was then used to embed the tissue. The resulting paraffin blocks were trimmed and cut as 8 pm-thick sections.

[0194] Staining-.

[0195] Hematoxylin and eosin (Cat:H-3502; Vector Laboratories, Burlingame, California), Masson’s Trichrome (ab150686; Abeam®, Waltham, MA) and Picro-sirius Red (ab150681 ; Abeam®, Waltham, MA) stains with standard protocols were used. For the cryosection samples the slides were first submerged into 1% PBS for 10 minutes. For the paraffin sections were hydrated prior to staining by placement in xylene for 20 minutes, followed by 10 minutes each of 100% EtOH, 95% EtOH, 70% EtOH, 50% EtOH, and 30% EtOH. Slides were then submerged in running tap water for 10 minutes. For immunofluorescent staining, both cryosectioned and paraffin section slides were then washed twice in Tween 20 (Sigma-Aldrich™, St. Louis, MO) and then one wash in PBS. Following blocking for 1 hour with Power Block (Biogenex™, Fremont, CA) the following primary antibodies were added: BS-5180R (anti-AxI), PA5-79300 (anti-gas6), Abeam ab5694 (anti-pros11), PA1 -26204 (anti-collagen type I).

[0196] Following primary antibody incubation slides were then incubated for 1 h with either Alexa Fluor 488, 594, or 647-conjugated anti-rabbit, anti-rat, or anti-mouse antibodies (Invitrogen, Waltham, MA). Finally, slides were mounted in Fluoromount-G mounting solution with or without DAPI (ThermoFisher Scientific™, Waltham, MA). The Leica CTR4000 microscope was used to collect Brightfield images were acquired, while fluorescent images were acquired with a LSM880 inverted confocal, Airyscan, AiryscanFAST, GaAsP detector upright confocal microscope.

[0197] Picrosirius Red Stained Histologic Analysis-.

[0198] Analysis of picrosirius red stained tissue sections from oral and skin took place using an in-house image-processing previously published algorithm.12The algorithm profiles 26 ultrastructural features to provide a quantitative comparison of extracellular matrices. Each group (n = 6) was randomly imaged at 50 separate locations at 40x. Color deconvolution following previously described methods50was performed to characterize each stain by absorbance in three RGB channels. Following ortho-normal transformation to determine each color’s contribution to the captured image, red and green images were produced, representing mature and immature ECM fibers, and analyzed as separate groups. A in house Matlab script was used to achieve analysis, including noise reduction, preferential selection for smooth regions with low variance, and “skeletonization” of images to characterize the fiber networks.

[0199] RNAscope:

[0200] RNAscope® 2.5 HD Assay in situ hybridization assay was conducted starting with a permeabilization step. Paraffin-embedded tissue sections of the oral and mucosa were pretreated to permeabilize cells and expose target RNA. Following this, RNAscope® and double-Z design probes were hybridized to the target sequence. Signals were then amplified using detection reagents and labeled fluorescent probes were added to bind to each amplifier region. Target probes, amplifiers and label probes were sourced from Advanced Cell Diagnostics, Hayward, CA. Samples were imaged using a bright field microscope with a Leica CTR4000 microscope.

[0201] Cell Culture: Mouse Fibroblast Oral and Facial Cell Line Cell Culture-.

[0202] Fibroblast oral and facial cell were produced from oral and facial tissue respectively from black six mice. Following digest cells were plated for 24 hours for cell attachment. Media was changed at 24 hours. Once confluency exceeded 70% cell were passaged. Cells used for experiments were between passages 2-5. Fibroblasts were cultured in DMEM + Glutamax media (ThermoFisher, Cat: 10569010) enriched with 10% fetal bovine serum (ThermoFisher, Cat: 10082147) and 1% Antibiotic-Antimycotic (ThermoFisher, Cat:15240062) at 37°C and 5% CO2.

