USE OF AXL, CCL19 AND/OR BMP-6 FOR PROMOTING WOUND HEALING

Abstract

The invention relates to the treatment of a wound, and in particular to uses of polypeptides (or genetic constructs or vectors encoding such peptides) to promote wound healing and/or reduce, prevent or inhibit scarring. The invention extends to pharmaceutical compositions comprising such polypeptides or constructs, for treating wounds, and for reducing scarring, and cosmetic formulations for improving the appearance of skin. The invention also extends to wound dressings, formulations and bandages comprising such polypeptides.

Claims

1. A method of treating a wound, or of preventing, reducing or inhibiting scar formation, the method comprising administering, to a subject in need thereof, a therapeutic amount of a polypeptide selected from a group consisting of AXL, CCL19 and BMP-6, or a biologically active variant or fragment thereof.

2. The method to claim 1, wherein the polypeptide is AXL or a biologically active variant or fragment thereof comprising the active domain of AXL, and optionally the polypeptide is soluble AXL, and optionally further comprising administration of CCL19, or a biologically active variant or fragment thereof and/or BMP-6, or a biologically active variant or fragment thereof.

3. (canceled)

4. (canceled)

5. The method to claim 1, wherein the polypeptide is CCL19, or a biologically active variant or fragment thereof.

6. The method to claim 1, wherein the polypeptide is BMP-6, or a biologically active variant or fragment thereof.

7. The method to claim 1, wherein the polypeptide is substantially as set out in SEQ ID NO: 1 or a variant or fragment thereof, and optionally as set out in SEQ ID NO: 3 or a variant or fragment thereof.

8. The method to claim 1, wherein the polypeptide is encoded by the nucleotide sequence substantially as set out in SEQ ID NO: 2 or a variant or fragment thereof, and optionally substantially as set out in SEQ ID NO: 4 or a variant or fragment thereof.

9. The method to claim 1, wherein the polypeptide is substantially as set out in SEQ ID NO: 10 or a variant or fragment thereof.

10. The method to claim 1, wherein the polypeptide is encoded by the nucleotide sequence substantially as set out in SEQ ID NO: 11 or a variant or fragment thereof.

11. The method to claim 1, wherein the polypeptide is substantially as set out in SEQ ID NO: 12 or a variant or fragment thereof.

12. The method to claim 1, wherein the polypeptide is encoded by the nucleotide sequence as set out in SEQ ID NO: 13 or a variant or fragment thereof.

13. The method to claim 1, wherein the treatment comprises re-epithelisation of epithelial tissue, and optionally wherein the rate of wound healing is increased and/or scar formation is prevented, reduced or inhibited.

14. (canceled)

15. The method to claim 1, wherein the wound is present on the skin.

16. (canceled)

17. A method of treating a wound, the method comprising administering, to a subject in need thereof, a therapeutic amount of a vector comprising a nucleic acid encoding a polypeptide selected from a group consisting of AXL, CCL19 and BMP-6, or a biologically active variant or fragment thereof.

18. (canceled)

19. A method according to claim 1, wherein the polypeptide is in a pharmaceutically acceptable composition, and the composition further comprises a pharmaceutically acceptable vehicle.

20. (canceled)

21. (canceled)

22. A device comprising a polypeptide selected from the group consisting of AXL, CCL19 and BMP-6, or a biologically active variant or fragment thereof, wherein the device is configured for the controlled spatio-temporal delivery of the polypeptide.

23. The device according to claim 22, wherein the device is a bandage comprising at least two layers comprising a polypeptide selected from the group consisting of AXL, CCL19 and BMP-6, or a biologically active variant or fragment thereof, wherein each layer comprises the same or different polypeptide, wherein the polypeptide comprised in a different layer is delivered to the wound site at a different time point, and optionally wherein the polypeptide is CCL19 in one layer and AXL in another layer, and CCL19 is delivered first, optionally for up to 2 days, and AXL is delivered after CCL19, optionally for the remainder of wound closure.

24. (canceled)

25. (canceled)

26. (canceled)

27. (canceled)

28. (canceled)

29. (canceled)

30. (canceled)

Description

[0155] For a better understanding of the invention, and to show how embodiments of the same may be carried into effect, reference will now be made, by way of example, to the accompanying Figures, in which:

[0156] FIG. 1 shows fibroblast sub-types found in human scalp skin;

[0157] FIG. 2 summarises known fibroblast responses during wound healing;

[0158] FIG. 3: A) Images of keratinocytes scratch wounds in DPFi CM, PFi CM, RFi CM and Epilife media. The red dotted line separates the cell area from the cell free area. B) DPFi promote significantly faster closure of keratinocyte scratch wounds compared to unconditioned Epilife medium. The effect is observed 4 hours after scratching, although only endpoint significance is shown on the graph for clarity. The Y-axis shows collective migration distance of all keratinocytes. N=3;

[0159] FIG. 4 shows cytokine array dataRaw Data Normalised. (A) Membrane 1 of the cytokine antibody array with DPFi CM from patient 1. (B) Membrane 1 of the cytokine antibody array with DPFi CM from patient 2. (C) Membrane 1 of the cytokine antibody array with PFi CM from patient 1. (D) Membrane 1 of the cytokine antibody array with PFi CM from patient 2. (E) Membrane 1 of the cytokine antibody array with RFi CM from patient 1. (F) Membrane 1 of the cytokine antibody array with RFi CM from patient 2. (G) Membrane 2 of the cytokine antibody array with DPFi CM from patient 1. (H) Membrane 2 of the cytokine antibody array with DPFi CM from patient 2. (I) Membrane 2 of the cytokine antibody array with PFi CM from patient 1. (J) Membrane 2 of the cytokine antibody array with PFi CM from patient 2. (K) Membrane 2 of the cytokine antibody array with RFi CM from patient 1. (L) Membrane 2 of the cytokine antibody array with RFi CM from patient 2;

[0160] FIG. 5 shows Volcano plots of cytokine array data identifies Axl, CCL19 and BMP6 as unique to DPFi compared to PFi; (N=2)

