WO2023215294A1

WO2023215294A1 - Complement pathway inhibition for wound healing

Info

Publication number: WO2023215294A1
Application number: PCT/US2023/020698
Authority: WO
Inventors: Michael T. Longaker; Hunter FRASER; Katya L. MACK; Heather E. TALBOTT; Michelle F. GRIFFIN; Jennifer B.L. PARKER
Original assignee: The Board Of Trustees Of The Leland Stanford Junior University
Priority date: 2022-05-02
Filing date: 2023-05-02
Publication date: 2023-11-09

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 a complement pathway inhibitor, e.g., a complement factor H active agent, to the wound to promote healing of the wound, e.g., to promote healing of the wound with more robust re-epithelialization and with reduced scarring. Also provided are kits including an amount of a complement inhibitor composition.

Description

COMPLEMENT PATHWAY INHIBITION FOR WOUND HEALING

ACKNOWLEDGEMENT OF GOVERNMENT RIGHTS

This invention was made with Government support under contract GM136659 awarded by the National Institutes of Health. The Government has certain rights in the invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

Pursuant to 35 U.S.C. §119(e), this application claims priority to the filing date of United States Provisional Application Serial No. 63/337,475 filed on May 2, 2022, the disclosure of which application is herein incorporated by reference.

INTRODUCTION

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.

SUMMARY

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.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 : MRL ear wounds uniquely heal in an accelerated fashion and via tissue regeneration. A. Gross photographs (first two columns) and hematoxylin and eosin (H&E) histology (third column) of ear (top panels) and dorsal (bottom panels) wounds in CAST (“normal healer”) and MRL (“super healer”) mice, showing that MRL ear wounds heal through regeneration of multiple tissue types (including cartilage) while ear wounds in CAST mice, and dorsal wounds in both strains, heal via fibrotic scarring. B. Wound closure curves for MRL and CAST ear (top panel) and dorsal (bottom panel) wounds. CAST ear wounds do not close to an appreciable extent, while MRL ear wounds are completely or near-completely healed over by 3-4 weeks postwounding. Splinted dorsal wounds heal at a similar rate in MRL vs. CAST mice, with re- epithelialization complete by postoperative day (POD) 14. C. Sampling scheme for RNA-seq libraries. MRL and CAST were crossed to produce F1 hybrids for allele-specific expression analysis. Each adult F1 mouse underwent both dorsal excisional and ear punch wounding. On POD 7, wounds were collected by excising a 1 mm ring of tissue around each wound site. Cell populations were isolated via fluorescence-activated cell sorting (FACS) and RNA was extracted for bulk RNAseq. D. Principal component analysis of allele-specific F1 RNAseq samples. Allele- specific samples separated into distinct clusters by tissue/wound context (ear vs. dorsal) and allele (MRL vs. CAST).

Figure 2: Analysis of differential allele-specific expression (diffASE) reveals patterns of c/s-regulation unique to MRL ear wounds. A. Schematic example of diffASE between MRL and CAST in ear and dorsal wounds. Blue and green solid boxes represent gene regulatory regions affecting transcription of the MRL or CAST allele, respectively, of a given gene; transcription levels from each allele are represented by blue and green wavy lines. In the context of a dorsal wound (where MRL and CAST phenotypes are similar), expression is the same from the MRL vs. CAST allele. In contrast, in ear wounds, the present of a context-specific (i.e. , wound- related) transcription factor (TF; grey circle) reveals ASE through differences in the sequence of the MRL vs. CAST regulatory elements (which respond differentially to the TF). Overall, this can be detected as a pattern of diffASE unique to ear wounds (exemplified in bottom panel bar graphs). B. Venn diagrams showing number of genes with ASE in ear wounds (blue region), dorsal wounds (yellow region), or both (overlapping region) in each analyzed wound cell type. C. Scatterplots for each cell type comparing distribution of allelic ratios between dorsal and ear wounds. Colored points represent genes with diffASE (gold points are genes with a larger difference between CAST and MRL alleles in the ear; blue points are genes with a larger difference between CAST and MRL alleles in the dorsum). D & E. Gene set enrichment analysis for genes with evidence of diffASE in fibroblasts, which are highly enriched for gene ontology (GO) categories (left (D)) and mutant phenotypes (right (E)) related to wound healing and development. Such enrichment patterns were unique to fibroblasts (not seen in endothelial or immune cells). Fibroblasts are the end cellular mediators of scarring/fibrosis. F. Specific genes associated with mutant phenotypes or GO terms related to responses to injury and wound healing with diffASE in fibroblasts. Yellow circles represent fold changes between alleles in the dorsum; blue circles represent fold changes in the ear.

Figure 3: Integration of diffASE with fine-mapping study results. A. LOD scores vs. chromosome position for ear hole closure from Cheverud et al. (Heredity, 2014; PMID 24569637). Red circles indicate positions of the genetic markers closest to genes identified as having diffASE in fibroblasts. B. Distribution of mean LOD scores of permuted gene sets (20,000 permutations). Red line indicates the mean LOD score of genetic markers closest to the fibroblast diffASE gene set; this mean score was significantly higher than that of random gene sets (*P = 0.0094). C. Three genes within the fine-mapped QTL intervals annotated with the GO term “wound healing” (G0:0042060) show diffASE in fibroblasts. Of these, Cfh had the largest magnitude of ASE in ear wound fibroblasts. Figure 4: Cfh treatment leads to partial regeneration and enhanced healing of dorsal wounds in wildtype mice. A. Schematic of MRL and CAST dermal fibroblast culture from dorsal and ear skin. B. Left, fluorescent histology of cultured MRL and CAST dorsal and ear fibroblasts with immunohistochemical (IHC) staining for complement factor H (CFH) and DAPI nuclear counterstain. Right, quantification of CFH expression across in vitro conditions. CFH expression was highest in MRL ear fibroblasts, consistent with diffASE gene expression results. *P < 0.05. C. Schematic of MRL and CAST dorsal and ear wounding for histology. D. Fluorescent histology (left) and quantification (right) of IHC staining of wounds for CFH (with DAPI nuclear counterstain) showing that CFH expression was specific to MRL ear wounds. *P < 0.05. E. Schematic of wildtype mouse dorsal splinted wounding with local wound treatment with either recombinant CFH protein or phosphate-buffered saline (PBS; vehicle control). F. Gross photographs of control (- CFH) and CFH-treated (+CFH) wounds showing a less prominent scar and more robust re- epithelialization in +CFH wounds. Black dotted outline indicates healed wound region. G. Picrosirius red connective tissue histology of -CFH and +CFH wounds and unwounded skin (UW). H. T-distributed stochastic neighbor embedding (t-SNE) plot of quantified extracellular matrix (ECM) ultrastructural parameters, based on picrosirius red histology (G), showing overall similarities/differences in ECM ultrastructure between wound and skin conditions. CFH-treated wounds had an ECM architecture intermediate between that of control scars (-CFH) and unwounded skin, indicating partial ECM regeneration in +CFH wounds. Each dot represents quantified parameters from one histologic image. I. Hematoxylin and eosin (H&E) histology of POD 14 wounds and skin showing more complete re-epithelialization and presence of putative regenerating dermal appendages (hair follicles or glands) in +CFH wounds, compared to lack of appendages in -CFH wounds which form a “bare area” of fibrotic scar. Yellow dotted lines indicate borders of healed wounds. J. Dermal thickness quantified from histology of wounds and skin, showing that +CFH wounds have decreased scar thickness (closer to the thickness of UW skin) compared to control (-CFH) wounds.

Figure 5: Dose responsiveness of reduced scar thickness with CFH treatment. Quantified scar thickness (first panel) and representative H&E histology (second through fourth panels) of wildtype mouse wounds treated with PBS (control) or CFH at varying doses (5 or 10 mg/mL). The reduction in scar thickness with CFH treatment was more pronounced with a higher dose of CFH.

Figure 6: MRL ear wounds uniquely heal in an accelerated and regenerative fashion. A. Gross photographs (first two columns) and hematoxylin and eosin (H&E) histology (third column) of ear wounds in CAST (“normal healer”) and MRL (“super healer”) mice, showing that MRL ear wounds heal through regeneration of multiple tissue types (including cartilage) while ear wounds in CAST mice heal via fibrotic scarring. B. Wound closure curves for MRL and CAST ear wounds. CAST ear wounds do not close to an appreciable extent, while MRL ear wounds are completely or near-completely healed over by 3-4 weeks post-wounding. C. Gross photographs (first two columns) and hematoxylin and eosin (H&E) histology (third column) of dorsal wounds in CAST (“normal healer”) and MRL (“super healer”) mice, showing that dorsal wounds in both strains, heal via fibrotic scarring. D. Wound closure curves for MRL and CAST dorsal wounds. Splinted dorsal wounds heal at a similar rate in MRL vs. CAST mice, with re-epithelialization complete by postoperative day (POD) 14.

Figure 7: RNA-seq of key wound cell types from CAST x MRL hybrid mice cluster by wound type and allele. A. Sampling scheme for RNA-seq libraries. MRL and CAST were crossed to produce F1 hybrids for allele-specific expression analysis. Each adult F1 mouse underwent both dorsal excisional and ear punch wounding. On POD 7, wounds were collected by excising a 1 mm ring of tissue around each wound site. Cell populations were isolated via fluorescence-activated cell sorting (FACS) and RNA was extracted for bulk RNAseq. B. Heatmap of the most variable genes (1 ,000) following regularized Iog2 transformation of allele-specific read counts. Hierarchical clustering groups samples by cell population (immune, endothelial, fibroblast), allele (MRL [‘M’] vs. CAST [‘C’] and wound site (ear [‘E’] vs. dorsal [‘D’]). C. Principal component analysis of allele-specific F1 RNAseq samples. Allele-specific samples separated into distinct clusters by tissue/wound context (ear vs. dorsal) and allele (MRL vs. CAST).

Figure 8: Analysis of differential allele-specific expression (diffASE) reveals patterns of c/s-regulation unique to MRL ear wounds. A. Schematic example of diffASE between MRL and CAST in ear and dorsal wounds. Blue and green solid boxes represent gene regulatory regions affecting transcription of the MRL or CAST allele, respectively, of a given gene; transcription levels from each allele are represented by blue and green wavy lines. In the context of a dorsal wound (where MRL and CAST phenotypes are similar), expression is the same from the MRL vs. CAST allele. In contrast, in ear wounds, the present of a context-specific (i.e. , wound- related) transcription factor (TF; grey circle) reveals ASE through differences in the sequence of the MRL vs. CAST regulatory elements (which respond differentially to the TF). Overall, this can be detected as a pattern of diffASE unique to ear wounds (exemplified in bottom panel bar graphs). B. Venn diagrams showing number of genes with ASE in ear wounds (blue region), dorsal wounds (yellow region), or both (overlapping region) in each analyzed wound cell type. C. Scatterplots for each cell type comparing distribution of allelic ratios between dorsal and ear wounds. Colored points represent genes with diffASE (gold points are genes with a larger difference between CAST and MRL alleles in the ear; blue points are genes with a larger difference between CAST and MRL alleles in the dorsum). D. Gene set enrichment analysis for genes with evidence of diffASE in fibroblasts, which are highly enriched for gene ontology (GO) categories (left) and mutant phenotypes (right) related to wound healing and development. Such enrichment patterns were unique to fibroblasts (not seen in endothelial or immune cells). Fibroblasts are the end cellular mediators of scarring/fibrosis. E. Specific genes associated with mutant phenotypes or GO terms related to responses to injury and wound healing with diffASE in fibroblasts. Yellow circles represent fold changes between alleles in the dorsum; blue circles represent fold changes in the ear.

Figure 9: Integration of diffASE with fine-mapping study results. A. LOD scores vs. chromosome position for ear hole closure from Cheverud et al. (Heredity, 2014; PMID 24569637). Red circles indicate positions of the genetic markers closest to genes identified as having diffASE in fibroblasts. B. Distribution of mean LOD scores of permuted gene sets (20,000 permutations). Red line indicates the mean LOD score of genetic markers closest to the fibroblast diffASE gene set; this mean score was significantly higher than that of random gene sets (*P= 0.0094). C. Cfh, which is associated with the gene ontology term for wound healing (G0:0042060) and falls within a fine-mapped region for ear closure, shows ear wound-specific ASE specific to fibroblasts in CAST x MRL hybrids. In fibroblasts, we see significant upregulation of the MRL allele relative to the CAST allele in ear wounds, in contrast to dorsal wounds where the expression of these alleles are similar. *diffASE q < 0.05.

Figure 10: Cfh treatment leads to partial regeneration and enhanced healing of dorsal wounds in wildtype mice. A. Schematic of MRL and CAST dermal fibroblast culture from dorsal and ear skin. B. Left, fluorescent histology of cultured MRL and CAST dorsal and ear fibroblasts with immunohistochemical (IHC) staining for complement factor H (CFH) and DARI nuclear counterstain. Right, quantification of CFH expression across in vitro conditions. CFH expression was highest in MRL ear fibroblasts, consistent with diffASE gene expression results. *P < 0.05. C. Schematic of MRL and CAST dorsal and ear wounding for histology. D. Fluorescent histology (left) and quantification (right) of IHC staining of wounds for CFH (with DAPI nuclear counterstain) showing that CFH expression was specific to MRL ear wounds. *P < 0.05. E. Schematic of wildtype mouse dorsal splinted wounding with local wound treatment with either recombinant CFH protein or phosphate-buffered saline (PBS; vehicle control). F. Gross photographs of control (-CFH) and CFH-treated (+CFH) wounds showing a less prominent scar and more robust re-epithelialization in +CFH wounds. Black dotted outline indicates healed wound region. G. Picrosirius red connective tissue histology of -CFH and +CFH wounds and unwounded skin (UW). H. T-distributed stochastic neighbor embedding (t-SNE) plot of quantified extracellular matrix (ECM) ultrastructural parameters, based on picrosirius red histology (G), showing overall similarities/differences in ECM ultrastructure between wound and skin conditions. CFH-treated wounds had an ECM architecture intermediate between that of control scars (-CFH) and unwounded skin, indicating partial ECM regeneration in +CFH wounds. Each dot represents quantified parameters from one histologic image. I. Hematoxylin and eosin (H&E) histology of POD 14 wounds and skin showing more complete re-epithelialization and presence of putative regenerating dermal appendages (hair follicles or glands) in +CFH wounds, compared to lack of appendages in -CFH wounds which form a “bare area” of fibrotic scar. Yellow dotted lines indicate borders of healed wounds. J. Dermal thickness quantified from histology of wounds and skin, showing that +CFH wounds have decreased scar thickness (closer to the thickness of UW skin) compared to control (-CFH) wounds.

