Skin substitute compositions and methods of making and using same - Patents.com

Abstract

Provided herein is a skin substitute composition comprising a stratified epidermis, the cells of which produce, e.g., secrete, recombinant growth factors and recombinant insulin. In some aspects, the disclosure further relates to methods of making the skin substitute, and to methods of using the composition for the treatment of a subject, e.g., for wound healing. TIFF2024531827000016.tif85128

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 233,196, filed August 13, 2021, the contents of each of which are incorporated by reference in their entirety for all purposes.

[0002] INCORPORATION BY REFERENCE TO SEQUENCE LISTING The contents of the electronic sequence listing (166312000140SEQLIST.xml; size: 65,924 bytes; and creation date: August 12, 2022) are incorporated herein by reference in their entirety.

[0003] Field The present disclosure relates in some aspects to a skin substitute composition comprising a stratified epidermis, the cells of which produce, e.g., secrete, recombinant growth factors and recombinant insulin. In some aspects, the present disclosure further relates to a method of making the skin substitute and a method of using the composition for the treatment of a subject, e.g., for wound healing. [Background technology]

[0004] background Skin substitute or skin equivalent compositions are useful for inducing wound healing in subjects in need thereof.However, the wound healing ability of such compositions is limited, and requires high subject compliance, such as by requiring several applications of the composition during wound healing.There is a need for an improved composition that induces strong wound healing with minimal subject intervention.Compositions and methods that meet such needs are provided herein. Summary of the Invention

[0005] overview In some aspects, a skin substitute is described herein. In some of the optional embodiments, the skin substitute comprises a stratified epidermis including a stratum basale, a stratum spinosum, a stratum granulosum, and a stratum corneum, and the cells of the stratified epidermis express recombinant growth factors and recombinant insulin. In some of the optional embodiments, the recombinant growth factors and recombinant insulin are secretable from the cells of the stratified epidermis. In some of the optional embodiments, the stratified epidermis is 100-200 μm thick. In some of the optional embodiments, the cells of the stratified epidermis expressing recombinant growth factors and recombinant insulin include cells of the stratum basale. In some of the optional embodiments, the recombinant insulin is or comprises recombinant human insulin.

[0006] In some of the embodiments, the recombinant insulin has (i) an amino acid sequence as set forth in SEQ ID NO:5, (ii) a functional variant of recombinant insulin having an amino acid sequence with at least 85%, at least 90%, or at least 95% sequence identity with SEQ ID NO:5; or (iii) a two-chain form of (i) or (ii) comprising an A chain and a B chain. In some of the embodiments, the A chain and the B chain are linked by a disulfide bond. In some of the embodiments, the recombinant insulin is encoded by a polynucleotide encoding (i) an amino acid sequence as set forth in SEQ ID NO:5, or (ii) a functional variant having an amino acid sequence with at least 85%, at least 90%, or at least 95% sequence identity with SEQ ID NO:5. In some of the embodiments, the recombinant insulin is an AspB10 insulin analog comprising a histidine to aspartic acid mutation at position 10 in the B chain of modified human proinsulin compared to the wild-type insulin set forth in SEQ ID NO:5.

[0007] In some of the embodiments, the skin substitute comprises a polynucleotide encoding proinsulin, the proinsulin comprising at least one furin recognition sequence in place of the endopeptidase Arg31-Arg32 cleavage site or the endopeptidase Lys64-Arg65 cleavage site. In some of the embodiments, the at least one furin recognition sequence is in place of the endopeptidase Arg31-Arg32 cleavage site and the endopeptidase Lys64-Arg65 cleavage site. In some of the embodiments, the at least one furin recognition sequence comprises the consensus sequence RXRR, where X is any amino acid (SEQ ID NO:8), or RXKR, where X is any amino acid (SEQ ID NO:9). In some of the embodiments, the at least one furin cleavage site is RTKR (SEQ ID NO:10) or RQKR (SEQ ID NO:42).

[0008] In some of the embodiments, the recombinant insulin has (i) an amino acid sequence as set forth in SEQ ID NO:6, (ii) a functional variant having an amino acid sequence with at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO:6; or (iii) a two-chain form of (i) or (ii) comprising an A chain and a B chain. In some of the embodiments, the A chain and the B chain are linked by a disulfide bond. In some of the embodiments, the recombinant insulin comprises a sequence as set forth in SEQ ID NO:6 or a two-chain form of (i) or (ii) comprising an A chain and a B chain. In some of the embodiments, the A chain and the B chain are linked by a disulfide bond. In some of the embodiments, the recombinant insulin comprises an A chain as set forth in SEQ ID NO:36 and a B chain as set forth in SEQ ID NO:41. In some of the embodiments, the A chain and the B chain are linked by a disulfide bond.

[0009] In some of any of the embodiments, the recombinant human insulin is encoded by a polynucleotide comprising a sequence having at least 85% or at least about 85%, at least 90% or at least about 90%, or at least 95% or at least about 95% sequence identity to the sequence set forth in SEQ ID NO: 2. In some of any of the embodiments, the recombinant human insulin comprises a sequence set forth in SEQ ID NO: 2. In some of any of the embodiments, the recombinant growth factor is selected from the group consisting of epidermal growth factor (EGF), erythropoietin (EPO), fibroblast growth factor (FGF), granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), hepatocyte growth factor (HGF), insulin-like growth factor (IGF), platelet-derived growth factor (PDGF), transforming growth factor alpha and beta, vascular endothelial growth factor (VEGF), epidermal growth factor receptor (EGFR), fibroblast growth factor receptor (FGFR), and any isoforms or alternatively spliced ​​variants thereof. In some of the optional embodiments, the recombinant growth factor is VEGF or an isoform or alternatively spliced ​​variant thereof.

[0010] In some of the embodiments, VEGF is encoded by a polynucleotide sequence having at least 85%, at least about 85%, at least about 90%, or at least about 90%, or at least about 95% or at least about 95% sequence identity to the sequence shown in SEQ ID NO:4. In some of the embodiments, VEGF is encoded by a polynucleotide sequence comprising the sequence shown in SEQ ID NO:4. In some of the embodiments, VEGF comprises a sequence having at least 85%, at least about 85%, at least about 90%, or at least about 90%, or at least about 95% or at least about 95% sequence identity to the sequence shown in SEQ ID NO:7, or a sequence thereof lacking a signal peptide. In some of the embodiments, VEGF comprises a sequence shown in SEQ ID NO:7, or a sequence thereof lacking a signal peptide.

[0011] In some of the embodiments, the skin substitute comprises VEGF, wherein the VEGF comprises a sequence having at least or at least about 85%, at least or at least about 90%, or at least or at least about 95% sequence identity to the sequence set forth in SEQ ID NO: 44. In some of the embodiments, the VEGF comprises a sequence set forth in SEQ ID NO: 44. In some of the embodiments, the recombinant growth factor and the recombinant insulin are encoded by a bicistronic expression cassette comprising a polynucleotide encoding the recombinant growth factor and a polynucleotide encoding the recombinant insulin, separated by a bicistronic element.

[0012] In some of the embodiments, the bicistronic element is an IRES. In some of the embodiments, the polynucleotide encoding the recombinant growth factor and the polynucleotide encoding the recombinant insulin are operably linked to a promoter. In some of the embodiments, the promoter is a constitutive promoter or an inducible promoter. In some of the embodiments, the promoter is a CAG promoter. In some of the embodiments, the polynucleotide encoding the recombinant growth factor is upstream of the polynucleotide encoding the recombinant insulin in the bicistronic expression cassette.