[0161] FIG. 6 shows AXL protein structure. In the inventors experiments, the extracellular domain of AXL was used, also known as soluble AXL (sAXL). Image taken from http://atlasgeneticsoncology.org/Genes/AXLID733ch19q13.html;

[0162] FIG. 7 shows cell front velocities for 3 cytokines concentrations for CCL19 (A), AXL (B), BMP6 (C) and IL6 (D);

[0163] FIG. 8 shows the effect of CCL19 (A), AXL (B), BMP6 (C) and IL6 (D) on keratinocytes wound closure. The Y-axis shows collective migration distance of all keratinocytes;

[0164] FIG. 9 shows a schematic summarising the role of fibroblasts and cytokines in wound healing;

[0165] FIG. 10 shows the evaluation of keratinocyte velocities across a wound with combinations of cytokines;

[0166] FIG. 11 shows cytokines AXL, CCL19, individually and together promote significantly faster closure of keratinocyte scratch wounds compared to unconditioned Epilife medium. The Y-axis shows collective migration distance of all keratinocytes. N=3;

[0167] FIG. 12 is a Venn diagram of significantly and differentially regulated transcripts in scratched keratinocytes exposed to AXL, DPFi conditioned medium or Epilife;

[0168] FIG. 13 shows A) the extracellular domain of AXL can bind Gas6 and B) the extracellular domain of AXL can bind itself. Taken from (Korshunov, 2012);

[0169] FIG. 14 shows A) examples of the punch within a punch wound closure over 6 days and B) daily delivery of AXL promotes the fastest wound closure in an ex vivo human wound. Wound closure with AXL is faster than with PDGF-BB, which is currently used to promote closure of chronic skin wounds;

[0170] FIG. 15 shows a schematic representation of the predicted AXL protein structure and showing a splice variant of the AXL polypeptide. The immunoglobulin (IgL) and fibronectin III (FNIII) domains are indicated with arrows. The amino acid sequence of AXL between the final FNIII domain and the transmembrane domain is shown to the right. The boxed 9 amino acids correspond to the differentially spliced AXL.

[0171] FIG. 16 shows an example of AXL and CCL19 temporally delivered via a layer-by-layer assembled bandage.

[0172] FIG. 17 shows the % of total COL1 fibres in unwounded skin, treated with the control Epilife, and treated with 2 g/ml sAXL. Significance is displayed in the graph (P0.05=*, P0.01=**, P0.001).

[0173] FIG. 18 shows the results of the soft agar colony formation assay to assess sAXL carcinogenicity in vitro.

[0174] FIG. 19 shows normalised intensity values of 2574 genes differentially expressed in KC in response to sAXL, DPFi CM and Epilife. (B) PCA plot showing variance on two components. Component 1 shows treatment variance whereas component 2 shows biological sample variance. (C) Four-way Venn of the upregulated and downregulated genes in sAXL and DPFi CM versus Epilife. (D) RT-PCR analysis on an in vitro wound assay. (E) Top pathways involved in regulating wound closure. Significance is displayed in the graph (P0.05=*, P0.01=**, P0.001=***) as determined by a one-way Anova and the error bars represent meanSD. In (A-C) N=2, n=3 and (D) N=2 and n=3.

[0175] FIG. 20 shows RT-PCR data from the edge of the wound of day 3 samples of ex vivo punch assays using EPHA4, SOS1, IL33 and CCL20 primers. Significance is displayed in the graph (P0.05=*, P0.01 =**, P0.001=***) as determined by a one-way Anova and the error bars represent meanSD. N=2 and n=3.

EXAMPLES

[0176] Materials and Methods

[0177] Human Skin Biopsies

[0178] For all the in vitro experiments, cells isolated from occipital scalp skin biopsies were used. These were taken from the occipital scalp of patients undergoing surgical proceedings after receiving informed consent, and using IC-REC approved consent forms. Tissue is held under ICHTB HTA license 12275, and used in the ICHTB approved project R15055.

[0179] For ex vivo experiments, human abdominal skin with adipose tissue was purchased from Caltag Medsystems LTD.

[0180] Isolation and Cell Culture of Fibroblasts

[0181] For cell isolation scalp skin was washed in Dulbecco's minimal essential medium (dMEM; Gibco Life Technologies) with 2% Antibiotics-Antimycotics (2% ABAM; Gibco Life Technologies) for 20 minutes for cleaning prior to dissection. Using a sterile Pasteur pipette, 8 small drops, and 1 larger drop of DMEM supplemented with 1xABAM are placed onto an inverted lid of a petri dish. Each small drop is used to hold a single end-bulb for inversion in. The drops are covered by placing the base of the petri dish inside the lid. The fat and connective tissue around the lower follicle is removed using scissors. Under a stereo microscope the end bulb of the hair follicle is visible; this is carefully cut off using sterile scissors and placed into dMEM with 1% ABAM. With a fine needle (27Gx3/4) the end of the hair follicle is fixed in place, while another needle is used to invert the end bulb structure and expose the dermal papilla containing dermal papilla fibroblasts (DPFi). The dermal papilla is then separated from the inverted end bulb and transferred into a 35 mm tissue culture dish covered in dMEM with 1% ABAM and 20% Fetal Bovine Serum (FBS; Gibco Life Technologies). The plates are placed in the incubator at 37 C., 5% CO2 and left undisturbed for 10 days during which time the papillae collapse and DFPi grow from the papilla in an explant.

[0182] With the remaining piece of skin the hypodermis is cut off to clean up the tissue. Using a scalpel blade to cut very close to the epidermis, the papillary and reticular dermis were separated into two pieces. Any remaining hair fibres in either piece of skin are removed with watchmaker's forceps. The pieces of skin are placed into separate 35 mm dishes and chopped into small pieces using scissors, and equally distributed around the dry dish. Once the tissue pieces have adhered to the base of the dish (usually 5 minutes later), dMEM containing 20% FBS and 1% ABAM is added to each dish to cover the tissue pieces and the dish is transferred to an incubator. After 10 days, cells have migrated from the reticular and papillary pieces of skin. These are termed reticular fibroblasts (RFi) and papillary fibroblasts (PFi) respectively.