Figure 11 : CFH treatment reduces scarring through Cxcl2 inhibition in dorsal skin wounds. A. Schematic of CFH and PBS treatment scRNA experiment. B. UMAP of all cells captured from scRNA-seq experiments colored by cell type. C. UMAP of Seurat defined fibroblast clusters. D. Pie charts showing proportion of fibroblast clusters at POD 7 in unwounded, PBS, and CFH treated wounds. E. Violin plot of Cxcl2 expression by Seurat cluster of all timepoints. F. Violin plot of Cxcl2 expression by post-operative day and treatment group. G. Immunostaining of CXCL2 in unwounded, PBS, and CFH treated wounds at POD 7 with quantification right (*p < 0.05). H. Schematic of CXCL2 receptor inhibitor (CXCLR2i) treatment experiment. I. H&E analysis of unwounded, PBS, and CXCLR2i treated wounds at POD 7. (Yellow dotted lines show wound borders) J. Representative Picrosirius red analysis of unwounded, PBS, and CXCLR2i treated wounds at POD 7 and UMAP quantification right. Scale bar; A, 150 |im, B, 250 |im, C, 40 urn.

Figure 12: T-distributed stochastic neighbor embedding (t-SNE) plots of quantified extracellular matrix (ECM) ultrastructural parameters, based on picrosirius red histology of dorsal (left) and ear (right) wounds, showing overall similarities/differences in ECM ultrastructure between wound and skin conditions. Each dot represents quantified parameters from one histologic image. Overlaid shaded regions highlight clustering of ECM properties by biological condition.

Figure 13: Sequencing coverage obtained from MRL genome sequencing. Histogram shows number of bases at each level of coverage.

Figure 14: Principal component analysis of RNA-seq data clearly separates samples by cell population/type (endothelial cells, fibroblasts, or immune cells). Figure 15: Differences in expression between CAST and MRL alleles (i.e., |logz fold change|) in ear and dorsal wounds in each cell population. Black circles represent medians.

Figure 16: Overlap of allele-specific expression (ASE) detected in different cell populations in ear (left) and dorsal (right) wounds, showing number of genes with significant ASE (FDR < 0.05 for MRL vs. CAST allelic expression) in each individual cell type or in multiple cell types (overlapping regions).

Figure 17: Genes with diffASE overlapping 19 QTL support intervals The -log(p-value) for diffASE for each gene (red and blue dots) are plotted versus genomic position for each cell type. Gray regions highlight QTL support intervals for which we identified a gene with significant diffASE. Red dots highlight genes with significant diffASE within a QTL interval.

Figure 18: Dose responsiveness of reduced scar thickness with CFH treatment. Quantified scar thickness (first panel (A)) and representative H&E histology (second through fourth panels (B)) of wildtype mouse wounds treated with PBS (control) or CFH at varying doses (5 or 10 mg/mL). The reduction in scar thickness with CFH treatment was more pronounced with a higher dose of CFH.

Figure 19: Allelic proportions (MRL reads/CAST reads per gene, summed across replicates) in (A) immune cells, (B) endothelial cells, and (C) fibroblasts. Allelic proportions center on 0.5 in each condition.

Figure 20: (A) Gross images of PBS, and CFH treated wounds at POD 3 and POD 7. (B) H&E analysis of PBS and CFH treated wounds at POD 3 and POD 7. (Yellow dotted lines show wound borders) (C) Picrosirius red analysis of unwounded, PBS, and CFH treated wounds at POD 3 (left) and POD 7 (right). (D) Immunostaining of PBS and CFH treated wounds at postoperative day 7 for CFH, Collagen type 1 and Vimentin (Green) with quantification right (*p < 0.05). Scale bar; (A) 3 mm, (B) 250 |im, (C) 150 |im.

Figure 21 : (A) UMAP of all cells captured from single-cell-RNAseq experiments colored by Seurat cluster. (B) Pie charts showing proportion of fibroblast clusters grouped by either cluster 0 (red) or cluster 1 -6 (black) at POD 3, POD 7 and POD 14 in PBS and CFH treated wounds. (C) Pie charts showing proportion of fibroblast clusters grouped by individual cluster at POD 3, POD 7, and POD 14 in PBS and CFH treated wounds. (D) Gene ontology pathway analysis of cluster 0 (top) and cluster 1 (bottom). (E) Bar graphs showing proportion of T cells (top) and Macrophages & Monocytes (bottom) across all timepoints and treatment groups.

Figure 22: (A) Immunostaining of PBS and CFH treated wounds at post-operative day (POD) 3 and 14 for CXCL2 (Green) and Collagen type I (Red). (B) Immunostaining of PBS and CFH treated wounds at post-operative day 7 for F480 and CD8. Scale bar (Green); (A) 150 urn. Figure 23: (A) Bar graphs showing number of interactions (left) and interaction strength (right) in CFH and PBS treated wounds by the Cell Chat platform. (B) Heat map showing differential number and strength of cell-cell communications in PBS compared to CFH treated wounds by the Cell Chat platform. (C) Heat map showing cell type signaling patterns in PBS compared to CFH treated wounds. (D) Relative information flow of specific pathways in CFH and PBS treated wounds.

Figure 24: (A) Gross images of PBS and CXCLR2i treated wounds at POD 3, 7 and 14. (B) H&E analysis of PBS and CXCLR2i treated wounds at POD 3 and 14. (Yellow dotted lines represent wound edges) Scale bar; (A) 3 mm, (B) 250 |im.

Figure 25: Reads mapped per library.

Figure 26: The number of genes with sufficient read counts (30 reads per wound type, allele) to be analyzed for allele-specific expression in each cell type.

Figure 27: ASE results from DESeq2 at FDR<0.1 and FDR<0.05.

Figure 28: Directionality and magnitude for genes with allele-specific expression in both ear and dorsal wounds.

Figures 29A-29B: Genes with differential allele-specific expression in fibroblasts associated with mutant phenotypes related to abnormal response to injury (annotated to mutant phenotypes based on ModPhEA).

Figures 30A-30B: Genes associated with differential allele-specific expression in fibroblasts associated with the GO terms “response to wounding" and “wound healing” (annotated with PANTHER, GO Ontology database released 2019-12-09).

DEFINITIONS

As used herein in its conventional sense, the term “fibroblast” refers to a cell responsible for synthesizing and organizing extracellular matrix. Two fibroblast lineages include Engrailed-1 lineage-negative fibroblasts (ENFs) and Engrailed-1 lineage-positive fibroblasts (EPFs). The EPF lineage includes all cells that express Engrailed-1 at any point during their development, and all progeny of those cells.

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.

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.

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” 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.

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.

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.

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.

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 T ransforming Growth Factor-0, a secreted protein that exists in at least three isoforms called TGF-0I , TGF-02 and TGF-03 (referred to collectively as TGF-0). TGF-0 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.

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.

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

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.

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.

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.

The term “hair follicle formation” or "induction of hair follicle formation" as used herein in its conventional sense refers to a phenomenon in which dermal papilla cells induce epidermal cells to form the structure of the hair follicle.

The term “hair growth” or "induction of hair growth" as used herein in its conventional sense refers to a phenomenon in which hair matrix cells of the hair follicle differentiate and proliferate thereby forming the hair shaft, and dermal sheath cells act on the hair matrix or outer root sheath (ORS) to elongate the hair shaft from the body surface. In some cases, hair growth includes generating one or more new hair follicles. In some cases, hair growth includes generating one or more new hairs.

As used herein in its conventional sense, the term “alopecia” refers to a disease in which hair is lost. It can be due to a number of causes, such as androgenetic alopecia, trauma, radiotherapy, chemotherapy, iron deficiency or other nutritional deficiencies, autoimmune diseases and fungal infection. The loss of hair in alopecia is not limited just to head hair but can happen anywhere on the body. Alopecia is often accompanied by fading of hair color. Alopecia is often accompanied by deterioration of hair quality such as hair becoming finer or hair becoming shorter. With regard to types of alopecia, there are alopecia areata, androgenetic alopecia, postmenopausal alopecia, female pattern alopecia, seborrheic alopecia, alopecia pityroides, senile alopecia, cancer chemotherapy drug-induced alopecia, alopecia due to radiation exposure, trichotillomania, postpartum alopecia, etc. The types of alopecia are further described in U.S. Patent No. 9808511 , the entirety of which is incorporated by reference herein.

Alopecia areata is an auto-immune disease that can cause hair to fall out suddenly. Alopecia areata is alopecia in which coin-sized circular to patchy bald area(s) with a clear outline suddenly occur, without any subjective symptoms or prodromal symptoms, etc. in many cases, and subsequently when spontaneous recovery does not occur they gradually increase in area and become intractable. It may lead to bald patches on the scalp or other parts of the body. Hair growth in the affected hair follicles is reduced or completely ceases. Alopecia areata is known to be associated with an autoimmune disease such as a thyroid disease represented by Hashimoto's disease, vitiligo, systemic lupus erythematosus, rheumatoid arthritis, or myasthenia gravis or an atopic disease such as bronchial asthma, atopic dermatitis, or allergic rhinitis.

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 neovascularization 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.

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 C0₂ 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.

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.

DETAILED DESCRIPTION

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.

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.

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.

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.

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.

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.

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.

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 U.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.

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.

METHODS

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 some cases, the methods promote hair growth on a subject. In certain embodiments, aspects of the methods include administering an effective amount of a complement pathway inhibitor to a wound to promote healing of the wound. The methods may be applied to any cell or population of cells as described herein.

As summarized above, aspects of the methods may include administering an effective amount of a complement pathway inhibitor to a wound. Complement is an arm of the innate immune system that plays an important role in defending the body against infectious agents. The complement system comprises more than 30 serum and cellular proteins that are involved in three major pathways, known as the classical, alternative, and lectin pathways. The classical pathway is usually triggered by binding of a complex of antigen and IgM or IgG antibody to C1 (though certain other activators can also initiate the pathway). The C1 complex consists of the recognition molecule 01 q and the tetrameric protease complex 01 r₂s₂. 01 q consists of six heterotrimers each containing 01 qa, C1qb, and 01 qc chains. Activated C1 cleaves 04 and 02 to produce C4a and C4b, in addition to C2a and C2b. C4b and C2a combine to form 03 convertase, which cleaves 03 to form C3a and C3b. Binding of C3b to 03 convertase produces 05 convertase, which cleaves 05 into 05a and C5b. 03a, C4a, and 05a are anaphylatoxins and mediate multiple reactions in the acute inflammatory response. 03a and 05a are also chemotactic factors that attract immune system cells such as neutrophils. The alternative pathway is initiated by and amplified at, e.g., microbial surfaces and various complex polysaccharides. In this pathway, hydrolysis of 03 to C3(H₂O), which occurs spontaneously at a low level, leads to binding of factor B, which is cleaved by factor D, generating a fluid phase 03 convertase that activates complement by cleaving 03 into 03a and C3b. C3b binds to targets such as cell surfaces and forms a complex with factor B, which is later cleaved by factor D, resulting in a 03 convertase. Surface-bound 03 convertases cleave and activate additional 03 molecules, resulting in rapid C3b deposition in close proximity to the site of activation and leading to formation of additional 03 convertase, which in turn generates additional C3b. This process results in a cycle of 03 cleavage and 03 convertase formation that significantly amplifies the response. Cleavage of 03 and binding of another molecule of C3b to the 03 convertase gives rise to a 05 convertase. 03 and 05 convertases of this pathway are regulated by host cell molecules CR1 , DAF, MOP, CD59, and fH. The mode of action of these proteins involves either decay accelerating activity (i.e., ability to dissociate convertases), ability to serve as cofactors in the degradation of C3b or C4b by factor I, or both. Normally the presence of complement regulatory proteins on host cell surfaces prevents significant complement activation from occurring thereon. The 05 convertases produced in both pathways cleave 05 to produce 05a and C5b. C5b then binds to 06, 07, and 08 to form C5b-8, which catalyzes polymerization of 09 to form the C5b-9 membrane attack complex (MAC). The MAC inserts itself into target cell membranes and causes cell lysis. Small amounts of MAC on the membrane of cells may have a variety of consequences other than cell death. The lectin complement pathway is initiated by binding of mannose-binding lectin (MBL) and MBL-associated serine protease (MASP) to carbohydrates. The MB1 -1 gene (known as LMAN-1 in humans) encodes a type I integral membrane protein localized in the intermediate region between the endoplasmic reticulum and the Golgi. The MBL-2 gene encodes the soluble mannose-binding protein found in serum. In the human lectin pathway, MASP-1 and MASP-2 are involved in the proteolysis of C4 and C2, leading to a C3 convertase described above.

Complement activity is regulated by various mammalian proteins referred to as complement control proteins (CCPs) or regulators of complement activation (RCA) proteins (U.S. Pat. No. 6,897,290). These proteins differ with respect to ligand specificity and mechanism(s) of complement inhibition. They may accelerate the normal decay of convertases and/or function as cofactors for factor I, to enzymatically cleave C3b and/or C4b into smaller fragments. CCPs are characterized by the presence of multiple (typically 4-56) homologous motifs known as short consensus repeats (SCR), complement control protein (CCP) modules, or SUSHI domains, about 50-70 amino acids in length that contain a conserved motif including four disulfide-bonded cysteines (two disulfide bonds), proline, tryptophan, and many hydrophobic residues. The CCP family includes complement receptor type 1 (CR1 ; C3b:C4b receptor), complement receptor type 2 (CR2), membrane cofactor protein (MCP; CD46), decay-accelerating factor (DAF), complement factor H (fH), and C4b-binding protein (C4bp). CD59 is a membrane-bound complement regulatory protein unrelated structurally to the CCPs. Complement regulatory proteins normally serve to limit complement activation that might otherwise occur on cells and tissues of the mammalian, e.g., human host.

A variety of different complement inhibitors may be used in various embodiments of the invention. In general, a complement inhibitor can belong 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). In certain embodiments a complement inhibitor inhibits an enzymatic activity of a complement protein. The enzymatic activity may be proteolytic activity, such as ability to cleave another complement protein. In some embodiments, a complement inhibitor inhibits cleavage of C3, C5, or factor B. In some embodiments, a complement inhibitor acts on C3. In some embodiments, a complement inhibitor acts on a complement component that lies upstream of C3 in the complement activation cascade. In some embodiments, a complement inhibitor acts on the C1 complex. In some embodiments, a complement inhibitor acts on C1 q. In some embodiments, the complement inhibitor acts on 01 qb. In some embodiments, a complement inhibitor inhibits activation or activity of at least one soluble complement protein. In certain embodiments a complement inhibitor that inhibits at least the alternative pathway of complement activation is used. In certain embodiments a complement inhibitor that inhibits at least the classical pathway of complement activation is used. In certain embodiments a complement inhibitor that inhibits both the classical and the alternative pathway is used. In some embodiments a complement inhibitor that inhibits C3 activation or activity is used. In some embodiments, a complement inhibitor inhibits activation of at least one complement receptor protein expressed in the respiratory system. In certain embodiments the complement receptor protein is a receptor for C3a. In certain embodiments the complement receptor protein is a receptor for C5a. Further details regarding complement inhibitors that may be employed in embodiments of the invention are provided in U.S. Patent No. 1 1 ,013,782, the disclosure of which is herein incorporated by reference.