[0013] In some of the embodiments, the cells of the stratified epidermis secrete recombinant growth factors and recombinant insulin at levels that result in a greater improvement in one or more markers of vascularization remodeling than skin substitutes containing either recombinant growth factors or recombinant insulin alone. In some of the embodiments, the improvement in one or more markers of vascularization remodeling can be assessed with a tube formation assay. In some of the embodiments, the marker of vascularization remodeling is an increase in the number of nodes or unions, defined as the joining sites of at least three chords. In some of the embodiments, the marker of vascularization remodeling is an increase in the number of webs, defined as closed circuits surrounded by two or more nodes. In some of the embodiments, the marker of vascularization remodeling is an increase in the number of main segments, defined as chords that join two nodes together.

[0014] In some of the embodiments, the cells of the stratified epidermis secrete quantifiable levels of recombinant growth factors and recombinant insulin continuously. In some of the embodiments, the cells of the stratified epidermis secrete recombinant growth factors and recombinant insulin continuously for up to or about 2 days, up to or about 3 days, up to or about 4 days, up to or about 5 days, up to or about 6 days, up to or about 7 days, up to or about 8 days, up to or about 9 days, up to or about 10 days, up to or about 11 days, up to or about 12 days, up to or about 13 days, or up to or about 14 days. In some of the embodiments, the cells of the stratified epidermis secrete recombinant growth factors and recombinant insulin continuously for up to or about 1 week, up to or about 2 weeks, up to or about 3 weeks, up to or about 1-2 weeks, or up to or about 2-3 weeks.

[0015] In some of the embodiments, the cells of the stratified epidermis secrete quantifiable levels of recombinant growth factors and C-peptide that are detectable for up to or about 2 days, up to or about 3 days, up to or about 4 days, up to or about 5 days, up to or about 6 days, up to or about 7 days, up to or about 8 days, up to or about 9 days, up to or about 10 days, up to or about 11 days, up to or about 12 days, up to or about 13 days, or up to or about 14 days. In some of the embodiments, the cells of the stratified epidermis secrete quantifiable levels of recombinant growth factors and C-peptide that are detectable for up to or about 1 week, up to or about 2 weeks, up to or about 3 weeks, up to or about 1-2 weeks, or up to or about 2-3 weeks.

[0016] In some of the embodiments, the cells of the stratified epidermis secrete recombinant growth factors and recombinant insulin at levels that reduce the level of advanced glycation end products (AGEs) in the skin of the subject. In some of the embodiments, the cells of the stratified epidermis are differentiated from keratinocytes. In some of the embodiments, the keratinocytes are human keratinocytes. In some of the embodiments, the keratinocytes are HaCaT keratinocyte cells.

[0017] In some aspects, provided herein is a bicistronic expression cassette comprising a polynucleotide encoding a recombinant human growth factor and a recombinant insulin. In some of the embodiments, the encoded recombinant insulin (i) has an amino acid sequence as set forth in SEQ ID NO:5, or (ii) is a functional variant having an amino acid sequence with at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO:5. In some of the embodiments, the polynucleotide encoding the recombinant insulin (i) comprises an amino acid sequence as set forth in SEQ ID NO:5, or (ii) is a functional variant having an amino acid sequence with at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO:5.

[0018] In some of the embodiments, the encoded recombinant insulin is an AspB10 insulin analogue, which comprises a histidine to aspartic acid mutation at position 10 in the B chain of modified human proinsulin compared to the wild-type insulin shown in SEQ ID NO:5. In some of the embodiments, the polynucleotide encoding the recombinant insulin encodes a proinsulin comprising at least one furin recognition sequence in place of the endopeptidase Arg31-Arg32 cleavage site or the endopeptidase Lys64-Arg65 cleavage site. In some of the embodiments, the at least one furin recognition sequence is in place of the endopeptidase Arg31-Arg32 cleavage site and the endopeptidase Lys64-Arg65 cleavage site. In some of the embodiments, the at least one furin recognition sequence comprises the consensus sequence RXRR, where X is any amino acid (SEQ ID NO:8) or RXKR, where X is any amino acid (SEQ ID NO:9). In some of the optional embodiments, at least one furin cleavage site is RTKR (SEQ ID NO:10) or RQKR (SEQ ID NO:42).

[0019] In some of any of the embodiments, the encoded recombinant insulin (i) has an amino acid sequence set forth in SEQ ID NO:6, or (ii) is a functional variant having an amino acid sequence having at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO:6. In some of any of the embodiments, the encoded recombinant insulin comprises a sequence set forth in SEQ ID NO:6. In some of any of the embodiments, the polynucleotide encoding the recombinant insulin comprises a sequence having at least 85%, or at least about 85%, at least 90%, or at least about 90%, or at least 95%, or at least about 95% sequence identity to the sequence set forth in SEQ ID NO:2.

[0020] In some of the embodiments, the polynucleotide encoding the recombinant insulin comprises the sequence shown in SEQ ID NO: 2. In some of the embodiments, the encoded recombinant growth factor is selected from the group consisting of epidermal growth factor (EGF), erythropoietin (EPO), fibroblast growth factor (FGF), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), hepatocyte growth factor (HGF), insulin-like growth factor (IGF), platelet-derived growth factor (PDGF), transforming growth factor alpha and beta, vascular endothelial growth factor (VEGF), epidermal growth factor receptor (EGFR), fibroblast growth factor receptor (FGFR), and any isoforms or alternatively spliced ​​variants thereof.

[0021] In some of the embodiments, the recombinant growth factor is VEGF or an isoform or alternatively spliced ​​variant thereof. In some of the embodiments, the polynucleotide encoding the growth factor comprises a sequence having at least 85% or at least about 85%, at least 90% or at least about 90%, or at least 95% or at least about 95% sequence identity to the sequence shown in SEQ ID NO:4. In some of the embodiments, the polynucleotide encoding the growth factor comprises a sequence shown in SEQ ID NO:4. In some of the embodiments, the encoded VEGF comprises a sequence having at least 85% or at least about 85%, at least 90% or at least about 90%, or at least 95% or at least about 95% sequence identity to the sequence shown in SEQ ID NO:7, or a sequence thereof lacking a signal peptide.

[0022] In some of the embodiments, the encoded VEGF comprises the sequence set forth in SEQ ID NO:7 or a sequence thereof lacking the signal peptide. In some of the embodiments, the encoded VEGF comprises a sequence having at least 85%, or at least about 85%, at least 90%, or at least about 90%, or at least 95%, or at least about 95% sequence identity to the sequence set forth in SEQ ID NO:44. In some of the embodiments, the encoded VEGF comprises the sequence set forth in SEQ ID NO:44.

[0023] In some of the embodiments, the polynucleotide encoding the recombinant growth factor and the polynucleotide encoding the recombinant insulin are separated by a bicistronic element. In some of the embodiments, the bicistronic element is an IRES. In some of the embodiments, the polynucleotide encoding the recombinant growth factor and the polynucleotide encoding the recombinant insulin are operably linked to a promoter. In some of the embodiments, the promoter is the same. In some of the embodiments, the promoter is a constitutive promoter or an inducible promoter. In some of the embodiments, the promoter is a CAG promoter. In some of the embodiments, the polynucleotide encoding the recombinant growth factor is upstream of the polynucleotide encoding the recombinant insulin in a bicistronic expression cassette.

[0024] In some aspects, provided herein is a vector comprising the bicistronic expression cassette of any of the embodiments provided herein. In some of the embodiments, the vector is a viral vector. In some of the embodiments, the viral vector is an adenovirus vector. In some of the embodiments, the vector is a non-replicative type 5 adenovirus. In some of the embodiments, the non-replicative adenovirus lacks or is deleted in the E1 and E3 regions. In some of the embodiments, the bicistronic expression cassette is inserted in the E1 region.