[0183] Keratinocyte Isolation and Culture

[0184] Scalp skin is washed in dMEM with 2% ABAM for 20 minutes for cleaning prior to dissection. The adipose tissue is cut off the skin, and the rest of the tissue is placed in Dispase (Gibco Life Technologies) solution overnight at 4 C. After the overnight incubation, using sterile forceps, the epidermis is peeled off the dermis and was placed in 5 mL 1% Trypsin in a waterbath, at 37 C. The solution is shaken every 5 minutes to ensure that the cells are freed from the epidermis. The reaction is quenched using 5 mL Defined Trypsin Inhibitor (DTI; Gibco Life Technologies). A cell strainer with 40 m pore sized is used to remove any pieces of tissue. The cells are then centrifuged into a pellet at 200 g for 8 minutes. The supernatant is removed and Epilife (Gibco Life Technologies) with Epilife Defined Growth Supplement (EDGS; Gibco Life Technologies) and 1% ABAM are added to the cells. The cells, which are epidermal keratinocytes (KC) are then plated at a density of 5000 cells per cm2 in flasks that have been pre-coated using a coating matrix kit (Gibco Life Technologies), which contains collagen I.

[0185] Conditioned Media Collection

[0186] DPFi, PFi and RFi cells from human occipital scalp are seeded at a density of 6000 cells per cm2 in Dulbecco's minimal essential medium (dMEM; Gibco Life Technologies) supplemented with 10% Fetal Bovine Serum (FBS; Gibco Life Technologies). After 24 hours, the cells are washed two times with Phosphate Buffered Saline (PBS; Gibco life technologies) and Epilife (Gibco Life Technologies) supplemented with Epilife defined growth supplement (EDGS; Gibco Life Technologies), which is a KC growth media, is added to the cultures. Epilife media conditioned by the DPFi, PFi or RFi is collected 2 days later. The media is then filtered through a 0.22 m pore sized filter to remove cell debris and aliquoted and stored at 20 C. until used. Unconditioned Epilife media is subject to the same treatment and used as a control.

[0187] Keratinocyte Wound Healing Assays Using Fibroblast Conditioned Media

[0188] 6 well plates are prepared by coating them using the coating matrix kit (Gibco Life Technologies) as described in KC isolation and culture section. The assay is performed by wounding the cells using a p200 pipette.

[0189] KC are seeded at a density of 6000 cells per cm2 using Epilife supplemented with EDGS. When they reached confluency, a p200 pipette tip is used to scratch the middle of the well to create a wound in the cells. The cells are then washed two times with PBS. Conditioned media obtained from DPFi, PFi and RFi as well as a control with just Epilife supplemented with EDGS is placed onto scratched KC. Photographs are taken at 10 timepoints, from time 0 to 9, using a phase contrast microscope at 5 magnification. Images are analysed using Image J software.

[0190] Scratch Assay Analysis

[0191] The 9 hour measurement is used to provide information about migration and velocity. Ten images of scratch wounds closing are taken, at equal time intervals (1 hour), between the starting and end point. Optimally, the wound is not closed by the last timepoint, as migration stops when the gap reaches confluency. In order to quantify the characteristics of cell migration, images were analysed with the image processing software Image J.

[0192] The images are loaded onto the software, and the scale is set for the correct magnification of the images. Then for each timepoint, the gap is measured and calculated in m. The difference in m.sup.2 of the area covered by the keratinocytes, is calculated by subtracting the total wounded area of each timepoint from the first to ensure consistency between results.

[0193] The cell front velocity of each wound is calculated as follows:

[0194] 1. The total area the image is calculated in m.sup.2.

[0195] 2. Then, the total area is multiplied by the speed of the gap closure calculated as a % per hour. 3. The total area % is divided by the length of the picture in m to calculate how many m per hour the front is migrating.

[0196] 4. As there are two cell fronts, the m per hour is divided by 2, to obtain the normalized cell front velocity in m per hour.

[0197] The difference in m of the distance covered by the keratinocytes is analysed using a two-way Anova to determine significance, and plotted as a line graph.

[0198] Human Cytokine Antibody Array

[0199] RayBio C-Series human cytokine antibody array C1000 (RayBiotech) is used to analyse the conditioned media obtained from DPFi, PFi and RFi to determine the components of the medias. The protocol and the reagents used are ones provided by the kit supplier. All the solutions are prepared according to the manufacturer's instructions.

[0200] Antibody arrays are carefully removed from the plastic packaging and each membrane was placed (printed side up) into a well of the incubation tray provided. One membrane is used per conditioned media analysed. 2 ml of blocking buffer is pipetted into each well and incubated for 30 minutes at room temperature. The blocking buffer is then aspirated from each well. 1 ml of conditioned media is placed into each well and incubated overnight at 4 C. on a rocking plate. The next day, the conditioned media is aspirated from each well. 2 ml of 1 Wash Buffer I is added into each well and incubated for 5 minutes at room temperature. This is repeated two more times for a total of 3 washes using fresh buffer aspirating out the buffer completely each time. Then, 2 ml of 1 Wash Buffer II is added into each well and incubated for 5 minutes at room temperature. This is repeated one more time for a total of 2 washes using fresh buffer and aspirating out the buffer completely each time. 1 ml of the pre-prepared Biotinylated Antibody Cocktail is into each well and incubated overnight at 4 C. The next day, 2 ml of 1 HRP-Streptavidin is added into each well and incubated overnight at 4 C. HRP-Streptavidin is aspirated from each well. The membranes are then washed with 2 ml of 1 Wash Buffer I and incubated for 5 minutes at room temperature. This is repeated two more times for a total of 3 washes using fresh buffer and aspirating out the buffer completely each time. Then, 2 ml of 1 Wash Buffer II is added into each well and incubated for 5 minutes at room temperature. This is repeated one more time for a total of 2 washes using fresh buffer aspirating out the buffer completely each time. The membranes are transferred, printed side up, onto a sheet of tissue paper lying on a flat surface. Excess wash buffer is removed by blotting the membrane edges with another piece of paper. The membranes are transferred, printed side up, onto a plastic sheet provided, lying on a flat surface. Into a single clean tube, equal volumes (1:1) of Detection Buffer C and Detection Buffer D are added and mixed well with a pipette. The Detection Buffer mixture is then gently pipetted onto each membrane and incubated for 2 minutes at room temperature. Exposure should ideally start within 5 minutes after finishing the last step and completed within 10-15 minutes as chemiluminescence signals will fade over time. Another plastic sheet is placed on top of the membranes by starting at one end and gently rolling the flexible plastic sheet across the surface to the opposite end to smooth out any air bubbles. The membranes are sandwiched between the two plastic sheets. The sandwiched membranes are transferred to the chemiluminescence imaging system to expose for 1 minute.