In some instances, the complement inhibitor is a small molecule agent that exhibits the desired 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.

In some cases, the complement inhibitor is a protein or fragment thereof or a protein complex. In some cases, the complement inhibitor is an antibody binding agent or derivative thereof. 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. In some cases, the Complement inhibitor is an enzyme or enzyme complex. In some cases, the Complement inhibitor includes a phosphorylating enzyme, e.g., a kinase. In some cases, the Complement inhibitor is a complex including a guide RNA and a CRISPR effector protein, e.g., Cas9, used for targeted cleavage of a nucleic acid. In some cases, the complement inhibitor is a nucleic acid. The nucleic acids may include DNA or RNA molecules. In certain embodiments, the nucleic acids modulate, e.g., inhibit or reduce, the activity of a gene or protein, e.g., by reducing or downregulating the expression of the gene. The nucleic acid may be a single stranded or double-stranded and may include modified or unmodified nucleotides or non-nucleotides or various mixtures and combinations thereof. In some cases, the complement inhibitor includes intracellular gene silencing molecules by way of RNA splicing and molecules that provide an antisense oligonucleotide effect or an RNA interference (RNAi) effect useful for inhibiting gene function. In some cases, gene silencing molecules, such as, e.g., antisense RNA, short temporary RNA (stRNA), doublestranded RNA (dsRNA), small interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), tiny non-coding RNA (tncRNA), snRNA, snoRNA, and other RNAi-like small RNA constructs, may be used to target a protein-coding as well as non-protein-coding genes. In some case, the nucleic acids include aptamers (e.g., spiegelmers). In some cases, the nucleic acids include antisense compounds. In some cases, the nucleic acids include molecules which may be utilized in RNA interference (RNAi) such as double stranded RNA including small interfering RNA (siRNA), locked nucleic acid (LNA) inhibitors, peptide nucleic acid (PNA) inhibitors, etc. In some cases the complement inhibitor is a nucleic acid encoding a protein or active fragment thereof, e.g., a factor H protein or active fragment thereof.

Complement inhibitors that may be employed in embodiments of the invention include, but are not limited to: Cinryze, Berinert, Ruconest, Sutimlimab, Pegcetacoplan, Eculizumab, Ravulizumab, Avacopan, etc.

In some instances, the complement inhibitor is a Factor H polypeptide or nucleic acid coding sequence therefor. Unless otherwise specified, the term "Factor H" or "FH" as used herein refers to both plasma-derived and recombinant Factor H. Factor H is a protein component of the alternative pathway of complement encoded by the complement factor G gene (for example, CFH; NM000186; GenelD:3075; UniProt ID P08603; Ripoche et al., Biochem. J. 249:593-602 (1988)). Factor H is translated as a 1 ,213 amino acid precursor polypeptide which is processed by removal of an 18 amino acid signal peptide, resulting in the mature Factor H protein (amino acids 19- 1231 ). As used in the present invention, Factor H encompasses any natural variants, alternative sequences, isoforms or mutant proteins that can be found in a plasma sample, for example a human plasma sample. Examples of Factor H mutations found in the human population include, without limitation, Y402H; V62I; R78G; R127L; .DELTA.224; Q400K; C431 S; T493R; C536R; I551T; R567G; C630W; C673S; C673Y; E850K; S890I; H893R; C915S; E936D; Q950H; Y951 H; T956M; C959Y; W978C; N997T; V1007I; V1007L; A1010T; T1017I; Y1021 F; C1043R; N1050Y; I1059T; Q1076R; R1078S; D1 119G; V1134G; Y1142D; Q1143E; W1 157R; C1163W; W1183L; W1 183R; T1184R; L1189R; S1191 L; G1194D; V1 197A; E1 198A; F1199S; R1210C; R1215G; R1215Q; YPTCAKR1225:1231 FQS; and P1226S. As will be appreciated, the Factor H used in the methods and compositions described herein may be plasma-derived or recombinant and may further comprise one or more different variants (including full-length and truncated forms). Variants of plasma-derived Factor H and methods for producing plasma-derived Factor H are known in the art and are described for example in WO 2007/149567; W02007/066017; W02008/113589; WO201 1/011753; U.S. Pat. No. 7,745,389, each of which is hereby incorporated by reference in its entirety for all purposes and in particular for all teachings related to the production of Factor H, particularly plasma-derived Factor H. A wide variety of Factor H polymorphisms are known in the art and described for example in W02000/52479; WO/2006/062716; U.S. Pat. No. 7,351 ,524; U.S. Pat. No. 7,745,389 (each which is herein incorporated by reference in its entirety for all purposes and in particular for all teachings related to Factor H and variants of Factor H), which also describe recombinant forms of these Factor H polypeptides and methods for producing the same. Many of these variant forms of Factor H are known as "protective" variants that show greater activity in limiting complement activation than plasma-derived Factor H. In non-limiting embodiments, the factor H active agent is as described in United States Published Patent Application Publication No. 20140336121 as well as U.S. Patent Nos. 1 1 ,007,254 and 10,233,235, the disclosures of which are herein incorporated by reference.

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 factor polypeptides provided above. By nucleic acid composition is meant a composition comprising a sequence of DNA 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., factor H or an 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.

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.

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.

The DNA 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-.beta.-ol 3- urethanyl)-N',N'-dimethylethylene diamine). When the DNA of interest is introduced using a liposome, in some instances one first determines in vitro the optimal values for the DNA: lipid ratios and the absolute concentrations of DNA 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.

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).

Instead of administration of a polypeptide of interest, e.g., factor H, 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.

As used herein, “an effective amount of a complement inhibitor” refers to an amount of a complement inhibitor suitable to promote healing of a wound. In some cases, an effective amount of a complement inhibitor includes one or more unit doses of the complement inhibitor 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 a complement inhibitor composition includes a single dose, e.g., a single injection, of the complement inhibitor. The complement inhibitor may include any suitable amount of complement inhibitor such as, e.g., an effective amount of a complement inhibitor. In some cases, the effective amount of a complement inhibitor does not delay wound closure or the wound closure rate. In some cases, the complement inhibitor includes an effective amount of a complement inhibitor 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, or 1 mg/ml to 5 mg/ml. The effective amount of the complement inhibitor 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.

In some embodiments, the complement inhibitor is administered as a pharmaceutically acceptable composition in which one or more complement inhibitors 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 complement inhibitors. In some cases, the complement inhibitor composition includes, e.g., a derivative of complement inhibitor. “Derivatives” include pharmaceutically acceptable salts and chemically modified agents.

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.

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.

The complement inhibitor composition may be stored at any suitable temperature. In some cases, the complement inhibitor composition is stored at temperatures ranging from 1^e C to 30 ^eC, from 2- C to 27 ^eC, or from 5^eC to 25 ^eC. The complement inhibitor composition may be stored in any suitable container, as described in detail below.

The complement inhibitor composition may be administered to a wound in a dermal location a subject. In some cases, the complement inhibitor 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 Complement inhibitor composition deposited thereon, as well as microneedles that include internal reservoirs that are configured to receive a complement inhibitor composition therein and disperse the complement inhibitor 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.

The complement inhibitor composition may be administered at any suitable time. In some cases, the complement inhibitor composition is administered to a wound immediately after formation of the wound in a subject. In some cases, the complement inhibitor 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 complement inhibitor 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.

In certain embodiments, aspects of the methods may include administering an effective amount of a Wnt signaling pathway inhibitor to a wound. Wnt signaling plays an important role in a variety of biological processes, such as cell proliferation, differentiation, organogenesis, tissue regeneration, and tumorigenesis. Wnt signaling can be broadly divided into p-catenin-dependent and p-catenin-independent signaling. Physiological activation of Wnt signaling is mediated by the binding of secreted WNT ligands to LRP5/6 coreceptors and frizzled (FZD) receptors. In the p- catenin-dependent signaling arm, binding of WNT ligands to FZD receptors results in inhibition of phosphorylation-mediated protein degradation of [3-catenin, leading to stabilization and accumulation of [3-catenin to drive transactivation of Wnt target genes. Wnt signaling is also modulated by a variety of factors. FZD receptors are ubiquitinated by E3 ubiquitin ligase ring finger proteins (RNF43/ZNRF3), targeting the FZD receptors for degradation and thus dampening Wnt signaling. R-spondin ligands are cysteine-rich glycoproteins that bind to cognate leucine-rich repeat-containing G-protein coupled receptors (LGR3/5/6 receptors) and RNF43/ZNRF3 to prevent FZD receptor ubiquitination and subsequent degradation, thus potentiating and amplifying Wnt signaling.

A variety of different Wnt signaling inhibitors may be used in various embodiments of the invention. In general, a Wnt signaling inhibitor can belong 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).

In some embodiments, a Wnt signaling inhibitor inhibits secretion of Wnt ligands. In some embodiments a Wnt signaling inhibitor binds Wnt ligands to inhibit binding of Wnt ligands to FZD receptors. In some embodiments a Wnt signaling inhibitor binds to FZD receptors to inhibit binding of Wnt ligands to FZD receptors. In some embodiments a Wnt signaling inhibitor reduces stabilization or induces degradation of [3-catenin. Wnt signaling inhibitors that may be employed in the embodiments of the invention include but are not limited to: WNT974, OMP-18R5, PRI-724, etc. Further details regarding Wnt signaling inhibitors that may be employed in embodiments of the invention are provided in (Jung, Youn-Sang, and Jae-ll Park. “Wnt signaling in cancer: therapeutic targeting of Wnt signaling beyond [3-catenin and the destruction complex.” Experimental & Molecular Medicine 52.2 (2020): 183-191 ), the disclosure of which is herein incorporated by reference.

In some instances, the Wnt signaling inhibitor is an inhibitor of leucine-rich repeatcontaining G-protein coupled receptor 6 (LGR6). In some embodiments, a LGR6 inhibitor acts on LGR6. In some embodiments, a LGR6 inhibitor inhibits association of LGR6 with R-spondins. In some embodiments, a LGR6 inhibitor inhibits association of LGR6 with E3 ubiquitin ligase ring finger proteins. In some embodiments, a LGR6 inhibitor inhibits association of LGR6 with FZD receptors. Inhibitors of leucine-rich repeat-containing G-protein coupled receptors (e.g. anti-LGR or anti-R-spondin antibodies) are known in the art and described for example in U.S. Pat. No. 8,802,097, U.S. Pub. Pat. App. 2018/0099047 A1 , U.S. Pub. Pat. App. 2018/0099047 A1 ,U.S. Pub. Pat. App. 2018/0312604 A1 , and U.S. Pub. Pat. App. 2021/0228682 A1 , the disclosures of which are herein incorporated by reference. In certain embodiments, aspects of the methods may include administering an effective amount of a CXC motif chemokine ligand 2 (CXCL2) or CXC motif chemokine receptor 2 (CXCR2) inhibitor to a wound. Chemokines are a subset of chemotactic and immune-cell modulating cytokines that play a role in immune homeostasis and the inflammatory response. Chemokines can be grouped into four sub-families based on the position of the first two cysteines (C) in the primary sequence: C, CC, CXC, and CX3C; where 'X’ is a non-conserved amino acid. CXC chemokine receptors are members of the G-protein-coupled receptor family and signal through a G-protein activated second messenger system.

A variety of different CXCL2/CXCR2 inhibitors may be used in various embodiments of the invention. In general, a CXCL2/CXCR2 inhibitor can belong 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).

In some embodiments, the CXCL2/CXCR2 inhibitor acts on CXCL2. In some embodiments, the CXCL2/CXCR2 inhibitor acts on CXCR2. In some embodiments, the CXCL2/CXCR2 inhibitor acts on CXCL2 to inhibit binding of CXCL2 to CXCR2. In some embodiments, the CXCL2/CXCR2 inhibitor acts on CXCR2 to inhibit binding of CXCL2 to CXCR2.

In some instances, the CXCL2/CXCR2 inhibitor is a CXCR2 antagonist. In some embodiments, the CXCR2 antagonist is a small molecule. CXCR2 antagonists are known in the art and described for example in U.S. Pat. No. 11 ,040,960 and WO 2022/098822 A1 , the disclosures of which are herein incorporated by reference.

CXCR2 antagonists that may be employed in the embodiments of the invention include but are not limited to: AZ 10397767 (Tocris), etc.

In some instances, the methods may include administering a secreted phosphoprotein 1 (SPP1 ) protein or fragment thereof or nucleic acid coding sequence therefor to a wound. SPP1 , also known as osteopontin (OPN), is a multifunctional protein that possesses cytokine, chemokine, and signal transduction functions and plays a role in intercellular communication and the extracellular matrix. Methods of expressing and purifying recombinant SPP1 are known in the art and described for example in Weng, Shunyan, et al. “Expression and purification of non-tagged recombinant mouse SPP1 in E. coli and its biological significance.” Bioengineered 5.6 (2014): 405-408, the disclosure of which is herein incorporated by reference.

In some instances, the methods may include administering a secretory leukocyte protease inhibitor (SLPI) protein or fragment thereof or nucleic acid coding sequence therefor to a wound. SLPI is a serine protease inhibitor that plays a role in regulating innate and adaptive immunity and wound repair. Methods of making and using recombinant protein protease inhibitors, such as SLPI, are known in the art and described for example in U.S. Pat. No. 7,709,446.

In some instances, the methods may include administering a thrombospondin 4 (THBS4) protein or fragment thereof or nucleic acid coding sequence therefor to a wound. THBS4 is a member of the thrombospondin family of calcium binding extracellular proteins that play a role in cell proliferation, cell adhesion, and cell migration. Methods of making, expressing, and purifying recombinant THBS4 are known in the art and described for example in Lawler, Jack, et al. "Characterization of Human Thrombospondin-4 (*).'' Journal of Biological Chemistry 270.6 (1995): 2809-2814.

In certain embodiments, aspects of the methods may include administering an effective amount of an adenosine A2b receptor (ADORA2B) inhibitor to a wound. ADORA2B is a member of the adenosine binding P1 receptor family that is known to play a role in pro-inflammatory, antiinflammatory, and immunosuppressive responses. Inhibitors of ADORA2B are known in the art and described for example in U.S. Pat. No. 11 ,072,597 and U.S. Pat. No. 11 ,161 ,850, the disclosures of which are herein incorporated by reference.