[0025] In some aspects, a method for producing a skin substitute is provided herein. In some of any of the embodiments, the method includes: 1) differentiating keratinocytes into stratified epidermis, the stratified epidermis comprising basal layer, spinous layer, granular layer, and stratum corneum; and 2) introducing the bicistronic expression cassette of any of the embodiments provided or the vector of any of the embodiments provided into the cells of the stratified epidermis to produce a skin substitute, the skin substitute comprising recombinant growth factor and recombinant insulin. In some of any of the embodiments provided, the introducing step is by transduction of the viral vector of any of the embodiments provided.

[0026] In some aspects, a method for producing a skin substitute is provided herein. In some of any of the embodiments, the method includes: 1) differentiating keratinocytes into stratified epidermis, the stratified epidermis including basal layer, spinous layer, granular layer, and stratum corneum; and 2) transducing the cells of the stratified epidermis with the viral vector of any of the embodiments provided to produce a skin substitute, the skin substitute including growth factors and insulin.

[0027] In some aspects, the present invention provides a method for producing a skin substitute.In some of any of the embodiments, the method includes: 1) differentiating keratinocytes into stratified epidermis, the stratified epidermis includes basal layer, spinous layer, granular layer, and stratum corneum; and 2) transducing the cells of stratified epidermis with an adenoviral vector encoding modified proinsulin and growth factor to produce a skin substitute, the skin substitute includes growth factor and insulin.

[0028] In some of the embodiments, at the time of introduction or transduction, cells of the stratified epidermis express occludin and claudin. In some of the embodiments, cells of the basal layer are introduced or transduced. In some of the embodiments, prior to differentiation in step 1), the method comprises culturing the keratinocytes in low calcium medium for 2 weeks, 3 weeks, 4 weeks, 5 weeks, or 6 weeks to cultivate the basal layer. In some of the embodiments, prior to differentiation in step 1), the method comprises culturing the keratinocytes in low calcium medium for 4 weeks or about 4 weeks to cultivate the basal layer.

[0029] In some of the embodiments, the low calcium medium comprises a calcium concentration of 0.01 to 0.1 mM at the time of seeding the cells or during the culturing step. In some of the embodiments, the low calcium medium comprises a calcium concentration of up to or about 0.05 mM at the time of seeding the cells or during the culturing step. In some of the embodiments, the low calcium medium comprises a calcium concentration that is about 0.03 mM at the time of seeding the cells or during the culturing step.

[0030] In some of the embodiments, the low calcium medium further comprises epidermal growth factor (EGF) and bovine pituitary extract (BPE). In some of the embodiments, the low calcium medium comprises up to or about 1 ng / ml EGF and up to or about 100 μg / ml BPE at the time of seeding the cells or during the culturing step. In some of the embodiments, the low calcium medium comprises up to or about 0.2 ng / ml EGF and up to or about 30 μg / ml BPE at the time of seeding the cells or during the culturing step. In some of the embodiments, the keratinocytes are human keratinocytes. In some of the embodiments, the keratinocytes are HaCaT keratinocyte cells.

[0031] In some optional embodiments, step 1) comprises culturing keratinocytes on an extracellular matrix substrate. In some optional embodiments, the extracellular matrix substrate is collagen. In some optional embodiments, the extracellular matrix substrate is certified for human use. In some optional embodiments, the keratinocytes are cultured at a density of 5×10 on the extracellular matrix substrate. 6 / ml to 50 x 10 6 In some optional embodiments, the cell density is between 10×10 6 pieces / ml, 20×10 6 pieces / ml, 30×10 6 / ml or 40 x 10 6 / ml, or approximately 10 x 10 6pieces / ml, 20×10 6 pieces / ml, 30×10 6 / ml or 40 x 10 6 In some optional embodiments, the cell density is 20×10 6 / ml or approximately 20 x 10 6 In some of the optional embodiments, the extracellular matrix substrate is coated onto a Transwell insert.

[0032] In some of the optional embodiments, the culturing in step (1) is for about 23-28 days. In some of the optional embodiments, the culturing in step (1) includes a first incubation in a low calcium medium and a second incubation in a high calcium medium. In some of the optional embodiments, the first incubation in the low calcium medium is for about 3-5 days and the second incubation in the high calcium medium is for about 20-23 days. In some of the optional embodiments, the low calcium medium includes 0.01-0.1 mM calcium. In some of the optional embodiments, the high calcium medium includes 1.0-3.0 mM calcium. In some of the optional embodiments, the low calcium medium includes 0.03 mM calcium and the high calcium medium includes 2.4 mM calcium.

[0033] In some of the optional embodiments, the low calcium medium and the high calcium medium further comprise EGF and BPE. In some of the optional embodiments, the low calcium medium and the high calcium medium comprise 0.05 ng / mL to 1 ng / ml EGF and 1 μg / ml to 100 μg / ml BPE. In some of the optional embodiments, the low calcium medium and the high calcium medium comprise 0.2 ng / ml or about 0.2 ng / ml EGF and 30 μg / ml or about 30 μg / ml BPE. In some of the optional embodiments, the high calcium medium further comprises hydrocortisone.

[0034] In some of the optional embodiments, the high calcium medium comprises 0.1-1.0 μg / ml hydrocortisone. In some of the optional embodiments, the high calcium medium comprises 0.4 μg / ml or about 0.4 μg / ml hydrocortisone. In some of the optional embodiments, the low calcium medium is a serum-free medium. In some of the optional embodiments, the high calcium medium is a serum-free medium.

[0035] In some of the optional embodiments, an air-liquid interface is introduced when culturing keratinocytes in high calcium medium during the second incubation, where the cells of the basal layer are exposed to the high culture medium but not to the gaseous environment. In some of the optional embodiments, the low calcium medium is replaced daily during the first incubation. In some of the optional embodiments, the high calcium medium is replaced daily during the second incubation.

[0036] In some of the embodiments, after step 2), the method can further include formulating the skin substitute with a cryoprotectant. In some of the embodiments, the cryoprotectant includes human albumin and glucose. In some of the embodiments, the method provided further includes freezing the skin substitute after step 2). In some of the embodiments, the method provided can further include performing a quality control evaluation on the skin substitute. In some of the embodiments, the quality control evaluation is performed before formulating the skin substitute with a cryoprotectant. In some of the embodiments, up to or about 24 hours elapse between the completion of step 2) and the quality control step. In some of the embodiments, the quality control step includes detecting one or more polypeptides selected from the group consisting of proinsulin, modified proinsulin, insulin, insulin variants, growth factors, and variants thereof.

[0037] In some of the embodiments, the method provided can further include placing the skin substitute on an absorbent gauze. In some of the embodiments, the keratinocytes include immortalized keratinocytes. In some of the embodiments, the keratinocytes include cells from HaCaT, NM1, or NIKS cell lines, and / or cells derived from HaCaT, NM1, or NIKS cell lines.

[0038] In some aspects, provided herein is a skin substitute produced by any of the methods provided. In some aspects, provided herein is a cryopreserved skin substitute comprising the skin substitute of any of the embodiments provided and a cryoprotectant. In some of the optional embodiments, the cryoprotectant comprises human albumin (0.02 g / mL) and D-glucose (0.09 g / mL).