[0201] Human Cytokine Array Analysis

[0202] The protein analyser plugin for Image J is used to analyse the cytokine array antibody membranes. Images can be loaded individually onto the software and the analysis is performed using the Array Analysis Menu followed by the Array Analysis function. This action proposes a method of background subtraction and builds a graphical interface for the dot matrix analysis. The visualisation can then then optimised by activating some options available from the graphical interface. Once the mask is set and recognises the membrane, a grid will form, and the matric can be measured automatically as a table of values.

[0203] Once the arrays are individually analysed using the Array Analysis tools, the Group Pattern menu then allows the user to obtain a global view of a set of arrays. The parent folder is set to contain the analysed arrays. This folder is selected containing the array analyses by the Masterize from Analysis Repertories function. This function looks for result tables coming from the Array Analysis functions, in the parent folder. The tool explores any sub-levels, and builds a master image, or pattern, associated to a master table presenting all the results. The program exhibits two default master representations:

[0204] 1. The default master pattern presents the arrays as they came from the analysis, with the visualization scaled between zero and the maximum values encountered in each array.

[0205] 2. The initial normalized pattern presents a normalization between zero and the maximum value found in the master. This representation gives the most natural aspect of the modelled pattern compared to the initial images.

[0206] The masters were then normalised using the internal references provided by the manufacturer on the membrane as positive and negative controls, by using the Group Pattern Menu and Set Internal Control and References. Each value is normalized following this formula:

Dot Value norm=(Dot Valuemean(Controls))/mean(References).

[0207] Once the normalised values are obtained, the following three correlations can be made: DPFi vs PFi, DPFi vs RFi and PFi vs RFi by calculating the fold change (log2) and the p-value (log10) by using t-test with unequal variance. The cut off values of p-value<0.05 and fold change ranging from 0.5 to 0.5 are set

[0208] Optimisation of Cytokine Concentrations and Cytokine Scratch Assay

[0209] The following four recombinant human cytokines were chosen to assess their effect on keratinocyte migration; AXL receptor tyrosine kinase (AXL; R&D systems), Chemokine ligand 19 (CCL19; Biolegend), Bone morphogenic protein 6 (BMP6; Biolegend) and Interleukin 6 (IL6; Gibco, life technologies). The specific activity of each cytokine is specified as a range by the supplier and three concentrations at the top, bottom and middle of this range were tested to obtain the concentration that yields the fastest wound gap closure using the KC scratch assay as previously described. The optimal cytokine concentrations were then used individually or in combination with one another, in the in vitro scratch wound assay. The following combinations were assessed: AXL, BMP6, CCL19, AXL+BMP6, AXL+CCL19, BMP6+CCL19, AXL+CCL19+BMP6, and IL6.

[0210] Recombinant human PDGF-BB (Biolegend) was also purchased to assess its effect on KC reepithelialisation. This cytokine has been optimised in human fibroblasts in culture before and its maximum effect was recorded to be 5 ng/ml, therefore this concentration was used going forward.

[0211] Human Ex Vivo Wound Model

[0212] Human abdominal skin with adipose tissue is obtained with informed consent. Subcutaneous fat is removed to obtain a sheet of epidermis with a thin dermis below. A series of 2 mm diameter partial thickness wounds are made using a biopsy punch, and the epidermis and papillary dermis are removed from these punches using fine scissors. Surrounding these 2 mm punches, a series of 8 mm full wounds are made, to create a series of wounds within a punch to assess wound closure of the 2 mm wound within the 8 mm punch. These 8 mm punches are then transferred to the top of a non-woven gauze and a 0.45 m nylon membrane (Millipore) in a 6 well plate. 1.5 ml of William's E media (Life Technologies) supplemented with 1% P/S, 2 mM L-Glutamine (Gibco), 10 g/ml Insulin (Sigma) and 10 ng/ml Hydrocortisone (Sigma) is added onto the non-woven gauze in each well. In order to test the effect of the different cytokines in wound closure, 6 conditions are tested simultaneously, with at least 6 technical replicates for each condition. The conditions tested are AXL, CCL19, PDGF-BB, IL6, CCL19+AXL and Epilife supplemented with EDGS. 5 l of solutions of these cytokines in Epilife with EDGS (control) are pipetted daily into the centre of the wound. Media is also changed daily with excess media being removed from the well and replaced with 1 ml fresh media. In the experiment as shown in FIG. 17, conditions were tested simultaneously (sAXL and Epilife), with 6 technical replicates for each condition. 5 l of solutions of 2 ug/ml of sAXL (R&D systems) in Epilife complete media and the control Epilife complete media were pipetted daily into the centre of the wound. Media was changed daily with excess media being removed from the well and replaced with 1 ml fresh media. 6 days after wounding, the ex vivo skin was embedded in OCT and was sectioned at 80 m thick sections to be imaged via SHG.

[0213] Images are taken of the wounds every 24 hours with a stereo microscope until wound closure is achieved (usually 5-10 days). The images are analysed using Image J. A two-way ANOVA can be used to analyse the difference in mm between pictures (indicating closure) and comparisons between conditions are made at individual time points using the same test in Graphpad Prism 6.0.