[0203] Gels:

[0204] Mouse Gels:

[0205] Mouse fibroblasts were used to generate fibroblast-populated collagen I hydrogels as per a previously published protocol. In short, a solution of 2mg / mL collagen I (Advanced Biomatrix, Cat:5005), 0.8x MEM (ThermoFisher, Cat:11430030) in 16mM HEPES (Sigma, CAT: 83264), and suspended mouse adipocytes (concentration 500,000 cells / mL) in DMEM (ThermoFisher, Cat: 10569010) were pipetted into cruciform-shaped PDMS (DOW, Cat:2646340) molds and allowed to gelate before removing the mold. Hydrogels were constrained by metal pins and medium containing DMEM + Glutamax media (ThermoFisher, Cat:10569010) enriched with 10% fetal bovine serum (ThermoFisher, Cat:10082147), 1% Antibiotic-Antimycotic (ThermoFisher Cat:15240062). Strain was achieved through movement of metal anchor pins in each of the four arms of the hydrogel. Strain quantification was confirmed by Fiji analysis of TiO2 dye marks on the central region of each hydrogel. Media containing small molecule mechanotransduction inhibitors to FAK (PF573228 [10uM], Tocris, Cat: 3239) Axl ([10nM] MedChem express), or Vehicle Control (PBS) was added. Media was replaced every other day. Hydrogels were kept for 2 days. For ICC analysis of gel contents, gels were fixed whole in 4%PFA (ChemCruz, Cat:281692) after being washed in PBS. For FACS and PCR analysis, cells were harvested by enzymatic digestion using Collagenase Type I (ThermoFisher, Cat: 17018029) at a concentration of 1500U / ml in DMEM (ThermoFisher, Cat:10569010) for 25 minutes at 37°C. Enzyme activity was stopped using FBS enriched media. Cells were spun, and the pellet was processed for FACS or resuspended in TRIzol for PCR.

[0206] Immunocytochemistry Processing for Gels:

[0207] Following 24-hour fixation, gels were carefully dissected into 2mm x 2mm pieces and placed into wells of a 96 well plate. Gentle agitation was provided to the plates during staining via a platform shaker at room temperature. Following washing in PBS for 30 minutes, gels were then permeablised with 0.1 % TritonX-100 in PBS (ThermoFisher, Cat:85111 ) for 30 minutes. Gels were then blocked with PowerBlock (BioGenex, Cat:HK085-GP) for 1 hour. Following blocking, primary antibody was made up in a 1 :1 solution of 0.1% TritonX-100 and PowerBlock. Primary staining lasted 1 hour, followed by two 15-minute washes in 0.025% Tween 20 in PBS (Sigma, Cat:P1379) and a single 15-minute wash in PBS. Secondary antibody containing Alexa Fluor 488, 594, or 647 against the primary antibody was added to a 1 :1 solution of 0.1% TritonX-100 and Powerblock. Secondary antibody staining lasted 45 minutes, followed by two 30-minute washes in 0.025% Tween 20 in PBS (Sigma, Cat:P1379) and a single 30-minute wash in PBS. Finally, samples were mounted on a slide with Fluoromount-G mounting solution with DAPI (ThermoFisher Cat:00495952). Fluorescent images were taken with an LSM880 inverted confocal microscope. shRNA Knockdown ofAxI in Gels:

[0208] Short hairpin RNA (shRNA) knockdown of AxI gene expression in mouse fibroblasts was performed per manufacturer guidelines (Santa Cruz, US). Mouse primary fibroblasts were collected as described in Cell Culture and populated into collagen 1 hydrogels as described above. Twenty-four hours after the formation of the gels, shRNA knockdown, scrambled control, or vehicle control was delivered. Following knockdown, gels were subjected to 0 or 10% equibaxial strain. Two days after strain, gels were harvested for FACS analysis or fixed in 4% PFA for Immunocytochemistry. Other than replacing traditional culture conditions in flasks / wells with a hydrogel populated system, no other modifications were made to the manufacturer's protocol.