In certain embodiments, aspects of the methods may include administering an effective amount of a junctional adhesion molecule-like (JAML) inhibitor. JAML is a member of the junctional adhesion molecule (JAM) family of proteins that facilitate tight-junction assembly and are known to play a role in development, angiogenesis, inflammation, and cancer. Inhibitors of JAML are known in the art and described for example in U.S. Pat. No. 9,605,083, the disclosure of which is herein incorporated by reference.

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 complement inhibitor 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.

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 certain embodiments, the methods provided herein promote hair growth on a subject, as described in detail below. In certain embodiments, the methods provided herein treat a subject for alopecia, e.g., by promoting hair growth in areas of hair loss, as described in detail below. 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 complement inhibitor 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 complement inhibitor 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.

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.

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), neodymium yttrium 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.

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.

Scar Reduction

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 a Complement inhibitor 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 a Complement inhibitor composition to the wound to prevent scarring of the wound, e.g., according to any of the embodiments described herein. The level or amount of scarring may be assessed and measured according to any convenient metric. The levels of scarring, e.g., in a wound treated with a complement inhibitor composition during healing or a healed wound treated with a complement inhibitor composition, may be assessed relative to a control, e.g., a wound or healed wound not treated with a complement inhibitor 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%.

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 Complement inhibitor composition. In certain embodiments, the method produces a scar-less 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 Complement inhibitor 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 Complement inhibitor 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 Complement inhibitor 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 a Complement inhibitor 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 a Complement inhibitor 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 a Complement inhibitor 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.

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, 1 1% 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, 1 1 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 a Complement inhibitor composition. 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 a Complement inhibitor composition.

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.

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.

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, 1 % 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.

According to some embodiments, the methods increase the number of hairs at a dermal location compared to a control. In some cases, the methods increase the number of hairs 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 increase the number of hairs 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. 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 a Complement inhibitor composition is effective to modulate messenger RNA (mRNA) levels expressed from scar-related genes. According to another embodiment, an effective amount of a Complement inhibitor 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-p1 (TGF- 1 ), 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 mucin-like hormone receptor-like 1 (EMR1 ), CD26, YAP, fibronectin, or one or more of the sma / 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.

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 a Complement inhibitor composition.

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.

Hair Growth

In certain embodiments, the methods provided herein promote hair growth on a subject in a dermal location. In some embodiments, the subject may have alopecia and/or have been diagnosed with alopecia. In certain embodiments, the methods are methods for treating a subject for alopecia, e.g., by promoting hair growth in a dermal location of hair loss. In certain embodiments, the methods include forming a wound in a dermal location of a subject where hair growth is desired, e.g., according to any of the embodiments described herein, and administering an effective amount of a complement inhibitor 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 may include forming a wound in a dermal location where hair growth is desired of a subject, e.g., according to any of the embodiments described herein, and administering an effective amount of a complement inhibitor composition to the wound.

In certain embodiments, the methods provided herein promote hair growth on a subject. The methods may induce or promote hair growth at any suitable dermal location in a subject. In certain embodiments, the methods promote or induce hair growth in a dermal location devoid of dermal appendages, e.g., hair follicles, sweat glands, etc. In some cases, the dermal location is hairless. In some cases, the dermal location includes a scar. In certain embodiments, the methods promote or induce hair growth in a dermal location having dermal appendages. In some cases, the dermal location includes hair. The dermal location may be located at any portion of the body where hair may naturally grow such as, e.g., the scalp, face, legs, arms, etc. In certain embodiments, the dermal location is present on a hairless area of the scalp of a subject. In certain embodiments, the dermal location includes the entire surface of the scalp of a subject.

The level of hair growth may be assessed and measured according to any convenient metric. The levels of hair growth may be assessed relative to a control, e.g., a dermal location characterized by hair loss, a dermal location devoid of dermal appendages, a wound not treated with a complement inhibitor composition, or healed wound not treated with a Complement inhibitor composition. In certain embodiments, hair growth is determined by detecting the presence of new hairs appearing in a dermal location. In this method, hair growth may be confirmed when tips of the new hairs appear on the treatment area. Hair growth may also be determined by detecting hair follicle formation and/or measuring an increase in length of the hair follicles. In some cases, hair growth includes generating one or more new hair follicles. Hair growth may also be determined by measuring a change in the hairline. In some cases, the change in the hairline is determined by measuring the change in distance between any point on the hairline and the browline of the subject’s head. In some cases, the methods decrease the amount of hair falling out compared to a control. In some cases, the methods prevent the progress of hair loss. In certain embodiments, there is no recurrence of hair loss permanently or for a period of time after performing the methods including, e.g., one month or more, two months or more, three months or more, half a year or more, one year or more, two years or more, three year or more, five years or more, or ten years or more.

According to some embodiments, the methods decrease the amount of hair loss compared to a control. In some cases, the methods decrease the amount of hair loss 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 the amount of hair loss 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 a complement inhibitor composition.

According to some embodiments, the methods increase the number of hair follicles at a dermal location, e.g., treated with a complement inhibitor composition, compared to a control. In some cases, the methods increase the number of hair follicles 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 increase the number of hair follicles 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 a complement inhibitor composition.

According to some embodiments, the methods increase the number of hairs at a dermal location compared to a control. In some cases, the methods increase the number of hairs 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 increase the number of hairs 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 a complement inhibitor composition.

COMBINATION THERAPY

For use in the subject methods, the complement inhibitor(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.

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.

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®).

Further details regarding YAP inhibitors and methods of using the same are provided in United States Patent Application Serial No. 17/626,699; the disclosure of which is herein incorporated by reference.

In some instances, aspects of the methods may include administering an effective amount of a complement inhibitor 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; the disclosure of which is herein incorporated by reference.

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 complement inhibitor is administered.

KITS

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 a complement inhibitor 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 promoting hair growth. In some cases, the kits are suitable for practicing embodiments of the methods for ameliorating scar formation. In some cases, the kits are suitable for practicing embodiments of the methods for treating a subject for alopecia.

The complement inhibitor composition may be present in any suitable amount. In some cases, the kit includes an effective amount of a complement inhibitor composition, e.g., according to the embodiments described above. The complement inhibitor composition may be present in any suitable container that is compatible with the complement inhibitor composition. By “compatible” is meant that the container is substantially inert (e.g., does not significantly react with) the liquid and/or reagent(s) of the Complement inhibitor composition 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. The container for holding the complement inhibitor composition may be configured to hold any suitable volume of the complement inhibitor composition. In some cases, the size of the container may depend on the volume of Complement inhibitor composition to be held in the container. In certain embodiments, the container may be configured to hold an amount of complement inhibitor 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 complement inhibitor 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 complement inhibitor 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 complement inhibitor composition) ranging from 0.1 ml to 200 ml.

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.

As described above, embodiments of the container can be compatible with the complement inhibitor 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.

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.

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 a Complement inhibitor 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 Complement inhibitor 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 a Complement inhibitor 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.

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.

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.

The subject methods further find use in applications for promoting hair growth. The subject methods may find use in applications where increased hair growth in a particular dermal location is desired, e.g., a region of substantial hair loss. In certain embodiments, the methods find use in treating hair loss and conditions involving hair loss as a side effect. The methods may be used to treat hair loss from a variety of conditions, such as, but not limited to hormonal changes during pregnancy and childbirth, disease (hyper- and hypo-thyroidism, lupus, trichotillomania), medication, chemotherapy, dietary deficiencies, stress, alopecia, trauma, radiotherapy, iron deficiency or other nutritional deficiencies, autoimmune diseases and fungal infection. In certain embodiments, the subject methods find use in treating a subject for alopecia.

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

EXAMPLES

I. Novel Genetic Analysis of MRL Mice Reveals that Complement Inhibition by Factor H Reduces Scarring

A. Background:

MRL mice are uniquely able to regenerate ear punch wounds, while dorsal wounds heal via scarring. Identifying genes underlying this regeneration could provide novel therapeutic insights, but previous methods could not robustly identify specific genes driving strain-specific complex traits. In recent studies, outbreeding a strain with a trait of interest (here, ear regeneration) and assessing hybrid offspring for spatial/tissue-specific differences in relative gene expression from the two parent alleles (differential allele-specific expression; “diffASE”) could reveal genes underlying that phenotype. We hypothesized that diffASE could represent a novel approach to identify pro-regenerative genes in MRL mice.

B. Methods:

MRL and CAST mice were cross-bred and offspring underwent dorsal excisional and ear punch wounding. POD7 wounds were harvested and dermal immune (CD45+), endothelial (CD31 +), and fibroblast (Lin-) cells were FACS-isolated and underwent bulk RNAsequencing. Reads were mapped to the MRL or CAST genome based on strain-specific variants. Genes with significantly differing expression (FDR<5%, Benjamini-Hochberg method) from the MRL vs. CAST allele and dorsal vs. ear wounds were identified.

C. Results:

Consistent with prior reports, MRL ear wounds regenerated by POD28, while MRL dorsal and CAST ear and dorsal wounds formed fibrotic scars. For all cell types, substantially more genes in ear (vs. dorsum) had significantly different expression from MRL vs. CAST allele (e.g., 2531 vs. 159 unique genes in immune cells), consistent with ear-restricted phenotypic divergence. In fibroblasts, such genes were enriched for wound-related pathways (e.g., cell adhesion) and phenotypes. Fibroblast diffASE genes significantly overlapped with regions identified by a previous “fine-mapping” genomic study of MRL ear regeneration (*P=0.0094). Cfh (complement factor H), a negative complement modulator, had MRL-specific upregulation in ear fibroblasts and was within a fine-mapped region. Treating typically scarring MRL dorsal wounds with recombinant factor H (10 mg/mL) resulted in significantly reduced scar thickness (*P=0.0050) and less dense/fibrotic ECM on picrosirius red staining.

D. Conclusions:

In MRL hybrid mice, key wound cells exhibited markedly greater allele-specific gene expression in ear wounds (regenerative) than dorsal wounds (scarring), consistent with MRL- specific gene regulation leading to ear regeneration. Our results are supported by a previous “fine- mapping” study that identified genomic regions functionally associated with MRL ear regeneration. Integrating our results with this study revealed a role for complement-inhibitory factor H in wound regeneration, indicating a therapeutic approach to reduce scarring. Our results indicate that probing differential allele-specific gene expression may represent a powerful approach for identifying genes underlying complex mammalian traits.

II. NOVEL GENETIC ANALYSIS OF MRL MICE REVEALS THAT COMPLEMENT INHIBITION BY FACTOR H REDUCES SCARRING

A. Introduction

MRL mice have tissue-specific healing properties: ear punch wounds regenerate, while dorsal wounds scar. However, the genes responsible for MRL ear regeneration remain unknown.

In recent studies, outbreeding a non-mammalian strain with a trait of interest and assessing offspring for spatial/tissue-specific differences in relative gene expression from the parent alleles (differential allele-specific expression; “diffASE”) revealed genes underlying that phenotype. We hypothesized that diffASE could be applied to identify MRL pro-regenerative genes.

B. Methods

1 . Mice:

F1 hybrid mice were generated by crossing MRL and CAST mice. 2. Wound Models:

Dorsal wounding: a full-thickness 6mm region of skin was excised and splinted to prevent rapid wound contraction. Ear wounding: a 2mm thumb-style punch was used to create through-and-through injuries.

3. Cell Isolation:

POD7 wounds were harvested and digested. Cells were sorted via FACS to obtain three key wound cell populations:

1 . Bulk immune cells (CD45+)

2. Endothelial cells (CD31 +)

3. Fibroblasts (Lin-)

Each cell type was subjected to bulk RNA-seq (n > 3 replicates per cell type).

4. Sequencing Analysis:

Variant calling was performed to identify MRL- and CAST-specific variants. Reads were mapped to MRL or CAST genomes based on these variants. Genes with significantly differing expression (FDR<5%) from MRL vs. CAST alleles and dorsal vs. ear wounds were identified.

5. In vivo Complement Factor H Studies:

Excisional splinted wounds were treated with recombinant mouse complement factor H (Cfh) or vehicle control (PBS). Scar thickness was assessed based on average measurements from H&E histology images.

C. Results

1 . MRL ear wounds regenerate by POD28, while MRL dorsal wounds and CAST ear and dorsal wounds scar

Figure 1 : MRL ear wounds uniquely heal in an accelerated fashion and via tissue regeneration. A. Gross photographs (first two columns) and hematoxylin and eosin (H&E) histology (third column) of ear (top panels) and dorsal (bottom panels) wounds in CAST (“normal healer”) and MRL (“super healer”) mice, showing that MRL ear wounds heal through regeneration of multiple tissue types (including cartilage) while ear wounds in CAST mice, and dorsal wounds in both strains, heal via fibrotic scarring. B. Wound closure curves for MRL and CAST ear (top panel) and dorsal (bottom panel) wounds. CAST ear wounds do not close to an appreciable extent, while MRL ear wounds are completely or near-completely healed over by 3-4 weeks postwounding. Splinted dorsal wounds heal at a similar rate in MRL vs. CAST mice, with re- epithelialization complete by postoperative day (POD) 14. C. Sampling scheme for RNA-seq libraries. MRL and CAST were crossed to produce F1 hybrids for allele-specific expression analysis. Each adult F1 mouse underwent both dorsal excisional and ear punch wounding. On POD 7, wounds were collected by excising a 1 mm ring of tissue around each wound site. Cell populations were isolated via fluorescence-activated cell sorting (FACS) and RNA was extracted for bulk RNAseq. D. Principal component analysis of allele-specific F1 RNAseq samples. Allelespecific samples separated into distinct clusters by tissue/wound context (ear vs. dorsal) and allele (MRL vs. CAST). The results show that MRL ear wounds exhibit accelerated healing (vs. wildtype strain, CAST) and regenerate normal ear tissue. Furthermore, dorsal wounds in both M L and CAST result in scars with absent hair/glands and dense, fibrotic matrix.

2. “diffASE” analysis in MRL/CAST hybrids shows greater differential gene expression in ear vs. dorsal wounds, indicating that ear-specific gene cis-regulation underlies MRL ear regeneration.