[0214] Transcriptional Analysis to Determine the Effect of AXL and DPFi CM on Keratinocyte Gene Expression

[0215] 3 wells of a 6 well plate were prepared by coating them using the coating matrix kit (Gibco Life Technologies) as previously described. KC are seeded at a density of 6000 cells per cm2 using Epilife supplemented with EDGS. When they reached confluency, a p200 pipette tip is used to scratch the well in 4 different regions (in a hashtag pattern), to create a wound in the cells. The cells are then washed two times with PBS. Conditioned media obtained from DPFi, AXL, and control with just Epilife supplemented with EDGS are placed on wounded cells. After 6 hours, media is removed, cells are washed in PBS, then RNA is collected using the RNeasy Plus Micro Kit (Qiagen). RNA is used to synthesized first-strand complementary DNA (cDNA) which is then converted to double-stranded cDNA, and used as a template for in vitro transcription generating cRNA. The cRNA is then transferred for hybridization and scanning onto the GeneChip Human Genome U133 Plus 2.0 Array.

[0216] Microarray Computational Analysis

[0217] Raw data from the microarray are analysed using the commercial software package Genespring GX 14.9 (Agilent Technologies Inc.). The intensity values of the samples are normalised and summarised using RMA algorithm. Parametric tests, with the p-value set at 0.05 are performed to determine significant differential expression between samples. Entities are chosen on a fold change cut off of >=2. Venn diagrams enable identification of genes which are uniquely upregulated or down regulated in keratinocytes after exposure to AXL, but not Epilife or DPFi conditioned medium. Pathway analysis on these specific genes is performed in Ingenuity.

[0218] Second Harmonic Generation (SHG)

[0219] To compare changes in the total collagen type 1 fibres, 80 m sections of ex vivo skin were imaged. 30 m-deep tile scans (6 Z-stack steps) of approximately 3,000 m2,000 m were obtained by the Second Harmonic Generation (SHG) by imaging the healed dermis. All images were first processed by adjusting the min-max at (0, 3000). Four regions of interest (ROIs) of 600 m450 m were then selected from tile scans within the previously wounded area or control area (unwounded). Stacks were then separated into single images. An interactive learning and segmentation toolkit called ilastik was applied to facilitate further analysis. As part of the pixel classification workflow, two features: fibres, and background (no signal), were selected and used for training of a set of 6 images. The probability mask was then applied to 20 images (single Z-stack steps) of each time point in a batch processing mode. The inventors then used the FIJI Macro Recorder to automatize the separation of segmented channels and quantification of the pixel area. Based on the pixel area covered by the two features, the total proportion of collagen in the dermis was calculated.

[0220] Soft Agar Colony Formation Assay

[0221] The method used for this assay has been previously described in detail by Borowicz et al 2014. Briefly, using a 6 well plate, a bottom layer of agar was plated by adding 1:1 ratio of 2 DMEM 10% FBS and 1% noble agar solution. The plates were covered, and the agar mixture was left to solidify at room temperature in the cell culture hood for 30 minutes. Once the lower layer of agar has solidified, the upper agar layer was prepared. The cells were seeded at 10000 cells/well and were resuspended in 1 DMEM 10%FBS and 0.6% agar in a 1:1 ratio and added on top of the solidified lower agar layer. The upper layer was left to solidify at room temperature in the cell culture hood for 30 min before placing into a 37 C. humidified cell culture incubator. A layer of medium was maintained over the upper layer of agar which contained the different concentrations of sAXL or the control media. 100 l of medium was added twice weekly for 21 days. After 21 days the cells were stained by adding 200 l of nitroblue tetrazolium chloride solution per well and incubating plates overnight at 37 C. The plates were then imaged to visualise colony formation.

[0222] Transcriptional Analysis

[0223] 6 well plates were prepared by coating them using the coating matrix kit. KC from two patients were seeded at a density of 6000 cells/cm2 using Epilife supplemented with EDGS. At confluency, a p200 pipette tip was used to scratch the well in 4 different regions (in a hashtag), to create a wound in the cells. The cells were then washed two times with PBS to remove debris. CM obtained from DPFi, sAXL, and control with just Epilife supplemented with EDGS were added on the wounded cells. After 6 hours, media was removed, cells were washed in PBS, then RNA was collected using the RNeasy Plus Micro Kit (Qiagen). RNA was used to synthesize first-strand complementary DNA (cDNA) using Nugen Ovation V2. This was then converted to double-stranded cDNA, and used as a template for in vitro transcription to generate cRNA using the Nugen Encore Biotin Module. The cRNA was then transferred for hybridization and scanning onto the GeneChip Human Genome U133 Plus 2.0 Array. Raw data from the microarray was analysed using the commercial software package Genespring GX 14.9 (Agilent Technologies Inc.). The intensity values of the samples were normalised and summarised using RMA algorithm. Parametric tests, with the p-value set at 0.05 were performed to determine significant differential expression between samples. Entities were chosen on a fold change cut off of >=2. Venn diagrams enabled identification of genes which were uniquely upregulated or down regulated in KC after exposure to sAXL and DPFi CM, but not Epilife. Pathway analysis on these specific genes was performed using Ingenuity Pathway Analysis (IPA; Agilent).

[0224] mRNA Extraction, Reverse Transcription and RT-PCR

[0225] RNA extraction was performed using a QiaShredder and RNeasy Mini kit (Qiagen) following manufacturer's instructions to obtain RNA from fresh tissue, DPFi, PFi and RFi. cDNA was synthesised using OligoDT primers and SuperScript III (Life Technologies). For the RT-PCR, PowerUP SYBR Green Master Mix (2X; Life Technologies) was used with primers designed using the UCSC database. RT-PCRs were run on an ABI 7500 Fast RealTime PCR with the cycles as follows: 2 minutes at 50 C. and 2 minutes at 95 C. followed by 35 cycles of 15 seconds at 95 C. and 1 minute at 60 C. Expression analysis was performed relative to GAPDH using the ddCT algorithm, with expression in fresh tissue used as a baseline comparison (value=1). RT-PCR was performed using cDNA from two biological replicates, and the relative expressions were consistent in both patients. Statistical analysis was performed using one-way Anova test.