[0209] ELISA:

[0210] Media samples from mouse fibroblasts-populated collagen 1 hydrogels was tested using Abeam mouse (ab210579, USA) according to manufacturer's protocol. A spectrophotometer captured absorbances, and concentrations of protein target were calculated and modeled using Prism.

[0211] Single-Cell Sequencing:

[0212] Skin and oral wounds were harvested at POD 1 , 2, 4, and 7 mechanically digested with sharp dissecting scissors as shown above (n = 10 per condition). Tissue was then added to an enzymatic digestion consisting of Collagenase II (ThermoFisher, Cat:17101015) and IV (ThermoFisher, Cat:17104019) in DMEM-F12 (GIBCOTM, Fischer Scientific, Hampton, NH). Samples were added to an orbital shaker at 150 rpm for 90 minutes at 37eC. FACS buffer was added to quench the digest and samples were filtered through 70 pm cell strainers. Samples were then centrifuged at 1500 for 5 minutes at 4°C. Following antibody labelling as described above the cells were sorted for fibroblasts positively. Briefly, a lineage gate for hematopoietic (CD45, Ter-119), endothelial (CD31 , Tie2), and epithelial (CD326, CD324) cell markers was used as a negative gate to isolate fibroblasts (Lin-), which were sorted into Wnt1 negative (Tomato+ GFP- Lin-) and Wnt1+ (Tomato- GFP+ Lin-). All other cells were also collected as lineage -i-ve for Wnt1 negative (Tomato+ GFP- Lin-) and Wnt1+ (Tomato- GFP+ Lin-).

[0213] Cell suspensions of oral and skin across the different timepoint were then labeled with TotalSeq Series B hashtag oligonucleotide-labeled antibodies (BioLegend). Samples were first centrifuged and resuspended with 0.04% UltraPure BSA (Thermo Fisher, Waltham, MA); cell counts were completed. A second filtration then step took place using 40 pm cell strainers. Quality control and single cell RNAseq were performed on unsorted cells using the 10x Chromium Single Cell platform (Single Cell 3’ v3, 10x Genomics, USA) at the Stanford Functional Genomics Facility (SFGF), Stanford University, Palo Alto.

[0214] Base calls were converted to reads using the Cell Ranger (10X Genomics; version 3.1) implementation mkfastq and then aligned against the Cell Ranger mm10 reference genome, available at: http: / / cf.10xqenomics.com / supp / cell-exD / , using Cell Ranger’s count function with SC3Pv3 chemistry and 5,000 expected cells per sample, as previously described. Hashtag oligos (HTOs) for samples were demultiplexed using Seurat’s implementation HTODemux as previously described. A maximum percent mitochondrial RNA cutoff of 15% was employed.

[0215] Unique molecular identifiers (UM Is) from each cell barcode were retained for all downstream analysis, normalized with a scale factor of 10,000 UMIs per cell, and subsequently natural log transformed with a pseudocount of 1 using the R package Seurat (version 4.0.5). The first 15 principal components of the aggregated data were then used for uniform manifold approximation and projection (UMAP) analysis. Cell annotations were ascribed using SingleR (version 3.11 ) against the Mouse-RNAseq reference dataset, available at https: / / rdrr.io / aithub / dviraran / SingleR / man / mouse.rnasea.html. Cell-type marker lists were generated using Seurat’s native FindMarkers function with a log fold change threshold of 0.25 using the ROC test to assign predictive power to each gene. The 200 most highly ranked genes from this analysis for each cluster were used to perform gene set enrichment analysis in a programmatic fashion using EnrichR (version 2.1 ). The scRNA sequencing data generated during this study has been sent to the NCBI’s Gene expression Omnibus for deposition and will be accessible on request.

[0216] Cel IChat Receptor-Ligand Analysis:

[0217] To evaluate the potential for interactions between different cell types in the dataset, the recently developed CellChat platform was applied. This was implemented using the scRNA-seq Seurat object in R. Cells were binned according to the SingleR-defined cell type classifications, with fibroblast cells subset based on their location within either the scarring or regenerative pseudotime arms. Default parameterizations were used throughout, and Secreted Signaling, ECM-Receptor, and Cell-Cell Contact relationships were considered.