Figure 2: Analysis of differential allele-specific expression (diffASE) reveals patterns of c/s-regulation unique to MRL ear wounds. A. Schematic example of diffASE between MRL and CAST in ear and dorsal wounds. Blue and green solid boxes represent gene regulatory regions affecting transcription of the MRL or CAST allele, respectively, of a given gene; transcription levels from each allele are represented by blue and green wavy lines. In the context of a dorsal wound (where MRL and CAST phenotypes are similar), expression is the same from the MRL vs. CAST allele. In contrast, in ear wounds, the present of a context-specific (i.e., wound- related) transcription factor (TF; grey circle) reveals ASE through differences in the sequence of the MRL vs. CAST regulatory elements (which respond differentially to the TF). Overall, this can be detected as a pattern of diffASE unique to ear wounds (exemplified in bottom panel bar graphs). The schematic shows that strain-specific (MRL) and tissue-specific (ear) gene regulation leads to altered gene expression specific to that tissue and allele, which is detected as diffASE (differential allele-specific gene expression). B. Venn diagrams showing number of genes with ASE in ear wounds (blue region), dorsal wounds (yellow region), or both (overlapping region) in each analyzed wound cell type. C. Scatterplots for each cell type comparing distribution of allelic ratios between dorsal and ear wounds. Colored points represent genes with diffASE (gold points are genes with a larger difference between CAST and MRL alleles in the ear; blue points are genes with a larger difference between CAST and MRL alleles in the dorsum). D. Gene set enrichment analysis for genes with evidence of diffASE in fibroblasts, which are highly enriched for gene ontology (GO) categories (left) and mutant phenotypes (right) related to wound healing and development. Such enrichment patterns were unique to fibroblasts (not seen in endothelial or immune cells). Fibroblasts are the end cellular mediators of scarring/fibrosis. F. Specific genes associated with mutant phenotypes or GO terms related to responses to injury and wound healing with diffASE in fibroblasts. Yellow circles represent fold changes between alleles in the dorsum; blue circles represent fold changes in the ear.

As shown, all cell types had significantly more genes with diffASE (i.e. , expression from MRL allele + expression from CAST allele) in ear vs. dorsal wounds, consistent with ear-specific phenotypic divergence. However, only fibroblast genes were enriched for involvement in wound-related processes.

3. Integration with a previous functional genomic study of MRL ear regeneration reveals Complement Factor H as a pro-regenerative gene

Figure 3: Integration of diffASE with fine-mapping study results. A. LOD scores vs. chromosome position for ear hole closure from Cheverud et al. (Heredity, 2014; PMID 24569637). Red circles indicate positions of the genetic markers closest to genes identified as having diffASE in fibroblasts. B. Distribution of mean LOD scores of permuted gene sets (20,000 permutations). Red line indicates the mean LOD score of genetic markers closest to the fibroblast diffASE gene set; this mean score was significantly higher than that of random gene sets (*P = 0.0094). C. Three genes within the fine-mapped QTL intervals annotated with the GO term “wound healing” (G0:0042060) show diffASE in fibroblasts. Of these, Cfh had the largest magnitude of ASE in ear wound fibroblasts.

Figure 4: Cfh treatment leads to partial regeneration and enhanced healing of dorsal wounds in wildtype mice. A. Schematic of MRL and CAST dermal fibroblast culture from dorsal and ear skin. B. Left, fluorescent histology of cultured MRL and CAST dorsal and ear fibroblasts with immunohistochemical (IHC) staining for complement factor H (CFH) and DAPI nuclear counterstain. Right, quantification of CFH expression across in vitro conditions. CFH expression was highest in MRL ear fibroblasts, consistent with diffASE gene expression results. *P < 0.05. C. Schematic of MRL and CAST dorsal and ear wounding for histology. D. Fluorescent histology (left) and quantification (right) of IHC staining of wounds for CFH (with DAPI nuclear counterstain) showing that CFH expression was specific to MRL ear wounds. *P < 0.05. E. Schematic of wildtype mouse dorsal splinted wounding with local wound treatment with either recombinant CFH protein or phosphate-buffered saline (PBS; vehicle control). F. Gross photographs of control (- CFH) and CFH-treated (+CFH) wounds showing a less prominent scar and more robust re- epithelialization in +CFH wounds. Black dotted outline indicates healed wound region. G. Picrosirius red connective tissue histology of -CFH and +CFH wounds and unwounded skin (UW). H. T-distributed stochastic neighbor embedding (t-SNE) plot of quantified extracellular matrix (ECM) ultrastructural parameters, based on picrosirius red histology (G), showing overall similarities/differences in ECM ultrastructure between wound and skin conditions. CFH-treated wounds had an ECM architecture intermediate between that of control scars (-CFH) and unwounded skin, indicating partial ECM regeneration in +CFH wounds. Each dot represents quantified parameters from one histologic image. I. Hematoxylin and eosin (H&E) histology of POD 14 wounds and skin showing more complete re-epithelialization and presence of putative regenerating dermal appendages (hair follicles or glands) in +CFH wounds, compared to lack of appendages in -CFH wounds which form a “bare area” of fibrotic scar. Yellow dotted lines indicate borders of healed wounds. J. Dermal thickness quantified from histology of wounds and skin, showing that +CFH wounds have decreased scar thickness (closer to the thickness of UW skin) compared to control (-CFH) wounds.

As shown, genes with diffASE overlapped significantly with genomic regions functionally associated with inheritance of the MRL ear regeneration trait. Treating MRL dorsal wounds with Cfh significantly reduced scar thickness.

D. Discussion

In MRL/CAST hybrid mice, greater allele-specific gene expression occurred in ear (regenerative) than dorsal (scarring) wounds. This finding is consistent with ear-restricted phenotypic divergence and thus supports our diffASE analysis methods.

Integration with a previous large-scale genomic study revealed significant overlap with genomic regions functionally implicated in MRL regeneration, further supporting the utility of diffASE in identifying functionally relevant genes. This analysis also revealed a role for complement factor H in MRL ear regeneration, which is supported by our preliminary in vivo wound data, indicating a therapeutic approach to reduce scarring.

Ill Novel Genetic Analysis of MRL Mice Reveals that Complement Inhibition by Factor H Reduces Scarring

A. Introduction

Fibrosis, or the replacement of functional tissue with non-functional connective tissue, can result from tissue damage to any organ in the human body. In the skin, fibrosis occurs as scarring and has major consequences for skin form and function. Scars lack the structures (e.g., hair, glands) of normal skin, compromising skin’s normal barrier system and its ability to thermoregulate, and are weaker and less flexible than uninjured skin. Healing via scarring has major consequences for human health: scarring can cause disfigurement, functional loss, and reduced quality of life. Despite the substantial clinical burden scars impose, there are no current therapies that induce scar-free healing in humans.

In contrast to humans, some other species possess the ability to regenerate skin after injury without scar formation. The Murphy Roths Large (MRL) mouse represents a valuable biomedical model for studying mammalian wound regeneration. While injuries to mammalian skin and other organs typically heal via formation of fibrotic scar tissue, MRL and its progenitor strain, the Large (LG/J) mouse, have been reported to regenerate multiple tissue types without fibrosis. The most well-studied example of MRL regeneration is that of ear punch wounds: while through- and-through ear wounds remain open and fail to regenerate the excised tissue in most mouse strains, MRL mice fully heal these wounds via initial formation of a blastema-like structure and subsequent regeneration of key tissue types including cartilage and hair-bearing skin. However, the molecular mechanisms underlying enhanced wound healing in the MRL ear remain poorly understood. To date, nine quantitative trait locus (QTL) mapping studies have been performed to identify associations between genomic regions and the ear closure phenotype, but these studies so far have failed to identify specific genes or pathways driving regenerative healing, with ear closure-associated QTL spanning dozens or hundreds of individual genes.

Identification of tissue- and behavior-specific c/s-regulatory divergence, through analyses of allele-specific gene expression in hybrids, has previously revealed genes and pathways underlying complex traits. MRL regeneration is wound site-specific - while ear wounds regenerate, dorsal wounds form fibrotic scars similar to other mouse strains- providing the opportunity to apply a similar approach to elucidate genes driving MRL ear regeneration. Here, we capitalize on mouse strain- and site-specific differences in healing to identify divergence in c/s-regulation of gene expression associated with MRL ear regeneration through allele-specific expression (ASE) analysis. Through single-cell RNAseq (scRNA-seq) we demonstrated that CFH treatment induces regeneration in dorsal wounds through CXCL2 inhibition. Lastly, chemical inhibition of CXCLR-2 in dorsal wounds mimicked the regenerative phenotype of MRL ear wounds. Collectively, our results highlight the power of this approach in dissecting complex phenotypes in mammals and implicate the complement pathway as a possible therapeutic target for improving wound healing and reducing scarring.

B. Methods

/. Mice

Mice were housed and maintained in sterile micro-insulators at the Stanford University Comparative Medicine Pavilion in accordance with Stanford University Administrative Panel on Laboratory Animal Care (APLAC) guidelines (APLAC-21308). Food and water were provided ad libitum. MRL/MpJ (MRL), CAST/EiJ (CAST), and C57BL/6J mice were obtained from The Jackson Laboratory (Bar Harbor, ME). Male MRL and female CAST mice were bred to produce CAST x MRL F1 hybrid (F1) offspring. Adult (postnatal day [P]60) female F1 mice were used for RNA-seq experiments, and both female and male P60 mice were used for all other experiments.

//. Dorsal and ear wounding

Mice underwent ear punch wounding and dorsal splinted excisional wounding following established protocols without modification. Briefly, mice were anesthetized using 1.5-2% isoflurane. All surgical tools were autoclaved prior to the procedure. For ear wounds, the skin was prepped using alcohol wipes. Punch wounds 2mm in diameter were created using a thumb-type metal ear punch (Fisherbrand). For mice wounded for histologic and wound curve analysis, one wound was created per ear, roughly in the middle of the pinna. For mice wounded for RNA-seq analysis, in order to obtain sufficient cells for analysis while minimizing the number of mice required, three wounds were created per ear, spaced at least 2mm apart. For dorsal wounds, hair was removed from the entire dorsum using an electric shaver followed by depilatory cream. The dorsal skin was then prepped using three sequential alternating swabs of betadine and 70% ethanol. Using sharp surgical scissors, two 6mm-diameter full-thickness excisional wounds were made per mouse, roughly at the level of the scapulae and 4mm lateral to midline. Wounds were stented open by affixing silicone rings (1cm internal diameter) around the wound using adhesive and eight simple interrupted sutures (6-0 nylon, Ethicon). Wounds were dressed using Tegaderm (3M) and dressings were changed every other day until harvest.

For dose response experiments with CFH-treated dorsal wounds (see Fig. 18), which were performed in CAST mice, recombinant mouse complement factor H protein (R&D Systems) was resuspended in phosphate-buffered saline (PBS) at a concentration of either 5 or 10 ng/mL, then 50 piL of CFH at these concentrations, or PBS (vehicle control), were injected locally into the wound base and surrounding dermis immediately following wounding (POD 0) and again at POD 7. For CFH scar prevention experiments (Fig. 10), which were done in C57BL/6J mice (due to poor anesthesia tolerance and excessive fighting leading to high morbidity/mortality with initial wounding experiments in CAST mice), recombinant CFH was resuspended at 30 pg/mL and 30 pL of CFH solution or PBS were injected on POD 0, 2, 4, 6. For wound curve analysis, wounds were photographed every other day for the first two weeks of healing, then (for ear wounds) weekly for two additional weeks. For ear wounds, a circular stencil was placed over wounds prior to photographing in order to provide a consistently sized reference for measurements; for dorsal wounds, the silicone splints served as a size reference. Area of the stencil/splint and remaining wound area at each timepoint were measured in Photoshop (Adobe) and used to calculate remaining wound area as a percentage of original wound size (normalized to size of stencil/splint for each photograph).

For CXCR2i scar prevention experiments (see Fig. 11 ), which were done in C57BL/6J mice, the CXCR2 antagonist AZ 10397767 (Tocris) was resuspended in DMSO at 802 ng/mL and 30 pL of CXCR2 solution or PBS were injected on POD 0, 2, 4, and 6. Hi. Tissue histologic analyses

Tissue for histologic analysis was harvested and fixed by incubation in 10% neutral buffered formalin for 16-18 hours at 4 ^SC. Following fixation, tissue was processed for paraffin or OCT embedding by standard procedures. Briefly, for paraffin embedding, tissue underwent sequential dehydration (ethanol), clearing (xylene), and infiltration by paraffin wax. For OCT embedding, tissue was incubated in 30% sucrose/PBS for two weeks at 4 ^SC, OCT for 1 day at 4 ^eC, then embedded in OCT blocks by freezing in a dry ice/tert-butanol bath. All wounds were bisected and embedded cut-side-down. Tissue sections were cut using a microtome (paraffin) or cryostat (OCT) at 8 pm thickness. Hematoxylin and eosin (H&E) and picrosirius red staining (using Piero Sirius Red Stain Kit, Abeam) were performed on paraffin sections, using standard protocols without modification. Dermal thickness was measured from H&E histology images; Photoshop (Adobe) was used to measure the dermis (from the bottom of the epidermis to the top of the subcutaneous tissue), and a minimum of nine measurements (three measurements per image from three individual histology images/sections) were averaged per wound. Machine learning analysis of ECM ultrastructure was performed as previously described using Matlab. Briefly, picrosirius red histology images were normalized, color deconvoluted, noise reduced, then binarized. Binarized images were filtered to select for fiber-shaped objects and the fiber network was skeletonized. Finally, 294 parameters of the digitized map (including fiber length, width, persistence, alignment, etc.) were measured. Dimensionality reduction of quantified fiber network properties by t-distributed stochastic neighbor embedding (t-SNE) was used to plot parameters for each image. Comparisons between conditions were based on visual assessment of t-SNE clustering from calculated ECM parameters. Matlab scripts containing our fiber quantification pipeline are available at the following Github repository: https://qithub.com/shamikmascharak/Mascharak-et-al-ENF. iv. FACS isolation of wound cell populations

To harvest a consistent region for RNA-seq analysis, wounds were excised with a 1 mm ring of tissue around each wound using a biopsy punch (4 mm punch for ear wounds; 8 mm punch for dorsal wounds). Wound tissue was incubated in ammonium thiocyanate (3.8% in Hank’s balanced salt solution [HBSS]) for 20 minutes at room temperature to dissociate the epidermis, then dermal tissue was separated from overlying epidermis and underlying cartilage (for ear wounds) under a surgical microscope. For each biological replicate, ear or dorsal wounds from three individual mice were pooled to obtain sufficient cell numbers for sequencing. Wounds were finely minced with sharp surgical scissors then enzymatically digested in collagenase type IV (1500 U/mL in Dulbecco’s Modified Eagle Medium [DMEM]) at 37 ⁹C, with agitation at 150 rpm, for 1 hour. After 1 hour, digestion was quenched by addition of equal volume of DMEM with 10% heat-inactivated fetal bovine serum (FBS), filtered through 70 pm followed by 40 pm nylon filters, then pelleted (200 x g, 5 min, 4 ^eC). Cell pellets were resuspended in FACS buffer (PBS with 1% FBS and 1% penicillin-streptomycin) then stained with the following antibodies: PE anti-CD45 (BioLegend #103105); APC anti-CD31 (Invitrogen #17-0311 -80); and eFluor 450-conjugated Lineage (Lin) antibodies anti-CD45 (ThermoFisher Scientific #48-0451 -82), anti-Ter-119 (ThermoFisher Scientific #48-5921 -82), anti-CD31 (BioLegend #303114), anti-Tie-2 (ThermoFisher Scientific #13-5987-82), anti-CD326 (ThermoFisher Scientific #48-5791 -82), and anti-CD324 (ThermoFisher Scientific #13-3249-82), for isolation of fibroblasts via lineage depletion as per previously published protocol.²³ DAPI (BioLegend; 1 :1000) was added as a viability stain. Live (DAPI ) singlet cells were sorted to obtain immune cells (PE/CD45⁺), endothelial cells (APC/CD31⁺), and fibroblasts (PB/Lin ), which were sorted directly into lysis reagent (QIAzol, QIAGEN), then stored at -20 ^eC until RNA purification. v. Bulk RNA-sequencing of wound cell populations