[0039] In some aspects, provided herein is a skin substitute comprising a skin substitute of any of the embodiments provided or a cryopreserved skin substitute of any of the embodiments provided and an absorbent gauze, wherein the cryopreserved skin substitute is placed on the absorbent gauze. In some of the embodiments, the absorbent gauze is Vaseline Petrolatum gauze. In some of the embodiments, the cryopreserved skin substitute has a size of about 40-50 cm. 2 , about 40~45cm 2 , or about 45-50cm 2 The size of the absorbent gauze is about 40 to 60 cm. 2 , about 45~60cm 2 , about 45~55cm 2 In some of the optional embodiments, the size of the cryopreserved skin substitute is 41 cm 2 or about 41cm 2 , 42cm 2 Or about 42cm 2 , 43cm 2 Or about 43cm 2 , 44cm 2 Or about 44cm 2 , 45cm 2 Or about 45cm2 , 46cm 2 Or about 46cm 2 , 47cm 2 Or about 47cm 2 The size of the absorbent gauze is about 47 cm 2 Or about 47cm 2 , 48cm 2 Or about 48cm 2 , 49cm 2 Or about 49cm 2 , 50cm 2 Or about 50cm 2 , 51cm 2 or about 51 cm 2 , 52cm 2 or about 52cm 2 , 53cm 2 or about 53cm 2 In some of any of the embodiments, the cryopreserved skin substitute of any of the embodiments provided or the skin substitute dressing of any of the embodiments provided can be sterile.

[0040] In some aspects, a container is provided herein that contains a skin substitute.In some of any of the embodiments, the container can contain any of the skin substitutes provided in any of the embodiments, any of the cryopreserved skin substitutes provided in any of the embodiments, or any of the skin substitute dressings provided in any of the embodiments.In some of any of the embodiments, the container is a bag.In some of any of the embodiments, the container is sterile and / or heat sealed.

[0041] In some aspects, provided herein is a package comprising the container of any of the provided embodiments. In some of the optional embodiments, the package is a bag. In some of the optional embodiments, the package is sterile and / or heat sealed.

[0042] In some aspects, provided herein is a method for preparing a skin substitute dressing. In some of any of the embodiments, the method includes placing the skin substitute of any of the embodiments provided or the cryopreserved skin substitute of any of the embodiments provided on an absorbent gauze. In some of any of the embodiments, the absorbent gauze is a Vaseline petrolatum gauze.

[0043] In some of the optional embodiments, the size of the cryopreserved skin substitute is about 40-50 cm. 2 , about 40~45cm 2 , or about 45-50cm 2 The size of the absorbent gauze is about 40 to 60 cm. 2 , about 45~60cm 2 , about 45~55cm 2 In some of the optional embodiments, the size of the cryopreserved skin substitute is 41 cm 2 or about 41cm 2 , 42cm 2 Or about 42cm 2 , 43cm 2 Or about 43cm 2 , 44cm 2 Or about 44cm 2 , 45cm 2 Or about 45cm 2 , 46cm 2 Or about 46cm 2 , 47cm 2 Or about 47cm 2 The size of the absorbent gauze is about 47 cm 2 Or about 47cm 2 , 48cm 2 Or about 48cm 2 , 49cm 2 Or about 49cm 2 , 50cm 2 Or about 50cm 2 , 51cm 2 or about 51 cm 2 , 52cm 2 or about 52cm 2 , 53cm 2 or about 53cm 2 It is.

[0044] In some aspects, provided herein are methods of promoting wound healing. In some of any of the embodiments provided, any of the methods provided include applying to a wound a skin substitute of any of the embodiments provided, a cryopreserved skin substitute of any of the embodiments provided, or a skin substitute dressing of any of the embodiments provided.

[0045] In some of the optional embodiments, the skin substitute prevents microbial infection. In some of the optional embodiments, the skin substitute is applied to acute and / or chronic wounds.

[0046] In some of the embodiments, the wound is selected from the group consisting of sores, open wounds, ulcers, and abscesses. In some of the embodiments, the skin substitute is applied to a wound in a diabetic patient. In some of the embodiments, the wound is a diabetic ulcer. In some of the embodiments, the wound is a diabetic foot ulcer. In some of the embodiments, the wound is a venous leg ulcer. [Brief description of the drawings]

[0047] [Figure 1A] FIG. 1A shows a schematic diagram depicting representative steps involved in the production of a skin substitute comprising a stratified epidermis. [Figure 1B] FIG. 1B shows a representative example of a paraffin-embedded, hematoxylin-eosin stained skin substitute after 25 days of culture on the substrate, showing the stratum corneum, stratum granulosum, stratum spinosum, and stratum basale formed by differentiated keratinocytes. [Diagram 2] FIG. 2 shows a schematic of an exemplary expression construct including a viral vector and growth factors, insulin, and regulatory elements. [Diagram 3] Figure 3A shows the mean levels (ng / mL) of C-peptide detected in an in vitro study of protein release from a skin substitute containing a stratified epidermis on days 1, 4, and 6. Figure 3B shows the mean levels (ng / mL) of VEGF detected in an in vitro study of protein release from a skin substitute containing a stratified epidermis on days 1, 4, and 7. Experiments were performed in triplicate (n=3). [Figure 4] Figures 4A-C show markers of wound healing in response to VEGF (MOI=12), insulin (MOI=24), or a combination of VEGF and insulin (MOI=12 and MOI=24, respectively) in an endothelial cell tube formation assay. Figure 4A shows the number of webs observed in response to negative and positive controls, VEGF, insulin, or VEGF+insulin. Figure 4B shows the number of nodes observed in response to negative and positive controls, VEGF, insulin, or VEGF+insulin. Figure 4C shows the number of main segments observed in response to negative and positive controls, VEGF, insulin, or VEGF+insulin. Experiments were performed in triplicate and data are shown as mean ± standard deviation. [Diagram 5] Figure 5A shows the percent open wound area over 21 days in healthy and diabetic rats treated with standard wound dressings and in diabetic rats treated with skin substitutes secreting both VEGF and insulin. There were a total of 7 rats in each group, and data are shown as mean ± standard deviation. Figure 5B shows representative images of wound initiation (day 1) and extent of wound closure (day 21) in the skin of healthy and diabetic rats treated with standard wound dressings or with VEGF / insulin skin substitutes. [Figure 6] Figure 6 shows representative images of paraffin-embedded hematoxylin and eosin-stained sections of rat wounds at 21 days after wound initiation in a healthy rat treated with a gauze dressing (left), a diabetic rat treated with a gauze dressing (center), and a diabetic rat treated with a VEGF / insulin skin substitute (right). [Figure 7] Figure 7A shows a comparison of wound area (cm2) between healthy pigs (n=3) and diabetic pigs (n=3) treated with gauze dressings and diabetic pigs (n=3) treated with VEGF / insulin skin substitutes for 28 days. Figure 7B shows a comparison of wound area (cm2) between diabetic pigs treated with gauze dressings and diabetic pigs treated with VEGF / insulin skin substitutes for 52 days. Data are presented as mean ± standard deviation. [Figure 8] FIG. 8 shows representative images of wounds in healthy pigs (top) and diabetic pigs (middle) treated with gauze dressings, and in diabetic pigs treated with VEGF / insulin skin substitute (bottom). [Figure 9] Figure 9 shows representative images of wound areas in diabetic pigs treated with gauze dressings (top) and diabetic pigs treated with VEGF / insulin skin substitute (bottom) on days 1 and 7 after wound initiation. [Figure 10] FIG. 1 shows glucose levels (mg / dL) before and 11 days after wound initiation in healthy and diabetic pigs treated with gauze dressings and diabetic pigs treated with VEGF / insulin skin substitute. Data are presented as mean ± standard deviation (n=3). [Figure 11] Figure 11 shows the levels of advanced glycation end products (AGEs) (ng / mg protein) in skin samples taken at wound initiation and wound healing from healthy and diabetic pigs treated with gauze dressings and diabetic pigs treated with VEGF / insulin skin substitute. Data are presented as mean ± standard deviation (n=3). [Figure 12] FIG. 12 shows a karyogram corresponding to the cytogenetic analysis of HaLow cells (HaCat cells grown in low calcium medium without fetal bovine serum) after expansion in culture. [Figure 13] Figure 13 shows a graph depicting the quantification of human epidermal growth factor (hEGF) expression in skin substitutes transduced with adenovirus expressing hEGF (Ad-CMV-hEGF). Non-transduced skin substitutes were included as experimental controls. Results are expressed as mean ± SEM of replicates. * p<0.05 compared to control. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0048] Detailed Description Provided herein is a composition of a skin substitute comprising a stratified epidermis, the composition being capable of secreting recombinant growth factors and recombinant insulin from cells of the stratified epidermis. In some aspects, provided herein is a method of producing a skin substitute comprising a stratified epidermis, the cells of which produce, e.g., secrete, recombinant growth factors and recombinant insulin. In other aspects, provided herein is a method of treating a subject in need of wound healing, such as a diabetic subject.