[0218] RNA Velocity Analysis:

[0219] RNA velocity analysis was also performed using the scVelo package to further evaluate trajectory analysis. scVelo uses a likelihood-based dynamical model to solve the full transcriptional dynamics of spliced and unspliced mRNA kinetics of each gene. RNA velocity analysis allowed for identification of transient cellular states in the dataset and to predict the directional progression of transcriptomic signatures along the identified trajectories. These predictions are based on gene-specific rates of transcription, splicing, and degradation of mRNA to estimate each cell’s position in their own underlying differentiation process.

[0220] Statistical Methods:

[0221] Statistical testing was performed in GraphPad Prism v9 unless otherwise stated. For two- group comparisons, unpaired t-tests were used. For multi-group analysis, one-way ANOVAs were used with Tukey’s post hoc corrections to compare groups; p<0.05 conferred statistical significance for all tests.

[0222] In at least some of the previously described embodiments, one or more elements used in an embodiment can interchangeably be used in another embodiment unless such a replacement is not technically feasible. It will be appreciated by those skilled in the art that various other omissions, additions and modifications may be made to the methods and structures described above without departing from the scope of the claimed subject matter. All such modifications and changes are intended to fall within the scope of the subject matter, as defined by the appended claims.

[0223] It will be understood by those within the art that, in general, terms used herein, and especially in the appended claims (e.g., bodies of the appended claims) are generally intended as “open” terms (e.g., the term “including” should be interpreted as “including but not limited to,” the term “having” should be interpreted as “having at least,” the term “includes” should be interpreted as “includes but is not limited to,” etc.). It will be further understood by those within the art that if a specific number of an introduced claim recitation is intended, such an intent will be explicitly recited in the claim, and in the absence of such recitation no such intent is present. For example, as an aid to understanding, the following appended claims may contain usage of the introductory phrases “at least one” and “one or more” to introduce claim recitations. However, the use of such phrases should not be construed to imply that the introduction of a claim recitation by the indefinite articles “a” or “an” limits any particular claim containing such introduced claim recitation to embodiments containing only one such recitation, even when the same claim includes the introductory phrases “one or more” or “at least one” and indefinite articles such as “a” or “an” (e.g., “a” and / or “an” should be interpreted to mean “at least one” or “one or more”); the same holds true for the use of definite articles used to introduce claim recitations. In addition, even if a specific number of an introduced claim recitation is explicitly recited, those skilled in the art will recognize that such recitation should be interpreted to mean at least the recited number e.g., the bare recitation of “two recitations,” without other modifiers, means at least two recitations, or two or more recitations). Furthermore, in those instances where a convention analogous to “at least one of A, B, and C, etc.” is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., “ a system having at least one of A, B, and C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). In those instances where a convention analogous to “at least one of A, B, or C, etc.” is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., “ a system having at least one of A, B, or C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). It will be further understood by those within the art that virtually any disjunctive word and / or phrase presenting two or more alternative terms, whether in the description, claims, or drawings, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase “A or B” will be understood to include the possibilities of “A” or “B” or “A and B.”

[0224] In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0225] As will be understood by one skilled in the art, for any and all purposes, such as in terms of providing a written description, all ranges disclosed herein also encompass any and all possible sub-ranges and combinations of sub-ranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as “up to,” “at least,” “greater than,” “less than,” and the like include the number recited and refer to ranges which can be subsequently broken down into sub-ranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 articles refers to groups having 1 , 2, or 3 articles. Similarly, a group having 1 -5 articles refers to groups having 1 , 2, 3, 4, or 5 articles, and so forth.

[0226] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it is readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

[0227] Accordingly, the preceding merely illustrates the principles of the invention. It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventors to furthering the art, and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the invention as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. Moreover, nothing disclosed herein is intended to be dedicated to the public regardless of whether such disclosure is explicitly recited in the claims.