[0226] Statistical Analyses

[0227] The number of replicates used for each experiment is indicated in their respective figure legends where N is the number of biological replicates and n is the number of technical replicates. Data are presented as the mean and standard deviation. Statistical significance was assessed using one-way ANOVA and a Tukey multiple-comparison post-hoc test unless otherwise stated. Differences were considered statistically significant if their p value0.05.

[0228] Results

Example 1

Hair Follicle Derived Fibroblasts Accelerate Wound Closure In Vitro

[0229] First, to assess whether sub-types of fibroblasts have differential paracrine effects on epidermal keratinocytes, the inventors collected keratinocyte medium (Epilife) which was conditioned by 3 sub-types of fibroblasts (DPFi, PFi and RFi) for 48 hours, filtered it to remove cell debris, and placed onto keratinocytes. Keratinocytes were scratched with a pipette, and the migration of cells into the scratch wound was then assessed. In this well established in vitro wound healing assay (13), the inventors found that RFi conditioned medium promoted significantly faster (p<0.05) wound closure compared to unconditioned keratinocyte medium. This supports the inventors' working hypothesis, that RFi release growth factors can promote re-epithelialisation. However, the most surprising result came with the DPFi conditioned medium, which promoted significantly faster (p<0.001) migration of keratinocytes across the scratch wound (FIG. 3) compared to controls and other fibroblast conditioned medias.

Example 2

Identification of Components of DPFi Medium Which Promote Wound Healing

[0230] To identify which factors are released by cells and contributing to the observed effects, the inventors then conducted cytokine arrays (FIG. 4) on keratinocyte medium conditioned by DPFi, RFi and PFi. The inventors then performed differential analysis to identify cytokines which were significantly released by DPFi compared to RFi or PFi (FIG. 5). The inventors identified 3 factors (AXL, CCL19, BMP6), which were released into the culture medium by DPFi at significantly higher levels than PFi. They also identified 10 factors released into the medium by DPFi at significantly higher levels than RFi, including AXL and CCL19 which were previously identified in the DPFi vs PFi cytokine array. BMP6 was released from DPFi at higher levels than from RFi, but did not pass the significance threshold. IL6, a well-known regulator of wound healing and epithelial migration (14, 15) was found at significantly higher levels in the RFi conditioned medium compared to the PFi, and the inventors, although not wishing to be bound by hypothesis, postulate that this may be contributing to the observed accelerated closure with RFi conditioned medium.

Example 3

Use of AXL in the Laboratory

[0231] For CCL19, BMP6 and IL6, it was easy to purchase a peptide. However, surprisingly, further research into AXL revealed that it is actually a tyrosine kinase receptor protein, and it was initially confusing as to why a transmembrane protein was on the cytokine array. The inventors have found that the extracellular domain of AXL is cleaved by ADAM10, leaving a small peptide product. The full structure of AXL is 894 amino acids long (FIG. 7A); it is a 140 kDa glycoprotein in the TAM receptor tyrosine kinase family with the gene located on chromosome 19q13.2 encoding 20 exons. The AXL gene is also known as UFO, ARK, JTK11 or TYRO7. Exons 1-10 encode the extracellular domain, which includes a signal peptide (aa 1-37), two immunoglobulin (Ig) domains (aa 37-124 for domain 1, 141-212 for domain 2), and two fibronectin type III (FNIII) domains (aa 224-322 for domain 1, 325-428 for domain) and is approximately 60-80 kDa (FIG. 6). Thus, as it was the extracellular domain of AXL which was detected on the cytokine array, the inventors purchased a peptide aa 33-440 of AXL for use in further experiments (FIG. 7). Exon 11 of AXL also encodes an extracellular region (aa438-451) that is subject to proteolytic cleavage along with exons 1-10 meaning the whole extracellular region of AXL is from aa1-451. Exons 12-20 compose the intracellular domain, which includes the tyrosine kinase domain (exons 13-20) (16). Not wanting to be bound to any particular hypothesis, the results described herein, and effect elicited by the soluble form of AXL, may be as a result of binding through one or both of the Ig domains, one of both of the FNIII domains, or either of the above combinations together (FIG. 7B).

Example 4

AXL Promotes Wound Closure in Scratch Assays

[0232] To further evaluate how the DPFi specific cytokines and IL6 might have a role in wound healing, the inventors assessed their effect on keratinocyte migration in a scratch wound individually and in combinations, compared to DPFi conditioned medium. The inventors used three concentrations, at the top, bottom and middle of the range suggested by the manufacturer, and determined maximal cell front velocity across a scratch wound for all three concentrations. The inventors identified an optimal concentration for use in further experiments (FIG. 8).

[0233] Surprisingly, when the inventors used the determined concentrations of cytokines in scratch wound assays, and assessed closure, they found that CCL19, AXL and BMP6 could all promote faster wound closure than DPFi, and significantly faster wound closure than Epilife control. IL6 on the other hand showed relatively little difference from the RFi conditioned medium in its ability to promote wound closure. This section is summarised in FIG. 9.

[0234] The inventors further assessed combinations of the cytokines together and, even more surprisingly, found that AXL by itself, CCL19 by itself, or AXL in combination with CCL19 were the best when they evaluated the maximum cell velocity front of keratinocytes crossing a scratch wound (FIG. 10). The inventors therefore used these individually, and in combination in the full scratch wound assay, plotting closure day by day and found, surprisingly, that CCL19 in combination with AXL significantly accelerated wound closure more than AXL or CCL19 by themselves (FIG. 11). Not wishing to be bound to any hypothesis, this effect could be promoted further by assessing temporal delivery of the cytokines. For example, CCL19 for 2 days, followed by AXL for the remainder of the wound closure. Not wanting to be bound to any particular hypothesis, each of these cytokines will activate distinct pathways which are important for wound closure. However, all the cytokines together at the same time may overload the cells.