[0049] Wound healing is a complex process and is impaired in certain patient populations, such as diabetic subjects. High blood glucose levels in diabetic subjects promote the formation of advanced glycation end products (AGEs). AGEs cause changes in both the structure and vasculature of the skin, resulting in delayed wound healing or increased time to wound closure compared to non-diabetic subjects. To further complicate the wound healing process, diabetic patients are more susceptible to infections, such as bacterial and / or fungal infections.

[0050] Certain skin substitute compositions have been approved by the FDA for wound healing applications, specifically for treating diabetic foot ulcers. Some limitations of currently available skin substitutes include high cost, the need for multiple applications, and limited effectiveness due to failure to promote or achieve proper scarring, which is a key component of the wound healing process. In contrast, the skin substitute compositions provided herein can promote wound healing, including scar formation, in diabetic subjects with few applications. In some cases, only one application is required to achieve wound healing (e.g., time to wound closure) comparable to that observed in non-diabetic subjects. In a further advantage, the skin substitutes provided herein can prevent microbial infection, thereby preventing any further complications to the wound healing process.

[0051] The skin substitute provided herein is composed of differentiated keratinocytes that form stratified epidermis. The cells of the basal layer of the stratified epidermis are transduced with recombinant polynucleotides that code for growth factors and insulin, facilitating secretion of mature forms of growth factors and insulin from the cells of the stratified epidermis. The levels of VEGF and insulin secreted by the skin substitute are lower than those required to cause reported tumor induction or systemic glucose reduction, respectively. Instead, the combination of VEGF and insulin at levels secretable by the skin substitute can potently promote angiogenesis to a greater extent than either VEGF or insulin alone. This combination can also reduce the amount of AGEs in the skin of a subject. Furthermore, the secretion levels of growth factors and insulin can be sustained for at least 7 days, thereby providing sustained release of this wound healing combination.

[0052] All publications, including patent documents, scientific articles and databases, referred to in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication was individually incorporated by reference. In the event that the definitions set forth herein conflict or are otherwise inconsistent with the definitions set forth in the patents, applications, published applications and other publications incorporated herein by reference, the definitions set forth herein shall take precedence over the definitions incorporated herein by reference.

[0053] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0054] I. Definition Unless otherwise defined, all terminology, notation, and other technical and scientific terms or terminology used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art to which the subject matter described in the claims pertains. In some cases, terms having commonly understood meanings are defined herein for clarity and / or ready reference, and the inclusion of such definitions herein should not necessarily be construed as representing a substantial difference beyond that commonly understood in the art.

[0055] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, "a" or "an" means "at least one" or "one or more." The various aspects and variations described herein are understood to include "consisting of" and / or "consisting essentially of" the various aspects and variations.

[0056] Throughout this disclosure, various aspects of the claimed subject matter are presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the claimed subject matter. Thus, the description of a range should be considered to specifically disclose all possible subranges as well as each individual numerical value included in the range. For example, when a range of values ​​is given, it is understood that each intermediate value between the upper and lower limits of the range, and any other specified or intermediate value in the specified range, is included within the scope of the claimed subject matter. The upper and lower limits of these smaller ranges may be independently included in these smaller ranges, which are also included within the scope of the claimed subject matter, subject to any specifically excluded limit in the specified range. If a specified range includes one or both of the limits, then a range excluding either or both of such included limits is also included within the scope of the claimed subject matter. This is true regardless of the breadth of the range.

[0057] The term "about" as used herein refers to the normal error range for each value well known to those skilled in the art. Reference herein to a value or parameter marked with "about" includes (describes) aspects directed to that value or parameter itself. For example, a description that refers to "about X" includes the description of "X". The term "about" can also include variations, which can be up to ±5%, but can also be ±4%, 3%, 2%, 1%, etc. Whether or not modified by the term "about", the claims include equivalents to the amount.

[0058] The term "expression", as used herein, refers to the process by which a polypeptide is produced based on a coding sequence of a nucleic acid molecule such as a gene. This process may include transcription, post-transcriptional control, post-transcriptional modification, translation, post-translational control, post-translational modification, or any combination thereof.

[0059] As used herein, a "subject" includes any living organism, such as humans and other mammals. Mammals include, but are not limited to, humans and non-human animals, including farm animals, sport animals, rodents, and pets.

[0060] As used herein, "operably linked" or "operatively linked" refers to the association of a component, such as a DNA sequence (e.g., a heterologous nucleic acid) with a regulatory sequence such that gene expression is permitted when the appropriate molecule (e.g., a transcriptional activator protein) is bound to the regulatory sequence. Thus, this means that the described components are in a relationship permitting them to function in their intended manner.

[0061] As used herein, "percent sequence identity (%)" and "percent identity" when used in relation to a nucleotide sequence (reference nucleotide sequence) or amino acid sequence (reference amino acid sequence) are defined as the percentage of nucleotide residues or amino acid residues in a candidate sequence that are identical to the residues in a reference sequence, respectively, after aligning the sequences to achieve maximum sequence identity percentage and introducing gaps, if necessary. Alignment to determine percent sequence identity can be achieved in a variety of ways within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine the appropriate parameters for aligning sequences, including any algorithms required to achieve maximum alignment over the entire length of the sequences being compared.

[0062] The term "vector" as used herein refers to a nucleic acid molecule capable of amplifying another nucleic acid to which it is linked. This term includes vectors as self-replicating nucleic acid structures as well as vectors that are integrated into the genome of a host cell into which it is introduced. Certain vectors are capable of directing the expression of a nucleic acid to which they are operably linked. Such vectors are referred to herein as "expression vectors". Among them, vectors are viral vectors, such as adenoviral vectors.

[0063] The term "skin substitute" as used herein refers to a material that temporarily or permanently replaces and / or augments one or more functions of skin, such as wound healing, depending on the properties of its composition. The structure of the skin substitute can include components similar in structure and function to mammalian epidermis and / or mammalian dermis.

[0064] The term "stratified epithelium" as used herein refers to epithelium that comprises two or more layers of epithelial cells.The multiple layers of epithelial cells can be identified by biochemical composition and by visual inspection using microscopy.For example, fully stratified epithelium can mimic the composition of human epidermis, including basal layer (basement membrane layer), spinous layer or spinous layer (spinous membrane layer), granular layer (granular membrane layer) and stratum corneum (horny layer).