RNA was purified from each cell sample using the miRNeasy Micro Kit (QIAGEN), then kept at -20 ^eC until sequencing. Samples were shipped on dry ice and library preparation and sequencing were performed by Admera Health (South Plainfield, NJ). Library preparation was with the SMART-Seq v4 Ultra Low Input Kit (Takara Bio) with PolyA Selection. Sequencing was done using Illumina HiSeq (2x150). vi. Sequencing of MRL and variant calling

To identify variant calls for allele-specific expression, we generated whole-genome data for the MRL inbred line. MRL tail DNA was extracted and purified using the Invitrogen PureLink Genomic DNA Mini Kit. The library was prepared using the KAPA Hyper Prep kit. The MRL genome was sequenced to moderate coverage (average of 25x for sites with at least one read) on the Illumina HISeq X platform (2x150 reads; Fig. S2). Genomic reads were then mapped to the M. m. domesticus mm10 (GRCm38) reference genome using bowtie2 v2.3.4 (argument: - very-sensitive)(Langmead and Salzberg, 2012). CAST/EiJ sequence data was obtained from Wellcome Trust Mouse Genome Project (https://www.sanqer.ac.uk/science/data/mouse- qenomes-project) in bam format, mapped to mm 10. SNP calling was performed using the Genome Analysis Toolkit v4.1 (GATK). Duplicate reads were marked with the Picard tool MarkDuplicates. GATK HaplotypeCaller was used to call variants between CAST/EiJ, MRL, and the mouse reference genome (mm10). We filtered variants for low quality calls (SNPs: QD < 2.0 || FS > 60.0 || MQ < 40.0 || MQRankSum < -12.5 || ReadPosRankSum < -8.0; Indels: QD < 2.0 || FS > 200.0 || ReadPosRankSum < -20.0), sites with a read depth of less than 5, and for any sites with heterozygous calls using GATK SelectVariants and bcftools (Danecek and McCarthy, 2017). This resulted in 19,472,153 SNPs, 1 ,171 ,415 of which were in exons. Variant calls where CAST and MRL differed from each other or both differed from the mm 10 reference were then used to create alternative references for MRL and CAST for mapping. SNP calls were inserted in mm10 and indels were masked. The concatenated MRL-CAST genome was used to identify reads mapping uniquely to each parental genome in F1 hybrids. vii. Mapping and allele-specific assignment

Raw reads were trimmed for adapter contamination with the TrimGalore (v0.5) wrapper for Cutadapt v1 .18. Trimmed reads were mapped to a concatenated MRL-CAST genome using STAR v2.5.4b, discarding any reads that did not map uniquely to one of the reference genomes (arguments: -outFilterMultimapNmax 1 -outFilterMultimapScoreRange 1 ). Requiring that reads map uniquely to one genome ensures we only consider reads overlapping a heterozygous site in F1 individuals. This resulted in an average of 41 ,215,441 , 60,938,290, 76,012,313 uniquely mapped reads for each immune, endothelial, and fibroblast library, respectively. Reads overlapping exonic regions were summed to generate a total count for each gene based on the Ensembl GRCm38 annotation. Cases where reads only mapped to one allele were discarded as they likely reflect SNP calling errors or genomic imprinting. Approximately 50% of reads mapped uniquely to both CAST and MRL, indicating little evidence of mapping bias (see Fig. 19). DESeq2 (v1.34.0; R computing environment, v4.1.2) was used to perform a variance stabilizing transformation for principal component analysis and perform regularized log₂ transformation of the count data (which minimizes differences between samples for rows with small counts and normalizes with respect to library size) for visual comparison of read count data in Fig. 7. viii. Identifying allele-specific expression

DESeq2 was used to identify allele-specific expression and condition-specific ASE (i.e., diffASE). Allele-specific expression analyses were restricted to genes with at least 30 reads in each condition (wound type, allele) and non-zero values for >4 alleles across individuals. We analyzed allele-specific reads from each cell population separately with DESeq2 with the model “-tissue + tissue:sample + tissue:allele” (where denotes an interaction term between two variables in DESeq2). Here, the term “tissue” is the wound site, for differences between ear and dorsal wounds. “Sample” refers to the sample pool the allele-specific sample pertains to, and the term accounts for variation among the different sample pools within wound site groups. “Allele” refers whether allele-specific reads are mapped preferentially to MRL or CAST, and the interaction between allele and tissue is used to estimate the MRL vs CAST allele ratios separately between wound sites. “DiffASE” genes are identified via a contrast between CAST/MRL ratios in ear and dorsal (DESeq2, Wald test). Consequently, significant cases represent scenarios in which the logs fold change of CAST/MRL differ between wound types. As read counts come from MRL and CAST come from the same sequencing library, library size factor normalization was disabled by setting SizeFactors = 1 (Love, 2017). A false discovery rate correction was applied using the Benjamini-Hochberg method for each comparison. R Code is available on Figshare (https://doi.Org/10.6084/m9.figshare.c.6025157). ix. Overlap with previous QTL mapping for ear hole closure

Marker locations and LOD scores for a model with additive and dominance values for ear wound closure are as described by Cheverud et al. 2014 (LOD and marker scores for analysis provided by J. Cheverud). Marker locations were converted from mm9 to mm10 using LiftOver. Autosomal genes were annotated to their closest genetic marker using BEDTools (Quinlan and Hall, 2010) (tool: “closest”) based on Ensembl mm10 gene start and end coordinates. Liftover coordinates of QTL support intervals defined by Cheverud et al. 2014 were used to identify overlap with diffASE genes within QTL using the BEDTools (tool: “intersect”). QTL mapping was performed using LG, the progenitor of MRL. MRL and LG mice share -75% of their genome, and both shared and unique QTL from these lines contribute to advanced wound healing. Consequently, comparisons with this study will be restricted to identifying QTL shared between the lines. However, this should only make our enrichment tests more conservative and for shared causal regions. x. Enrichment analyses

GO enrichment analyses were performed with PANTHER, using a foreground list of genes of interest vs. a background list of all genes with sufficient expression to be tested in a cell population (GO Ontology database released 2019-12-09). Mutant phenotype enrichment tests were performed with modPhEA, also using a foreground list of genes and a background list of all genes with sufficient expression to be tested in a cell population.

Enrichment of GO terms and mutant phenotypes for diffASE genes are available in File S1 . Enrichment for wound healing terms for fibroblasts was found for diffASE genes at both the FDR<0.05 and FDR<0.1 cut-offs. xi. Fibroblast cell culture

Fibroblast cells were isolated from MRL and CAST dorsal and ear skin for in vitro analysis. Following dissection of the dermis from the dorsum and ear, tissue was washed in PBS and finely minced using sterile scissors. Tissue was then digested in collagenase type IV (1500 U/mL in DMEM) at 37 ^eC, with agitation at 150 rpm, for 1 hour. Enzyme activity was quenched by addition of FBS-enriched media, and digested tissue was successively strained through 300pm followed by 100pm cell strainers. Filtered samples were then centrifuged at 1500 rpm for 5 minutes at 4 °C to obtain a cell pellet. Pelleted cells were resuspended and plated in fibroblast culture media (DMEM + Glutamax media [ThermoFisher, Cat: 10569010] enriched with 10% fetal bovine serum [ThermoFisher, Cat: 10082147] and 1 % Antibiotic-Antimycotic [ThermoFisher, Cat: 15240062]) then grown until confluency in tissue culture incubators kept at 37 °C and 5% CO₂. Cells used for experiments were between passages 2-4. For in vitro analysis of CFH expression, fibroblasts were seeded onto coverslips at 15,000 cells/coverslip for immunostaining (see section below). x/7. Immunostaining of cells and wounds

For both OCT wound section slides and cell-seeded coverslips, immunofluorescent staining was performed as follows. Samples were washed twice in Tween 20 (Sigma-Aldrich, St. Louis, MO) followed by one wash in PBS. Samples were then blocked for 1 hour with Power Block (Biogenex, Fremont, CA) prior to addition of anti-CFH primary antibody (LS bio, LS-C819285, 1 :200). Samples were washed then incubated for 1 hour with Alexa Fluor 488 anti-rabbit secondary antibody (Invitrogen, Waltham, MA). Finally, samples were mounted in Fluoromount- G mounting solution with or without DAPI (ThermoFisher Scientific, Waltham, MA). Fluorescent images were acquired with a LSM880 inverted confocal, Airyscan, AiryscanFAST, GaAsP detector upright confocal microscope. x/77. Single Cell RNA-sequencing of wound cells

Dorsal dermal wounds from C57BL/6J mice treated with CFH or PBS control were harvested at POD 3, 7, and 14, and mechanically digested with sharp surgical scissors (n=3 per condition). Tissue then underwent enzymatic digestion with Collagenase II (ThermoFisher, Cat: 17101015) and IV (ThermoFisher, Cat: 17104019) in DMEM-F12 (GIBCOTM, Fisher Scientific, Hampton, NH). These samples were placed on an orbital shaker for 90 minutes at 150 rpm at 37^eC. After adding FACS buffer to quench the digest, samples were passed through 70 pm cell strainers and centrifuged at 1500 rpm at 4^SC for 5 minutes. Samples were then passed through a second filtration step using 40pm cell strainers and then resuspended with 0.04% UltraPure BSA (Thermo Fisher, Waltham, MA). Following this cell counting was followed by scRNA-seq using the 10x Chromium Single Cell platform (Single Cell 3’ v3, 10x Genomics, USA) as previously described.

Using the Cell Ranger (10X Genomics; version 3.1 ) implementation mkfastq, base calls were converted to reads. These reads were then aligned against the mm10 reference genome (http://cf.10xgenomics.com/supp/cell-exp/), applying Cell Ranger’s count function with SC3Pv3 chemistry and 10,000 expected cells per sample, as previously described. Cutoffs of 2,500 maximum unique genes and a maximum percent mitochondrial RNA of 15% were employed. This resulted in 12995 cells, of which 3185 were annotated as fibroblasts.

For downstream analysis, each cell’s unique molecular identifiers (UMIs) were normalized with a scale factor of 10,000 UMIs per cell, and, using the Seurat R package (version 4.3.0), and the first 15 principal components used for uniform manifold approximation and projection (UMAP). Using SingleR (version 3.11 ), cell annotations were assigned to each cell using the Mouse-RNA- seq reference dataset (https://rdrr.io/qithub/dviraran/SingleR/man/mouse.rnasea.html). Using Seurat’s FindMarkers function, cell-type marker lists were created using a log fold change threshold of 0.25. Using the 200 most highly ranked genes within each cluster, EnrichR (version 2.1 ) was used to conduct gene set enrichment analysis.

The scRNA-seq data from this study is accessible on request from NCBI’s Gene expression Omnibus. xiv. CellChat Receptor-Ligand Analysis The CellChat platform was used to assess possible interactions between cell types in our scRNA-seq dataset. Using our scRNA-seq Seurat object in R, we implement this in conjunction with standalone CellChat Shiny App for its Cell-Cell Communication Atlas Explorer. SingleR- defined cell types were used to bin cells. Secreted Signaling, ECM-Receptor, and Cell-Cell Contact relationships were considered, and default parameterizations used throughout.

C. Results

/. Tissue- and strain- specific differences in wound healing

We first sought to robustly establish differences in healing phenotypes between two mouse strains: MRL, which regenerate ear wounds but heal dorsal wounds via scarring; and CAST/EiJ (CAST), which do not possess any known strain-specific regenerative ability. Ear wounds were generated using a 2 mm punch tool to create a through-and-through wound in the center of the pinnae. Full-thickness dorsal excisional wounds were created via a previously published protocol¹⁷; in this model, silicone splints are applied around wounds to prevent the rapid contraction that typically occurs in mice and instead yield healing via granulation and re- epithelialization with human-like kinetics. Consistent with previous work, MRL ear wounds had largely closed by 3-4 weeks after wounding, with regeneration of normal-appearing skin grossly and cartilage histologically, while CAST ear wounds failed to close to an appreciable extent and instead formed scar tissue over the exposed wound edge (Fig. 6A-B). In contrast, dorsal wounds healed at a comparable rate in the two strains, with re-epithelialization complete by postoperative day (POD) 14 (Fig. 6C-D). Both MRL and CAST dorsal wounds healed by forming fibrotic scars, which grossly and histologically appeared as “bare areas” devoid of dermal appendages (e.g., hair follicles) and with dense connective tissue (Fig. 6C). Analysis of wound ECM ultrastructure using a previously published image analysis pipeline confirmed that dorsal wounds in both CAST and MRL healed with ECM architecture that was quantitatively distinct from that of unwounded skin (consistent with fibrotic scar ECM); while CAST ear wounds also had a distinct ECM pattern, MRL ear wounds had ECM features that had a higher overlap with unwounded ear tissue, indicating regeneration at the tissue ultrastructural level (Fig. 12).

//. Extensive cis -regulatory divergence during wound healing between regenerative and non- regenerative mouse strains

Having confirmed that enhanced/regenerative healing is specific to both the strain (MRL) and anatomic site (ear), we sought to leverage this unique phenotypic pattern to identify genes responsible for regeneration versus fibrosis. As wound repair involves a series of transcriptional cascades triggered by injury, we hypothesized that MRL ear closure may be driven by wound site- specific c/s-regulatory activity. To identify cis- regulatory variation associated with wound healing, we crossed MRL with CAST mice to generate CAST x MRL F1 hybrids. In these first-generation hybrid offspring, alleles from both parents are present in the same cellular environment (i.e. , are subject to the same trans-regulatory influences); so, differences in expression between the two alleles can only be the result of c/s-regulatory differences. Thus, assaying allele-specific gene expression in F1 hybrid wounds allows for identification of injury-relevant c/s-regulatory differences between these two mouse strains. Further, comparing allele-specific expression between wounds in the ear - which exhibits regenerative healing specifically in MRL mice - and the dorsum - which heals with a scar in both MRL and CAST - may allow us to pinpoint causal c/s-regulatory differences driving the unique MRL ear regeneration phenotype.