Example 5

Role of Specific Cytokines in Wound Healing

[0235] After extensive research by the inventors, to their knowledge there hasn't been a connection previously made between AXL, the AXL cleaved peptide, nor AXL in combination with CCL19 in cutaneous wound healing or scar reduction.

[0236] As little is known about the role of the cleaved peptide of AXL, and the signalling pathways it is involved in, the inventors performed transcriptional analysis to further their understanding of it. The inventors took human keratinocytes in culture, and scratched them to create a scratch wound. Next, the inventors placed AXL (which is a component of DPFi conditioned medium), or DPFi conditioned medium, or control medium (Epilife) on cells for 6 hours before collecting RNA for analysis. After performing microarrays to identify the transcriptional profiles of cells, and identifying genes which were differentially expressed between conditions, the inventors identified 6 transcripts uniquely up regulated in AXL conditioned cells, and 9 transcripts which were down regulated (FIG. 12) (Table 1).

TABLE-US-00011 TABLE 1 15 transcripts identified as up or down regulated uniquely in keratinocytes containing AXL in media. Affymetrix FC AXL vs FC AXL vs Probe set ID Gene Symbol DPFi CM Epilife 204105_s_at NRCAM 2.78 4.55 209794_at SRGAP3 2.10 8.17 227497_at SOX6 2.30 3.03 227498_at SOX6 2.15 3.98 227943_at 2.00 2.51 230343_at CST3 2.01 8.71 205122_at MSANTD3- 2.17 4.32 TMEFF1///TMEFF1 205220_at HCAR3 2.12 3.17 210517_s_at AKAP12 2.80 6.07 211924_s_at PLAUR 2.32 9.57 216243_s_at IL1RN 2.03 4.30 227529_s_at AKAP12 3.05 2.37 1554086_at TUBGCP3 2.66 3.60 1555673_at KRTAP2-3///KRTAP2-4 2.22 2.48 1566764_at MACC1 2.35 6.61

[0237] The vitamin k dependent protein Gas6 is known to bind AXL and trigger autophosphorylation of the AXL cytoplasmic domain, which leads to further downstream processes such as migration, proliferation and reduced inflammation (21). It has also been suggested that AXL is able to undergo homophilic binding of its extracellular domains with AXL on neighbouring cells (FIG. 13). This is a ligand-independent type of receptor activation that occurs after overexpression of AXL (22, 23). Potentially, but not wanting to be bound to any particular hypothesis, addition of sAXL to the media is either neutralising GAS6 thereby inhibiting the AXL downstream processes or alternatively sAXL is acting as an AXL decoy and undergoes homophilic binding with membrane bound AXL on cells. Not wishing to be bound by any particular hypothesis, sAXL may either be inhibiting or activating full length AXL.

Example 6

Assessment of Wound Healing in a Human Skin Model

[0238] So far, the inventors had only assessed the role of AXL and CCL19 in 2D scratch wounds, and so they sought to evaluate their effect in an ex vivo human skin model, called a punch within a punch (FIG. 14A). Here, a small wound is created within a larger wound, and wound closure of the small wound is evaluated over the course of a few days (24). Epithelial migration over the wound bed can then be plotted as a function of time. Using this assay, the inventors evaluated the effect of AXL alone, CCL19 alone and in combination with AXL. They also evaluated IL6 as it has a documented role in wound healing along with PDGF-BB which is the active component in the aforementioned Regranex product. Surprisingly, the inventors found that AXL alone promoted the fastest wound closure in the ex vivo human skin wound, significantly faster than the control just 3 days after the start of the experiment (FIG. 14B). It is worth noting that AXL promoted faster wound closure than Regranex, which is currently still used in the USA to promote wound closure and thus the AXL represents a significant improvement over currently known wound treatments.

[0239] It is also important to note here that the punch within a punch assay wound was given fresh doses of cytokines daily. Not wanting to be bound to any particular hypothesis, when assessing modes of delivery, bandages assembled using a layer by layer technique, or specific cosmeceutical formulations would enable sustained delivery of cytokines over the course of a few days, which would be beneficial as a treatment option for chronic wounds. Lower concentrations of cytokines could be employed if delivery was sustained over longer time periods.

Example 7

Reduced Scar Formation with the Addition of sAXL Ex Vivo

[0240] The skin dermis is mainly composed of cells (such as fibroblasts and endothelial cells) and extracellular matrix (ECM). Interstitial collagens make up the majority of that ECM with Collagen I (COL1) being one of the main ECM protein in the skin dermis (Xue and Jackson 2015). After a cutaneous injury, the skin heals via a series of events known as haemostasis and inflammation, reepithelialisation and ECM remodelling. Dermal remodelling can take months to years to be completed. Previous research has shown that the content of COL1 is significantly altered in a scar tissue compared to unwounded controls, with significantly higher COL1 content in wounded patients even at 24 months post injury (Wang Cheng and Guo-an 2011). In order to quantify scar formation (COL1 content) in ex vivo skin, the inventors used Second Harmonic Generation (SHG) imaging (see methods section) to quantify percentage (%) of total COL1 fibres within the skin dermis of skin healed with and without our cytokine of interest (see methods section; FIG. 17). Analysis of the SHG images using image segmentation revealed that the volume of collagen is significantly greater in Epilife treated wounds (94% +/SD) in comparison to the both the unwounded area (85% +/SD) and sAXL treated wounds (89% +/SD). Since scar tissue contains higher amounts of total COL1, the significantly lower amounts of COL1 found in sAXL, and the similarity to unwounded skin, suggest that sAXL promotes a reduced scar phenotype in human skin.