[0065] II. Skin substitutes that produce growth factors and insulin Provided herein is a skin substitute comprised of stratified epidermis, where cells in the stratified epidermis produce, e.g., secrete, growth factors and insulin. In some embodiments, the growth factors and insulin are recombinant sequences that are heterologous to the cells of the stratified epidermis. In some embodiments, the skin substitute is comprised of keratinocytes. In some embodiments, the skin substitute is comprised of differentiated keratinocytes. In some embodiments, the skin substitute is comprised of immortalized keratinocytes and / or differentiated immortalized keratinocytes.

[0066] In some embodiments, the recombinant growth factors and recombinant insulin are secretable from cells of the skin substitute. In some embodiments, the skin substitute is comprised of a stratified epidermis including a stratum basale, a stratum spinosum, a stratum granulosum, and a stratum corneum, wherein the cells of the stratified epidermis express the recombinant growth factors and recombinant insulin. In some embodiments, the cells of the stratified epidermis that express the recombinant growth factors and recombinant insulin include cells of the stratum basale. In some embodiments, the stratified epidermis is about 50 μm to about 300 μm thick. In some embodiments, the stratified epidermis is about 100 μm to about 250 μm thick. In some embodiments, the stratified epidermis is about 100 μm to about 200 μm thick. In some embodiments, the stratified epidermis is at least 50 μm, 75 μm, 100 μm, 125 μm, 150 μm, 175 μm, 200 μm, 225 μm, 250 μm, 275 μm, or 300 μm thick, or has a thickness between any of the aforementioned values.

[0067] A. Growth factors In embodiments of the skin substitutes provided, the cells (e.g., basal cells) that make up the stratified epidermis produce and / or secrete recombinant growth factors. Exemplary recombinant growth factors are described herein. In some embodiments, the recombinant growth factors are secretable from the stratified epidermis. In some embodiments, the recombinant growth factors are secretable from the basal cells of the stratified epidermis. In some embodiments, the cells (e.g., basal cells) that make up the stratified epidermis also produce and / or secrete recombinant insulin, such as any of those described in Section B below.

[0068] Growth factors are known in the art.Growth factors include, for example, bone morphogenetic protein (BMP), epidermal growth factor (EGF), erythropoietin (EPO), fibroblast growth factor (FGF), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), hepatocyte growth factor (HGF), insulin-like growth factor (IGF), platelet-derived growth factor (PDGF), transforming growth factor α and β, and vascular endothelial growth factor (VEGF), and their isoforms or alternative splice variants.

[0069] In some embodiments, the growth factor is epidermal growth factor (EGF), erythropoietin (EPO), fibroblast growth factor (FGF), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), hepatocyte growth factor (HGF), insulin-like growth factor (IGF), platelet-derived growth factor (PDGF), transforming growth factors alpha and beta, vascular endothelial growth factor (VEGF), epidermal growth factor receptor (EGFR), fibroblast growth factor receptor (FGFR), and any isoforms or alternatively spliced ​​variants thereof.

[0070] In some aspects, the recombinant growth factor is encoded by a polynucleotide encoding a growth factor sequence that contains a signal peptide that facilitates secretion of the growth factor. In some aspects, the signal peptide is present in the precursor growth factor sequence and is cleaved to form the secretable mature growth factor. In some aspects, the signal peptide is the endogenous or native signal peptide of the growth factor. In some aspects, the signal peptide is a heterologous signal peptide from a different protein. In some aspects, the signal peptide is cleaved upon expression of the growth factor from the cells of the skin substitute. In some aspects, the secretable growth factor sequence lacks a signal peptide. In some aspects, the growth factor is secretable from the cells. In some aspects, the recombinant growth factor is secretable from the stratified epidermis. In some aspects, the cells of the stratified epidermis secrete the recombinant growth factor.

[0071] In some embodiments, the growth factor is VEGF-A or its isoform or alternatively spliced ​​variant. VEGF-A is an important mediator of angiogenesis and signals through the class IV tyrosine kinase receptor family of VEGF receptors (VEGFR). VEGF-A ligands bind to both VEGFR1 and VEGFR2, but they primarily signal through VEGFR2, leading to endothelial cell proliferation, survival, migration and vascular permeability. Separate VEGF-A isoforms result from alternative splicing. Any isoform or alternatively spliced ​​variant of VEGF-A that retains the ability to bind to VEGF-R (e.g., VEGFR2) is contemplated in the provided skin substitute. Usually, VEGF-A isoforms vary in length and are named VEGF xxx (where xxx represents the number of amino acids present in the final protein sequence).

[0072] Exemplary VEGF-A isoforms include vascular endothelial growth factor A (VEGF-A) polypeptides VEGF 206 (SEQ ID NO:11), VEGF-A isoform VEGF 189 (SEQ ID NO:19), VEGF-A isoform VEGF 183 (SEQ ID NO:20), VEGF-A isoform VEGF 148 (SEQ ID NO:21), VEGF-A isoform VEGF 145 (SEQ ID NO:22), VEGF-A isoform VEGF 165B (SEQ ID NO:23), VEGF-A isoform VEGF 121 (SEQ ID NO:24), VEGF-A isoform VEGF 111 (SEQ ID NO:25), VEGF-A isoform VEGF 165 (SEQ ID NO:7), VEGF-A isoform L-VEGF165 (SEQ ID NO:8), VEGF-A isoform L-VEGF 121 (SEQ ID NO:27), VEGF-A isoform L-VEGF 189 (SEQ ID NO:28), VEGF-A isoform L-VEGF 206 (SEQ ID NO:29), VEGF-A isoform 15 (SEQ ID NO:30), VEGF-A isoform 16 (SEQ ID NO:31), VEGF-A isoform 17 (SEQ ID NO:32), or VEGF-A isoform 18 (SEQ ID NO:33). It is also understood that the mature sequence thereof, which is cleaved and lacks the signal peptide when expressed and produced from a cell, is also included.

[0073] In some embodiments, cells constituting the stratified epidermis (e.g., basal cells) contain a polynucleotide encoding a recombinant human VEGF-A isoform having at least or at least about 80%, at least 85% or at least about 85%, at least 90% or at least about 90%, or at least 95% or at least about 95% sequence identity to the amino acid sequence shown in any one of SEQ ID NOs: 7, 11, and 19-33, and retaining binding to a VEGFR (e.g., VEGFR-2). In some embodiments, cells constituting the stratified epidermis (e.g., basal cells) contain a polynucleotide encoding a recombinant human VEGF-A isoform shown in any one of SEQ ID NOs: 7, 11, and 19-33. In some embodiments, the polynucleotide encodes a protein containing a signal peptide, which is proteolytically cleaved and removed such that the protein lacking the signal peptide is secreted, such as via a constitutive secretory pathway. In some embodiments, the cells of the stratified epidermis of the skin substitute produce, e.g., secrete, recombinant VEGF-A. In some embodiments, the basal cells of the stratified epidermis of the skin substitute produce, e.g., secrete, recombinant human VEGF-A isoforms.

[0074] In some embodiments, cells constituting the stratified epidermis (e.g., basal cells) comprise a VEGF-A isoform having an amino acid sequence having at least or at least about 80%, at least or at least about 85%, at least or at least about 90%, or at least or at least about 95% sequence identity to the amino acid sequence shown in any one of SEQ ID NOs: 7, 11, and 19-33, and retaining binding to a VEGFR (e.g., VEGFR-2). In some embodiments, cells constituting the stratified epidermis (e.g., basal cells) comprise a recombinant VEGF-A isoform shown in any one of SEQ ID NOs: 7, 11, and 19-33. In some embodiments, the protein lacks a signal peptide, which is proteolytically cleaved and removed such that the encoded protein lacks the signal peptide shown in any one of SEQ ID NOs: 7, 11, and 19-33 (e.g., lacks amino acid residues 1-26). In some embodiments, recombinant human VEGF-A is secreted, such as via a constitutive secretory pathway. In some embodiments, cells of the stratified epidermis of the skin substitute produce, e.g., secrete, recombinant VEGF-A. In some embodiments, basal cells of the stratified epidermis of the skin substitute produce, e.g., secrete, recombinant human VEGF-A isoforms.