In order to assess allele-specific gene expression across wound contexts, we performed bulk RNA-seq of key cell populations associated with wound healing. Adult F1 female mice were subjected to dorsal splinted excisional and ear punch wounding. On POD 7, all wounds were harvested. Ear and dorsal wound tissue were separately digested and subjected to fluorescence- activated cell sorting (FACS) to isolate three cell populations: immune cells (CD45⁺); endothelial cells (CD31⁺); and fibroblasts (Lin-, per published sorting strategy; see Methods for details). Due to cell number limitations, wounds from three individual mice were pooled per biological replicate. Cell samples were then subjected to bulk RNA-sequencing (Fig. 7A). At least three biological replicates were sequenced and analyzed for each cell type.

Across all tissue samples, we obtained a total of ~3.6 billion reads (Fig. 25). To enable the allele-specific assignment of RNA-seq reads, we performed whole-genome re-sequencing of MRL (~25X depth of coverage; Fig. 13). Genetic variants differing between MRL and CAST strains were used to preferentially assign RNA-seq reads to either the MRL or the CAST allele (Fig. 25). After filtering for genes with low coverage in either genotype or wound context, we were able to analyze >10,000 genes in each cell population (Fig. 26). Hierarchical clustering and principal component (PC) analysis of allele-specific expression data grouped samples strongly by cell type (fibroblasts, immune, endothelial) (Fig 7B, Fig. 14). PC analysis of individual cell populations clearly separated samples by wound site (dorsal vs. ear: PC1 for fibroblasts, 71% of variance explained; PC2 for immune and endothelial; 23% and 25% of variance explained, respectively) and allele (CAST vs. MRL: PC2 for fibroblasts, 14% of variance explained, PC1 for immune and endothelial, 43% and 42% of variance explained respectively; Fig. 7C).

We next sought to identify genes with expression patterns reflecting MRL ear-specific c/s- regulatory divergence, which could reflect functional involvement of these genes in driving regeneration rather than fibrosis for wounds in this tissue (Fig. 8A). Across all cell types, a greater number of genes exhibited significant allele-specific expression (ASE; FDR < 0.05 for MRL vs. CAST allelic expression with DESeq2) in ear wounds (5,121 genes; 32.7%) compared to dorsal wounds (2,655 genes; 17%), consistent with phenotypic divergence restricted to the ear (Fig. 8B, Fig. 27). This was most apparent in immune cells, where over four times as many genes had evidence of ASE in ear compared to dorsal wounds. Differences in expression between CAST and MRL alleles (i.e., |log₂ fold change|) were also larger on average in ear wounds in each cell population (Fig. 15; Wilcoxon rank sum test, all ear vs. dorsal pairwise comparisons p < 2.2 x I O'¹⁶).

Considering both ear and dorsal wounds, approximately 23% of genes demonstrated ASE in more than one cell population (Fig. 16). Within each cell population, while a substantial proportion of genes showed ASE in both wound contexts, many exhibited ASE unique to either ear or dorsal wounds (Fig. 8B), indicating the existence of both general and tissue-specific regulatory divergence between CAST and MRL during wound repair. For genes with ASE in both dorsal and ear wounds, the vast majority maintained the same directionality of allelic expression across wound sites (i.e., same allele up-/down-regulated in both ear and dorsal wounds; Fig. 28). Further, we found that allelic ratios were correlated between wound sites (i.e., log₂(CAST ear/MRL ear) vs. log₂(CAST dorsal/MRL dorsal); Pearson’s correlation, all comparisons p< 2.2 x 10 ¹⁶; Fig. 8C). Taken together, our findings of ASE were consistent with greater context-dependent regulatory divergence in ear wounds relative to dorsal wounds.

As MRL mice demonstrate a regenerative phenotype unique to the ear wound context and not seen in dorsal wounds (Fig. 6), we reasoned that the subset of genes with differential allelespecific expression (“diffASE”) between ear and dorsal wounds could include genes driving the regenerative healing phenotype specifically in MRL ear wounds. Across different wound settings, context-specific ASE may reflect wound site-specific activity of genes controlled by cis- regulatory elements with sequence differences between MRL and non-regenerating (e.g., CAST) mice (Fig. 8A). Comparing genes’ ASE measurements in ear and dorsal wounds, we identified 432 genes in immune cells, 91 in endothelial cells, and 235 in fibroblasts with diffASE between wound healing contexts (DESeq2 Wald-test, [CAST/MRL ear counts] vs. [CAST/MRL dorsal counts], FDR < 0.05; see Methods; Fig. 8C, Fig. 27). The majority of genes with diffASE were unique to a single cell population (732/745 genes total).

Examining genes with diffASE in each cell population, we found that those in fibroblasts were uniquely enriched for known mutant phenotypes and gene ontology (GO) terms associated with injury and wound repair (Fig. 8D). In contrast, wound healing-related terms were not highly enriched for either immune or endothelial cell diffASE genes, indicating that c/s-regulatory divergence in fibroblasts may play a particularly important role in driving divergent wound healing phenotypes in the MRL ear versus dorsum (Fig. 12). In fibroblasts, genes with diffASE were most highly enriched for the mouse mutant phenotype “abnormal response to injury” (MP:0005164, FDR-adjusted p-value = 1 .85 x 1 O'⁵) and were also enriched for the phenotypes “abnormal wound healing” (MP:0005164, FDR = 0.00086) and “abnormal blood vessel physiology” (MP:0000249, FDR = 0.00023). Additionally, the GO terms “response to wounding” (G0:000961 1 , FDR = 1.37 x 10^-4) and “wound healing” (G0:0042060, FDR = 2.37 x 10’³) showed greater than four-fold enrichment compared to a background set. Fibroblast diffASE genes were also enriched for GO and Reactome Pathway terms related to processes involved in scarring and regeneration. These included cell adhesion (G0:0007155, FDR = 1 .34 x 10'⁶) and integrin cell surface interactions (R- M MU-216083, FDR = 1.95 x 10²), which are implicated in activated mechanotransduction and pro-fibrotic changes in fibroblasts; and extracellular matrix organization (G0:0030198, FDR = 3.50 x 10^-3; and R-MMU-1474244, FDR = 9.52 x 10’³), a critical determinant of scarring versus regenerative wound properties, among others (Fig. 8D).

Further, we identified several genes associated with wound repair phenotypes or known pathways with large differences in allelic ratio between ear and dorsal wound fibroblasts (Fig. 8E, Fig. 29A-B, Fig. 30A-B). Some of these genes with MRL-specific upregulation in ear wounds had known functions consistent with promoting wound healing and/or decreasing scarring. For instance, Slpi (Secretory leukocyte protease inhibitor; ear wounds: log₂(CAST/MRL) = -2.98, q = 5.74 x 10'⁶; dorsal wounds: log2(CAST/MRL) = -0.036, q= 0.58) has important functions in wound healing, in part via regulating transforming growth factor-beta (TGF-P) activity, and S/p/'-null mice exhibit delayed wound repair and increased inflammation. Spp1 (Secreted phosphoprotein 1 , which encodes the protein osteopontin; ear: log₂(CAST/MRL) = -1 .91 FDR = 1 .083 x 10'³⁶; dorsal: log₂(CAST/MRL) = -0.18, FDR = 0.57) has been implicated in resolution of inflammation as well as matrix remodeling following skin injury, the latter being especially critical in determining scarring versus regenerative healing outcomes. Additionally, osteopontin knockout mice have impaired wound closure. Thbs4 (Thrombospondin 4, an ECM protein; ear: log2(CAST/MRL) = - 1.24, FDR = 6.55 x 10'³⁸; dorsal: log2(CAST/MRL) = -0.23, FDR = 0.048) has previously been shown to promote wound healing by stimulating fibroblast migration and keratinocyte proliferation and is reported to promote angiogenesis and reduce fibrosis, with mouse Thbs4 knockout associated with damaging cardiac inflammation and fibrosis. We also identified several genes with reported functions that promote fibrosis and/or impair injury repair, which exhibited upregulation from the CAST allele in ear wounds. For instance, Jami (Junctional adhesion molecule-like; ear: log₂(CAST/MRL) = 1.78, q = 4.24 x 10^-7; dorsal: log₂(CAST/MRL) = -0.04, q = 0.87) has been associated with inflammation and impaired injury repair in the intestine. Adora2b (Adenosine A2b receptor; ear: log₂(CAST/MRL) = 0.82, FDR = 0.00057; dorsal: log₂(CAST/MRL) = -0.12, FDR = 0.71 ) inhibition has been associated with reduced dermal fibrosis, consistent with a pro-scarring role for this gene. Collectively, many genes exhibiting diffASE preferentially in ear wounds had known functions consistent with the phenotypes observed in CAST versus MRL ear wounds (i.e., pro-fibrotic genes enriched from the CAST allele; pro-regenerative genes and genes promoting efficient wound repair enriched from the MRL allele). iv. Overlap with healing quantitative trait loci identifies candidate genes for regeneration

Next, we sought to integrate our results with prior functional studies of MRL ear regeneration. Specifically, having identified genes with wound site-specific cis- regulatory differences, we next asked whether those genes were located within previously mapped genomic intervals associated with enhanced ear punch closure. We capitalized on a recent QTL fine- mapping study for ear wound closure in LG/J (LG), the MRL progenitor line (LG x SM, F32 generation. LG shares -75% of its genome with MRL and exhibits similar regenerative healing of ear punch wounds. Consequently, overlap between these studies will be restricted to causal loci for regenerative healing that are shared between lines. To test whether our diffASE gene sets were enriched in genomic regions driving ear wound closure, we compared the LOD scores of genetic markers closest to genes with diffASE (Fig. 9A) against those of randomly permuted gene sets of the same size. While the presence of a significant LOD score proximal to a single gene does not necessarily implicate that gene, a shift towards a higher average LOD score for a group of genes suggests that this set of genes is collectively more likely to be associated with differences in wound phenotypes (i.e., regenerative versus non-regenerative). Our analysis revealed that genes with diffASE in fibroblasts had significantly higher average LOD scores compared to random sets (20,000 permutations, diffASE genes FDR < 0.05, p = 0.0094; diffASE FDR < 0.1 , p = 0.026; Fig. 9B). In contrast, genes with diffASE in immune or endothelial cells did not exhibit higher LOD scores on average (p > 0.05 for each comparison; 20,000 permutations). Further, genes with ASE in both ear and dorsal wound fibroblasts were not associated with higher LOD scores (p > 0.05), indicating that diffASE was uniquely useful in pinpointing causal wound healing genes. Next, to identify specific candidates for driving regenerative wound healing, we searched for genes with diffASE located within support intervals of significant wound closure QTL Across cell types, we identified a total of 27 genes with diffASE overlapping these QTL intervals (diffASE FDR < 0.05; fibroblasts, 9 genes; endothelial cells, 6 genes; immune cells, 12 genes; diffASE FDR < 0.1 , 40 genes; Fig. 17). Genes with diffASE in fine-mapped intervals were enriched for the GO term “wound healing” (GQ:0042060; Fisher’s exact test, p = 0.0032, 9.45-fold enrichment). Several genes within these intervals could be promising candidates based on their known functions or mutant phenotypes; specifically, two of the genes with diffASE in fibroblasts - Lgr6, and Cfh - were also associated with GO terms related to wound healing and regeneration. Lgr6 was downregulated in MRL ear wounds compared to CAST ear wounds. For wildtype dorsal wounds to mimic MRL ear wounds, a Lgr6 inhibitor would need to be added to wounds. Of these genes, Cfh (Complement factor H) also had the greatest difference in allelic ratios between ear and dorsal wounds, with expression from the MRL allele over four times that of the CAST allele on average in ear wounds, but no significant difference between alleles in dorsal wounds (Fig. 9C). The complement cascade is a part of the innate immune system that is involved in clearing microbes, immune complexes, and damaged self cells and is activated in response to tissue injury. In addition to Cfh, ear-specific ASE in wound fibroblasts was also observed in two other genes encoding complement components, C3 and C1qb, both of which have been directly implicated in wound repair. While the complement system plays a vital role in injury repair - for example, C1qb is important for angiogenesis - inappropriate or prolonged complement activation is also known to perpetuate damaging inflammation and cause cell death. However, prior studies conflict on whether complement activation or inhibition may promote wound healing, and the effects of complement modulation on scarring have not been investigated. v. Ectopic application of CFH reduces scarring and drives partial regeneration after wounding

Complement factor H is a central regulatory protein in this pathway that inhibits complement activation and which has previously been shown to prevent inflammation and fibrosis in the mouse kidney. Given these known functions, strong ear wound-specific ASE of Cfh, and its presence within a QTL interval for ear closure, we hypothesized that Cfh could be a driver of wound regeneration. We first sought to verify that our gene expression findings corresponded to differences at the protein level. First, we cultured fibroblasts from the ear and dorsum of both CAST and MRL mice, then performed immunostaining for CFH (Fig. 10A). CFH protein expression was absent in both ear and dorsal CAST fibroblasts, and was significantly greater in MRL ear than MRL dorsal fibroblasts (Fig. 10B), consistent with our finding of MRL ear-specific upregulation from RNA-seq. We next performed CFH staining on sections from POD 14 ear and dorsal wounds from CAST and MRL (Fig. 10C), which revealed that CFH protein expression was markedly upregulated in MRL ear wounds compared to all other conditions (Fig. 10D).

Given that CFH expression was strongly associated with regenerating conditions (MRL ear wounds), we next evaluated whether modulating CFH signaling could drive wound regeneration and reduce scarring. We treated dorsal wounds in another scarring wildtype mouse strain (C57BL/6J) with recombinant CFH protein (5pig/pil and 10pig/pil doses) or vehicle control (phosphate-buffered saline [PBS]), then evaluated wound outcomes (Fig. 10E; see Methods for full details and dosing). We found that the reduction in scar thickness with CFH treatment was dose-dependent, with more significant scar prevention observed at higher doses (Fig. 18; see Methods for details). Therefore, we selected the higher dose of 10pig/pil for the CFH in vivo experiments.