Example 8

Soft Agar Colony Formation Assay to Assess sAXL Carcinogenicity In Vitro

[0241] Transformation of normal cells into neoplastic cells occurs via a series of genetic alterations, leading to a cell population that is capable of proliferation in a three-dimensional environment. Anchorage-independent growth is the ability of neoplastic cells to grow independently of a solid surface. The soft agar colony formation assay (Method previously described by (Borowicz, Van Scoyk et al. 2014)) has been widely used to monitor cell transformation and anchorage-independent growth, by visualising colony formation after 3 weeks in culture. The inventors used this assay to identify whether different concentrations of sAXL could transform skin fibroblast cells from the dermis, into neoplastic ones. The inventor's results show that sAXL does not transform the cells into neoplastic cells at concentrations 2 g/ml to 32 g/ml, as the cells are not able to proliferate and form colonies in the three-dimensional environment. This was compared to a positive control of Suite-007 (human cancel cell line derived from the metastatic liver from Pancreatic ductal adenocarcinoma) cells which were able to form colonies in the soft agar assay in contrast to sAXL and the negative control that did not form any colonies. Here, the inventors have illustrated that the soft agar colony formation assay using sAXL at concentrations 2 g/ml to 32 g/ml does not promote transformation of normal cells into neoplastic cells (FIG. 18).

Example 9

Microarray Reveals that sAXL Promotes Keratinocyte Migration While Inhibiting Keratinocyte Differentiation

[0242] The inventors used a microarray to perform an unbiased transcriptional analysis where they compared sAXL, Dermal papilla fibroblast conditioned media (DPFi CM) and Epilife on scratch wound transcription in keratinocytes (KC) in vitro (FIG. 19A). Raw data from the microarray was analysed with a one-way Anova test identifying 2574 genes which were significantly and differentially regulated between conditions (FIG. 19A). Principal component analysis shows that sAXL and DPFi clustered more closely together than Epilife thus sharing less variance (FIG. 19B). Specifically, variance between Epilife media and both DPFi CM and sAXL was on the 1st principle component while variance between the biological repeats (P1 and P2) was on the 2nd principle component.

[0243] To help the inventors determine unique genes involved in accelerated wound closure in vitro, upregulated and downregulated gene lists of sAXL and DPFi CM vs Epilife were plotted in a Venn diagram (FIG. 19C). Using the Venn the inventors identified 1222 genes upregulated and 570 downregulated in both DPFi CM and sAXL treated KC in comparison to KC treated with the Epilife control. The inventors believe that these gene lists encompass genes which are enabling accelerated scratch wound closure as a result of their differential regulation (Table 1). In an attempt to identify the pathways activated in response to the genes uniquely upregulated/downregulated by DPFi CM and sAXL, the inventors used IPA software to identify signalling pathways activated or inhibited in the KC. Using the list of genes upregulated/downregulated in KC in both sAXL and DPFi CM, the inventors identified three main pathways that are activated; the Hippo pathway, Ephrin pathway and Epidermal Growth Factor (EGF) pathway (FIG. 19D). Activation of Yes-associated protein 1 (YAP1), a member of the Hippo pathway, can promote migration of cells while blocking KC differentiation. In addition, the EGF receptor (EGFR) was also upregulated in KC, predicted to promote cell cycle progression but simultaneously block KC differentiation. Ephrin A4 (EPHA4), a member of the Ephrin pathway, was the most highly upregulated gene in the KC in sAXL and is known to promote cell migration, cell movement and adhesion of epithelial cells.

[0244] To validate the transcriptional data, the inventors performed RT-PCR on EPHA4, SOS1, IL-33 and CCL20 (FIG. 19E and Table 1).

TABLE-US-00012 TABLE 1 Microarray top upregulated and downregulated genes. Gene list with the FC (Fold Change) of the top ten upregulated and downregulated genes in DPFi CM and sAXL compared to Epilife. Highlighted genes have been validated both in vitro and ex vivo. Gene FC FC name (sAXL vs Epilife) (DPFi CM vs Epilife) EPHA4 14.921973 10.557523 ATP6V0A2 11.761193 9.18081 DIDO1 11.577912 11.624034 FAM49B 11.449174 10.309539 SOS1 11.297932 12.510902 MAP7D3 10.468552 13.538113 GIT2 9.92446 10.028248 SENP2 9.882794 10.513346 DSCAM 9.637356 8.835972 NDUFAF4 9.1372 9.017144 IL33 10.3360815 15.048633 ICA1 10.473449 11.850388 C7orf57 11.098472 8.344663 LOC100129518///SOD2 11.761277 11.891947 ADAM9 12.056555 10.755545 OSMR 12.578187 10.769784 TOR1A 14.24639 11.107677 CCL20 15.549934 14.422149 CDCP1 18.541739 22.558874 IL13RA2 21.426365 22.007032

[0245] To determine if these genes would also be differentially regulated ex vivo, the inventors isolated RNA from the leading edge of the epidermis of the ex vivo punches treated with Epilife, sAXL or DPFi CM. Here, only the EphA4 results were able to be duplicated (FIG. 20), highlighting the Ephrin's pathway involvement in the wound healing process.

[0246] Conclusions

[0247] Not wishing to be bound to any particular hypothesis, in the context of a chronic wound where re-epithelisation is impaired, the inventors believe AXL will kick start the wound healing process, and thus promote closure where previously there was none. Chronic wounds have enhanced risk of wound site infection and therefore promoting re-epithelisation will also help to reduce infection. However, promoting re-epithelisation has advantages in other contexts, such as the reduction of scarring in normal wound closure and chronic wounds. Scarring is an inherent human property, which occurs due to impaired dermal re-modelling in the third phase of wound closure. However, chronic wounds with delayed re-epithelisation are characterised by extensive scarring, and there are clear links between scar formation and the time it takes for the wound to initially close. For example, re-epithelisation also occurs faster in oral wounds compared to skin wounds, and oral scars are few and far between. In vitro, oral keratinocytes migrate three times faster than skin keratinocytes in scratch wound assays. Thus, without wishing to be bound to any particular hypothesis, the inventors believe that targeting and accelerating the very first stage of wound healing, re-epithelisation, will have be useful both for the closure of chronic wounds, and in the reduction scar formation in the skin after injury.

[0248] In conclusion, and not wishing to be bound to any hypothesis, the inventors propose that the cleaved extracellular domain of AXL is a novel peptide which can be used to promote faster wound closure and reduce scarring of human skin by accelerating re-epithelisation.

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