[0075] In some embodiments, the recombinant human VEGF-A is encoded by a polynucleotide comprising a sequence having at least or at least about 80%, at least 85% or at least about 85%, at least 90% or at least about 90%, or at least 95% or at least about 95% sequence identity to the sequence shown in SEQ ID NO:4. In some embodiments, the recombinant human VEGF-A is encoded by a polynucleotide comprising a sequence shown in SEQ ID NO:4. In some embodiments, the recombinant human VEGF-A is encoded by a polynucleotide shown in SEQ ID NO:4. In some embodiments, the recombinant human VEGF-A comprises a sequence having at least or at least about 80%, at least 85% or at least about 85%, at least 90% or at least about 90%, or at least 95% or at least about 95% sequence identity to the sequence shown in SEQ ID NO:7, or a sequence thereof lacking a signal peptide. In some embodiments, the recombinant human VEGF-A comprises the sequence shown in SEQ ID NO:7 or a sequence thereof lacking the signal peptide. In some embodiments, the recombinant human VEGF-A is shown in SEQ ID NO:7 or a sequence thereof lacking the signal peptide. In some embodiments, the cells of the stratified epidermis of the skin substitute produce, e.g., secrete, recombinant human VEGF-A. In some embodiments, the basal cells of the stratified epidermis of the skin substitute produce, e.g., secrete, recombinant human VEGF-A.

[0076] In some embodiments, the recombinant VEGF is encoded by a polynucleotide encoding a growth factor sequence that contains a signal peptide that facilitates secretion of the VEGF. In some embodiments, the signal peptide is present in a precursor growth factor sequence and is cleaved to form a mature secretable growth factor. In some embodiments, the signal peptide is the endogenous or native signal peptide of the growth factor. In some embodiments, the signal peptide is a heterologous signal peptide from a different protein. In some embodiments, the signal peptide is The sequence shown as TIFF2024531827000002.tif4128. In some embodiments, the signal peptide is cleaved upon expression of VEGF from cells of the skin substitute. In some embodiments, the secretable VEGF sequence lacks a signal peptide. In some embodiments, the recombinant human VEGF-A comprises a sequence having at least 80% or at least about 80%, at least 85% or at least about 85%, at least 90% or at least about 90%, or at least 95% or at least about 95% sequence identity to the sequence shown in SEQ ID NO:44. In some embodiments, the recombinant human VEGF-A comprises a sequence shown in SEQ ID NO:44. In some embodiments, the recombinant human VEGF-A is shown in SEQ ID NO:44. In some embodiments, the VEGF is secretable from the cells. In some embodiments, the recombinant VEGF is secretable from the stratified epidermis. In some embodiments, the cells of the stratified epidermis secrete the recombinant VEGF.

[0077] In some embodiments, the growth factor is a member of the PDGF / VEGF family of proteins. In some embodiments, the growth factor is a vascular endothelial growth factor B (VEGF-B) polypeptide (e.g., SEQ ID NO:12), a c-fos-induced growth factor (FIGF) polypeptide (also called VEGF-D) (e.g., SEQ ID NO:13), a platelet-derived growth factor A (PDGF-A) polypeptide (e.g., SEQ ID NO:14), a platelet-derived growth factor B (PDGF-B) polypeptide (e.g., SEQ ID NO:15), or a placenta growth factor (PLGF) polypeptide (e.g., SEQ ID NO:16), and any isoforms or alternatively spliced ​​variants thereof.

[0078] In some embodiments, cells constituting the stratified epidermis (e.g., basal cells) comprise a recombinant human growth factor having an amino acid sequence having at least 80% or at least about 80%, at least 85% or at least about 85%, at least 90% or at least about 90%, or at least 95% or at least about 95% sequence identity to the amino acid sequence shown in any one of SEQ ID NOs: 12-16, or an alternatively spliced ​​form or isoform thereof. ... In some embodiments, the protein lacks a signal peptide, which is proteolytically cleaved and removed such that the encoded protein lacks the signal peptide set forth in any one of SEQ ID NOs:12-16 (see, e.g., Sequence Listing). In some embodiments, the recombinant human growth factor is secreted, such as via a constitutive secretion pathway. In some embodiments, cells of the stratified epidermis of the skin substitute produce, e.g., secrete, the recombinant human growth factor. In some embodiments, basal cells of the stratified epidermis of the skin substitute produce, e.g., secrete, the recombinant human growth factor.

[0079] B. Insulin In embodiments of the skin substitutes provided, the cells (e.g., basal cells) that make up the stratified epidermis produce and / or secrete recombinant insulin. Exemplary recombinant insulins are described herein. In some embodiments, recombinant insulin is secretable from the stratified epidermis. In some embodiments, recombinant insulin is secretable from the basal cells of the stratified epidermis. In some embodiments, the cells (e.g., basal cells) that make up the stratified epidermis also produce and / or secrete recombinant growth factors, such as any of those described in section A above.

[0080] Insulin is a hormone that controls glucose levels. Depending on the route of administration and dose, insulin can be available systemically or locally. In one example, systemic insulin is used as a treatment for glycemic control, for example in diabetic patients. In another example, local insulin activity does not affect systemic glucose levels. In some aspects, the cells of the skin substitutes provided herein produce insulin at levels that affect local glucose levels. In some aspects, the cells of the skin substitutes provided herein produce insulin at levels that do not affect systemic glucose levels.

[0081] Insulin is produced as a preproprotein that is processed into a two-chain form when expressed in cells. Normally, human insulin is translated as preproinsulin, a 110 amino acid precursor polypeptide that contains a 24 amino acid signal peptide that directs the protein to the endoplasmic reticulum (ER), where the signal sequence is cleaved, resulting in proinsulin (SEQ ID NO:5). Proinsulin is further processed to release the 31 amino acid C-peptide, or connecting chain peptide. In wild-type insulin, proinsulin is cleaved coordinately by endopeptidases (e.g., PC-2 and PC-3 endopeptidases) at the carboxyl side of two sites on human proinsulin, Arg31Arg32 (B-chain / C-chain peptide junction) and Lys64Arg65 (C-chain / A-chain peptide junction), to generate the A and B chains of mature insulin, the C peptide, and free basic amino acids. As an example, in wild-type human insulin, the resulting insulin contains a 21 amino acid A chain as shown in SEQ ID NO:36 (corresponding to amino acid residues 66-86 of the proinsulin polypeptide as shown in SEQ ID NO:5) and a 30 amino acid B chain as shown in SEQ ID NO:40 (corresponding to amino acid residues 1-30 of the proinsulin polypeptide as shown in SEQ ID NO:5), which are cross-linked by disulfide bonds. Normally, properly cross-linked human insulin contains three disulfide bridges: one between position 7 of the A chain and position 7 of the B chain, a second between position 20 of the A chain and position 19 of the B chain, and a third between positions 6 and 11 of the A chain.

[0082] In some embodiments, recombinant insulin is encoded by a polynucleotide encoding a proinsulin polypeptide, which results in a single-chain or two-chain form of the insulin polypeptide. In some embodiments, insulin is a single-chain polypeptide containing an A chain and a B chain. In some embodiments, the encoded insulin can be processed in keratinocytes into a two-chain form containing an A chain and a B chain linked by a disulfide bond or the like. In some embodiments, cells of the stratified epidermis of the skin substitute produce, e.g., secrete, recombinant insulin. In some embodiments, basal cells of the stratified epidermis of the skin substitute produce, e.g., secrete, recombinant human insulin. In some embodiments, the two-chain form containing an A chain and a B chain can be secreted from cells (e.g., basal cells) of the stratified epidermis.