Grossly, CFH-treated wounds had reduced scarring and more robust re-epithelialization by POD 14 compared to control wounds (Fig. 10F and Fig. 20A-B). Quantification of ECM ultrastructural parameters from picrosirius red histology (Fig. 10G and Fig. 20C) showed that CFH treatment yielded ECM intermediate between that of control scars and unwounded skin (Fig. 10H), indicating partial regeneration at POD 3, 7, and 14. On hematoxylin and eosin (H&E) histology, CFH-treated wounds had more complete re-epithelialization (confirming gross observations), significantly reduced dermal thickness (consistent with reduced scarring), and structures morphologically resembling early invaginating neogenic hair follicles, in contrast with control scars which remained “bare areas” devoid of any dermal appendages (Fig. 101-J and Fig. 20C). Immunostaining further demonstrated a decrease in collagen type 1 expression in CFH compared to PBS treated wounds (Fig. 20D). Finally, we also found that the reduction in scar thickness with CFH treatment was dose-dependent, with more significant scar prevention observed at higher doses (Fig. 18; see Methods for details). Collectively, these findings were consistent with CFH being sufficient to drive partial wound regeneration and significantly reduce scarring in mouse dorsal wounds, and indicate that this gene plays a similar, pro-regenerative role in MRL ear wounds. vi. Single cell-RNAseq revealed that CFH induces regeneration through CXCL-2 inhibition

Given that CFH treatment effectively reduced scar thickness in wild type mouse wounds, we questioned how CFH treatment induced regeneration in dermal wounds. We treated dorsal wounds in a wildtype mouse strain (C57BL/6J) with recombinant CFH protein or vehicle control (PBS), and subjected wounds at POD 3, 7, and 14 to single cell RNA-seq (scRNA-seq; Fig. 11 A). Eighteen transcriptionally defined clusters were identified by Louvain-based (Seurat) clustering including immune, endothelial, fibroblast, and epithelial cells (Fig. 21 A and Fig. 11 B). Seven transcriptionally distinct fibroblast clusters were present across both CFH and PBS treated wounds (Fig. 11C). Interestingly, cluster 0 fibroblasts were relatively increased in PBS-treated wounds at POD 7, but decreased in both CFH-treated wounds at POD 7 and unwounded skin (Fig. 11 D and Fig. 21 B-C). GO pathway analysis showed cluster 0 to be associated with “collagen synthesis” and “inflammatory signaling" terms (Fig. 21 D). In contrast, clusters 1 to 6 demonstrated similar fibroblast proportions and pathway analysis in CFH- compared to PBS-treated wounds (Fig. 11 D and Fig. 21 B-C).

We next assigned putative functions to each fibroblast cluster based on differential gene expression, and identified gene markers for each population. Cluster 0 had enriched expression of CXC motif chemokine ligand 2 (CXCL2), which also increased in PBS-treated wounds compared to CFH-treated wounds, particularly at POD 7 (Fig. 1 1 E-F). Immunostaining confirmed upregulation of CXCL2 in PBS- compared to CFH-treated wounds at POD 7 (Fig. 1 1 G and 22A).

CXCL2 is known to drive inflammatory cells to wound sites during the inflammatory phase of wound healing. CXCL2 is also pivotal in guiding neutrophils to infected and irradiated tissue. Given this known pro-inflammatory role of CXCL2, we assessed the inflammatory cells in CFH- and PBS-treated wounds. Macrophages/monocytes and T cells were both observed to be enriched in PBS- compared to CFH-treated wounds at POD 7 and POD 14 by scRNA-seq (Fig. 21 E). Immunostaining confirmed upregulation of macrophages and T cells in PBS- compared to CFH-treated wounds at POD 7 (Fig. 22B).

To further assess cell-cell communication in PBS- and CFH-treated wounds, cell signaling was evaluated based on scRNA-seq data using the CellChat platform (Fig. 23A-D). CFH treated wounds showed greater cell communication than PBS treated wounds (Fig. 23A). PBS-treated wounds demonstrated an upregulation of inflammatory cell signaling compared to those in CFH- treated wounds (Fig. 23B). PBS- and CFH-treated wounds showed distinct patterns of cell signaling (Fig. 23C). Interestingly CFH-treated wounds showed upregulation of signaling pathways including Galectin, Thy1 , and Cadherin compared to PBS wounds (Fig. 23D). These data indicate that CFH treatment reduced inflammatory cell signaling through CXCL2 downregulation by fibroblasts. v/7. Inhibition of CXCL2-R mimics the regenerative capacity of CFH treatment in dorsal wounds

Given that CXCL2 expression was strongly associated with PBS-treated wounds, we next evaluated whether modulating CXCL2 receptor through chemical inhibition could reduce scarring and mimic the effect by which CFH treatment reduced scarring of dorsal wounds. We treated dorsal wounds in a wildtype mouse strain (C57BL/6J) with CXCL2 receptor small molecule inhibitor (CXC2Ri) or vehicle control (PBS), then evaluated wound outcomes in WT mouse (Fig. 11 H). Gross images showed PBS wounds healed more slowly than CXC2Ri-treated wounds (Fig. 24A). On H&E histology, CXC2Ri treated wounds increased re-epithelization at POD 7 (Fig.1 11) and reduced scar thickness at POD 14 (Fig. 24B) compared to PBS treated wounds, resembling CFH-treated wounds. Furthermore, ECM analysis demonstrated that CXC2Ri-treated wounds displayed a more similar ECM architecture to unwounded skin than that of PBS-treated wounds (Fig. 11 J). Collectively, these data indicate that CXCL2R inhibition mimics the regenerative effect of CFH treatment in skin wound healing.

D. Discussion

Healing via fibrosis, rather than through regeneration, is a major cause of morbidity and an immense burden for healthcare systems worldwide, with over $20 billion spent annually on the treatment and management of scars in the United States alone. Instances of regeneration in nature, such as the striking example of regenerative mammalian wound repair that occurs in the ears of MRL mice, may provide valuable insights for therapeutically promoting regeneration and preventing fibrosis.

While QTL mapping has previously been used to identify genomic intervals associated with regenerative healing in the MRL strain, identifying specific candidates for functional followup has proven challenging, in part due to the large size of intervals identified. Our approach, using allele-specific gene expression to probe for site-specific cis-regulatory divergence, offers the advantage of not only interrogating potential drivers of regeneration at the single-gene level, but also being substantially less resource-intensive (e.g., <20 mice used, compared to multiple hundreds in prior studies) and thus more accessible. The strength of our methodology is supported by multiple interesting findings. Our approach found greater ASE in ear compared to dorsal wounds across all cell types studied, as well as an enrichment of wound repair pathways and genes associated with differential allele-specific expression in fibroblasts. This would not be expected if the genetic changes leading to MRL ear regeneration were entirely protein-coding, and instead indicates that the strain-specific phenotypic difference seen in ear wounds may reflect cis-regulatory divergence between CAST and MRL. Furthermore, our findings were supported by integrating our results with the QTL fine-mapping study by Cheverud et al. 2014. The overlap observed with this orthogonal method suggests that at least some of the cis- regulatory changes we identified underlie the MRL ear regeneration phenotype. Of note, while our findings highlighted fibroblasts as the most likely drivers of MRL ear wound regeneration, our and other studies have implicated alterations in diverse cell types (e.g., reduced inflammation mediated by immune cells, more rapid re-epithelialization mediated by keratin ocytes) in MRL ear regeneration. These could result from tissue-specific cis-regulatory differences directly affecting other cell types (such as immune or epithelial cells). However, they also likely result, at least in part, from cell-cell crosstalk mediated by fibroblasts. For instance, intimate fibroblast-keratinocyte crosstalk is critical for wound repair, and we have previously found that modulating pro-fibrotic fibroblast molecular processes can also induce changes in the overlying epidermis. Fibroblast-immune and immune-epithelial interactions have also been extensively reported in the context of injury repair. Thus, it is feasible that altered fibroblast phenotype in wounds could fundamentally drive many of the differences observed in regenerating MRL ear wounds; we found that fibroblast diffASE genes were enriched for pathways involved in modulating cartilage development and immune cell activity, further supporting this hypothesis.

Through diFFASE analysis we have identified several genes that may be associated with regeneration in ear wound compared to dorsal wounds. These included Spp1, Thbs4, and Clic4 (Fig. 8). All of these genes are associated with extracellular matrix organization and cell matrix signaling, indicate that regeneration in MRL ear wounds is associated with alterations in ECM-related processes. Finally, our genomic findings are also supported by the results of our experiments with CFH, which was identified by diffASE analysis and subsequently shown via in vivo wounding experiments to promote regeneration and reduce scarring. In addition to providing important functional validation for our methodological approach, this finding could have important translational implications, as no targeted molecular therapies currently exist to prevent human scarring.

CFH expression demonstrated the greatest difference in allelic ratios between ear and dorsal wounds. Our scRNA-seq data revealed that CFH treatment reduces skin scarring through CXCL2 inhibition (Fig 11 ). CXCL2 is a classical chemoattractant for neutrophils in wounds. A previous study has demonstrated that in CFH KO mice, clearance of Streptococcus pneumoniae infection is delayed and CXCL2 secretion is higher.

EMBODIMENTS

Notwithstanding the appended embodiments, the disclosure is also defined by the following embodiments:

1 . A method of promoting healing of a wound in a dermal location of a subject, the method comprising: administering an effective amount of a complement pathway inhibitor to the wound to promote healing of the wound.

2. The method according to Embodiment 1 , wherein the complement pathway inhibitor comprises an alternative pathway inhibitor.

3. The method according to any of Embodiments 1 or 2, wherein the alternative pathway inhibitor comprises a protein or nucleic acid coding sequence therefor.

4. The method according to Embodiment 3, wherein the protein comprises a Factor H protein.

5. The method according to Embodiments 1 or 2, wherein the complement pathway inhibitor comprises a small molecule.

6. The method according to any of the preceding embodiments, wherein the administering comprises injecting a composition comprising the complement pathway inhibitor below a topical dermal location of the subject.

7. The method according to any of the preceding embodiments, wherein the method further comprises administering a Complement inhibitor.

8. The method according to any of the preceding embodiments, wherein the method further comprises administering a YAP inhibitor.

9. The method according to Embodiment 8, wherein the YAP inhibitor is verteporfin.

10. The method according to any of the preceding embodiments, wherein the method further comprises administering a CXCR2 inhibitor.

11 . The method according to any of the preceding embodiments, wherein the method further comprises administering a LGR6 inhibitor.

12. The method according to any of the preceding embodiments, wherein the subject is an adult.

13. The method according to any of the preceding embodiments, wherein the method comprises regeneration of dermal appendages.

14. The method according to Embodiment 13, wherein the dermal appendages comprise hair follicles, sweat glands, and sebaceous glands.

15. The method according to any of the preceding embodiments, wherein the method further comprises forming the wound.

16. The method according to any of the preceding embodiments, wherein the wound is a surgical wound. 17. The method according to any of the preceding embodiments, wherein the method produces a healed wound with reduced levels of scarring compared to a control.

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

19. A method of promoting healing of a wound in a dermal location of a subject, the method comprising: administering an effective amount of a CXCR2 inhibitor to the wound to promote healing of the wound.

20. A method of promoting healing of a wound in a dermal location of a subject, the method comprising: administering an effective amount of a LGR6 inhibitor to the wound to promote healing of the wound.

21 . A method of ameliorating scarring during healing of a wound in a subject, the method comprising: administering an effective amount of a complement pathway inhibitor composition to the wound to ameliorate scarring of the wound.

22. The method according to Embodiment 21 , wherein the wound is a surgical wound.

23. The method according to Embodiments 21 or 22, wherein the method produces a scarless healed wound.

24. The method according to any of Embodiments 21 -23, wherein the administering comprises injecting the composition below a topical dermal location.

25. The method according to any of Embodiments 21 -24, wherein the complement pathway inhibitor comprises an alternative pathway inhibitor.

26. The method according to Embodiment 25, wherein the alternative pathway inhibitor comprises a protein or nucleic acid coding sequence therefor.

27. The method according to Embodiment 26, wherein the protein comprises a Factor H protein.

28. The method according to any of Embodiments 21 -27, wherein the method further comprises administering a Complement inhibitor.

29. The method according to any of Embodiments 21 -28, wherein the method further comprises administering a YAP inhibitor.

30. The method according to Embodiment 29, wherein the YAP inhibitor is verteporfin.

31 . The method according to any of Embodiments 21 -28, wherein the method further comprises administering a CXCR2 inhibitor. 32. The method according to any of Embodiments 21 -28, wherein the method further comprises administering a LGR6 inhibitor.

33. The method according to any of Embodiments 21 -32, wherein the subject is an adult.

34. The method according to any of Embodiments 21 -33, wherein the method comprises regeneration of dermal appendages.

35. The method according to Embodiment 34, wherein the dermal appendages comprise hair follicles, sweat glands, and sebaceous glands.

36. The method according to any of Embodiments 21 -35, wherein the method produces a scarless healed wound.

37. A method of ameliorating scarring during healing of a wound in a subject, the method comprising: administering an effective amount of a CXCR2 inhibitor composition to the wound to ameliorate scarring of the wound.

38. A method of ameliorating scarring during healing of a wound in a subject, the method comprising: administering an effective amount of a LGR6 inhibitor composition to the wound to ameliorate scarring of the wound.

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.

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.”

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.

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 , , 3, 4, or 5 articles, and so forth.

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.

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.

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. §112(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. §1 12(6) is not invoked.

Claims

WHAT IS CLAIMED IS:

2. The method according to Claim 1 , wherein the complement pathway inhibitor comprises an alternative pathway inhibitor.

3. The method according to any of Claims 1 or 2, wherein the alternative pathway inhibitor comprises a protein or nucleic acid coding sequence therefor.

4. The method according to Claim 3, wherein the protein comprises a Factor H protein.

5. The method according to Claims 1 or 2, wherein the complement pathway inhibitor comprises a small molecule.

6. The method according to any of the preceding claims, wherein the administering comprises injecting a composition comprising the complement pathway inhibitor below a topical dermal location of the subject.

7. The method according to any of the preceding claims, wherein the method further comprises administering a Complement inhibitor.

8. The method according to any of the preceding claims, wherein the method further comprises administering a YAP inhibitor.

9. The method according to Claim 8, wherein the YAP inhibitor is verteporfin.

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 a complement pathway inhibitor composition to the wound to ameliorate scarring of the wound.

18. The method according to Claim 17, wherein the wound is a surgical wound.

19. The method according to Claims 17 or 18, wherein the method produces a scarless healed wound.

20. The method according to any of Claims 17-19, wherein the administering comprises injecting the composition below a topical dermal location.

21 . The method according to any of Claims 17-20, wherein the complement pathway inhibitor comprises an alternative pathway inhibitor.

22. The method according to Claim 21 , wherein the alternative pathway inhibitor comprises a protein or nucleic acid coding sequence therefor.

23. The method according to Claim 22, wherein the protein comprises a Factor H protein.

24. The method according to any of Claims 17-23, wherein the method further comprises administering a Complement inhibitor.

25. The method according to any of Claims 17-24, wherein the method further comprises administering a YAP inhibitor.

26. The method according to Claim 25, wherein the YAP inhibitor is verteporfin.

27. The method according to any of Claims 17-26, wherein the subject is an adult.

28. The method according to any of Claims 17-27, wherein the comprises regeneration of dermal appendages.

29. The method according to Claim 28, wherein the dermal appendages comprise hair follicles, sweat glands, and sebaceous glands.

30. The method according to any of Claims 17-29, wherein the method produces a scarless healed wound.