[0228] The scope of the present invention, therefore, is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of present invention is embodied by the appended claims. In the claims, 35 U.S.C. §11 (f) or 35 U.S.C. §112(6) is expressly defined as being invoked for a limitation in the claim only when the exact phrase "means for" or the exact phrase "step for" is recited at the beginning of such limitation in the claim; if such exact phrase is not used in a limitation in the claim, then 35 U.S.C. § 112 (f) or 35 U.S.C. §112(6) is not invoked.

Claims

WHAT IS CLAIMED IS:1 . A method of promoting healing of a wound in a dermal location of a subject, the method comprising: administering an effective amount of an AXL signaling pathway activator to the wound to promote healing of the wound.

2. The method according to Claim 1 , wherein the AXL signaling pathway activator comprises an AXL receptor agonist.

3. The method according to any of Claims 1 or 2, wherein the AXL signaling pathway activator comprises a polypeptide or nucleic acid coding sequence therefor.

4. The method according to Claim 3, wherein the polypeptide is an AXL signaling pathway specific activator GAS6 protein.

5. The method according to Claim 3, wherein the polypeptide comprises an amino acid sequence having 70% or greater amino acid sequence identity to SEQ ID NO:1 .

6. The method according to Claim 3, wherein the polypeptide comprises an amino acid sequence having 70% or greater amino acid sequence identity to SEQ ID NO:2.

7. The method according to any of Claims 1 or 2, wherein the AXL signaling pathway activator comprises a peptidomimetic.

8. The method according to any of Claims 1 or 2, wherein the AXL signaling pathway activator comprises a small molecule.

9. The method according to any of the preceding claims, wherein the administering comprises injecting a composition comprising the AXL signaling pathway activator below a topical dermal location of the subject.

10. The method according to any of the preceding claims, wherein the subject is an adult.11 . The method according to any of the preceding claims, wherein the method comprises regeneration of dermal appendages.

12. The method according to Claim 11 , wherein the dermal appendages comprise hair follicles, sweat glands, and sebaceous glands.

13. The method according to any of the preceding claims, wherein the method further comprises forming the wound.

14. The method according to any of the preceding claims, wherein the wound is a surgical wound.

15. The method according to any of the preceding claims, wherein the method produces a healed wound with reduced levels of scarring compared to a control.

16. The method according to any of the preceding claims, wherein the method produces a scarless healed wound.

17. A method of ameliorating scarring during healing of a wound in a subject, the method comprising: administering an effective amount of an AXL signaling pathway activator composition to the wound to ameliorate scarring of the wound.

18. The method according to Claim 17, wherein the AXL signaling pathway activator comprises an AXL receptor agonist.

19. The method according to any of Claims 17 or 18, wherein the AXL signaling pathway activator comprises a polypeptide or nucleic acid coding sequence therefor.

20. The method according to Claim 19, wherein the polypeptide is an AXL signaling pathway specific activator GAS6 protein.21 . The method according to Claim 19, wherein the polypeptide comprises an amino acid sequence having 70% or greater amino acid sequence identity to SEQ ID NO:1 .

22. The method according to Claim 19, wherein the polypeptide comprises an amino acid sequence having 70% or greater amino acid sequence identity to SEQ ID NO:2.

23. The method according to any of Claims 17 or 18, wherein the AXL signaling pathway activator comprises a peptidomimetic.

24. The method according to any of Claims 17 or 18, wherein the AXL signaling pathway activator comprises a small molecule.

25. The method according to any of Claims 17-24, wherein the wound is a surgical wound.

26. The method according to any of Claims 17-25, wherein the method produces a scarless healed wound.

27. The method according to any of Claims 17-26, wherein the administering comprises injecting the composition below a topical dermal location.

28. The method according to any of Claims 17-27, wherein the subject is an adult.

29. The method according to any of Claims 17-28, wherein the method comprises regeneration of dermal appendages.

30. The method according to Claim 29, wherein the dermal appendages comprise hair follicles, sweat glands, and sebaceous glands.

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