[0083] In some embodiments, the recombinant insulin is regular insulin, which is a natural or wild-type insulin polypeptide. These include recombinant human insulin, as well as insulin from bovine, porcine, and other species. In some embodiments, the recombinant insulin is recombinant insulin of regular human insulin sold as Humulin® R, Novolin® R, and Velosulin®. In some embodiments, the recombinant insulin is recombinant insulin of regular porcine insulin sold as Iletin II®.

[0084] In some embodiments, the insulin is recombinant human insulin. In some embodiments, cells constituting the stratified epidermis (e.g., basal cells) contain a polynucleotide encoding a proinsulin precursor form of insulin. In some embodiments, the precursor of human insulin is human proinsulin. In some embodiments, cells constituting the stratified epidermis (e.g., basal cells) contain a polynucleotide comprising a human proinsulin amino acid sequence having at least 80% or at least about 80%, at least 85% or at least about 85%, at least 90% or at least about 90%, or at least 95% or at least about 95% sequence identity to the amino acid sequence shown in SEQ ID NO:5. In some embodiments, cells constituting the stratified epidermis (e.g., basal cells) contain a human proinsulin amino acid sequence having at least 80% or at least about 80%, at least 85% or at least about 85%, at least 90% or at least about 90%, or at least 95% or at least about 95% sequence identity to the amino acid sequence shown in SEQ ID NO:5. In some embodiments, the encoded proinsulin can be processed in keratinocytes into a two-chain form containing A and B chains linked, such as by a disulfide bond. In some embodiments, cells of the stratified epidermis of the skin substitute produce, e.g., secrete, recombinant insulin. In some embodiments, basal cells of the stratified epidermis of the skin substitute produce, e.g., secrete, recombinant human insulin. In some embodiments, the two-chain form containing A and B chains can be secreted from cells (e.g., basal cells) of the stratified epidermis.

[0085] In some embodiments, cells constituting the stratified epidermis (e.g., basal cells) comprise a polynucleotide encoding human proinsulin as set forth in SEQ ID NO:5. In some embodiments, cells constituting the stratified epidermis (e.g., basal cells) comprise human proinsulin as set forth in SEQ ID NO:5. In some embodiments, insulin is a single-chain polypeptide. In some embodiments, the proinsulin form is processed into a two-chain form containing an A chain and a B chain. In some embodiments, a two-chain recombinant form of insulin containing the A chain and the B chain of SEQ ID NO:5 is secretable from cells constituting the stratified epidermis (e.g., basal cells). In some embodiments, the encoded insulin is processed into a two-chain form containing the A chain as set forth in SEQ ID NO:36 and the B chain as set forth in SEQ ID NO:40. In some embodiments, cells of the stratified epidermis of the skin substitute produce, e.g., secrete, recombinant insulin. In some embodiments, the basal cells of the stratified epidermis of the skin substitute produce, e.g., secrete, recombinant human insulin. In some embodiments, a two-chain recombinant form of insulin containing the A chain shown in SEQ ID NO:36 and the B chain shown in SEQ ID NO:40 can be secreted from cells (e.g., basal cells) that make up the stratified epidermis.

[0086] In some embodiments, the recombinant insulin is a variant of human insulin, such as a functional variant or a species or allelic variant, or a truncated form of active human insulin. In some embodiments, variants of insulin, including allelic and species variants, variants encoded by splice variants, and other functional variants, such as insulin analogs or other derivatives or modifications, include polypeptides having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity to human insulin as set forth in SEQ ID NO:5 or to its processed insulin containing A and B chains, so long as the insulin binds to the human insulin receptor and initiates a signaling cascade that leads to increased glucose uptake and storage and / or decreased endogenous glucose production. In some embodiments, the encoded proinsulin can be processed in keratinocytes into a two-chain form containing A and B chains linked, such as by a disulfide bond. In some embodiments, cells of the stratified epidermis of the skin substitute produce, e.g., secrete, recombinant insulin. In some embodiments, basal cells of the stratified epidermis of the skin substitute produce, e.g., secrete, recombinant human insulin. In some embodiments, the two-chain form containing A and B chains can be secreted from cells (e.g., basal cells) of the stratified epidermis.

[0087] For example, recombinant insulin may include species variants of human insulin. These include, but are not limited to, insulin from bovine and porcine origin. Bovine insulin differs from human insulin at amino acids 8 and 10 in the A chain and amino acid 30 in the B chain (SEQ ID NO:17). Porcine insulin differs from human insulin only at amino acid 30 in the B chain, where, as in the bovine sequence, there is an alanine substitution instead of threonine (SEQ ID NO:18). In some embodiments, cells constituting the stratified epidermis (e.g., basal cells) comprise a polynucleotide encoding a proinsulin precursor form of bovine or porcine insulin, such as the proinsulin form of SEQ ID NO:17 (e.g., amino acids 25-105 of SEQ ID NO:17) or the proinsulin form of SEQ ID NO:18 (e.g., amino acids 25-105 of SEQ ID NO:18), or a sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the insulin shown in SEQ ID NO:17 or SEQ ID NO:18 or to the processed insulin thereof containing the A and B chains, so long as the insulin binds to the human insulin receptor and initiates a signaling cascade that results in increased glucose uptake and storage and / or decreased endogenous glucose production. In some embodiments, the B chain corresponds to amino acids 25-54 of SEQ ID NO:17 or SEQ ID NO:18, and the A chain corresponds to amino acids 85-105 of SEQ ID NO:17 or SEQ ID NO:18. In some embodiments, the encoded insulin is a single chain polypeptide containing the A and B chains shown in SEQ ID NO:17 or SEQ ID NO:18. In some embodiments, the encoded proinsulin is capable of being processed in keratinocytes into a two-chain form containing the A and B chains linked, such as by a disulfide bond.In some embodiments, the encoded insulin is processed into a two-chain form containing the A and B chains shown in SEQ ID NO:17 or SEQ ID NO:18. In some embodiments, cells of the stratified epidermis of the skin substitute produce, e.g., secrete, recombinant insulin. In some embodiments, basal cells of the stratified epidermis of the skin substitute produce, e.g., secrete, recombinant human insulin. In some embodiments, a two-chain recombinant form of insulin containing the A and B chains of SEQ ID NO:17 or SEQ ID NO:18 can be secreted from cells (e.g., basal cells) that make up the stratified epidermis.

[0088] Insulin variants also include insulin analogs that contain one or more amino acid modifications compared to human insulin. Exemplary insulin analogs (A and B chains) include fast-acting and slow-acting analog types or superactive insulin (see, for example, Vajo et al. 2001 Endocrine Reviews 22:706-717). Fast-acting insulin analogs are modified forms of insulin that usually contain one or more amino acid changes. The analogs are designed to reduce the self-association of the insulin molecule, with the aim of increasing the absorption rate and onset of action compared to regular insulin. For example, insulin analogs include, but are not limited to, glulisine (LysB3, GluB29), HMR-1 153 (LysB3, IleB28), HMR-1423 (GlyA21, HisB32), insulin aspart (AspB28), insulin lispro (LysB28, ProB29) and AspB10. In all the above examples, the nomenclature of the analogues is based on describing the amino acid substitution at a particular position on the A or B chain of insulin, numbering from the N-terminus of the chain, with the remainder of the sequence being that of native human insulin.

[0089] In some embodiments, the recombinant insulin is insulin AspB10. Insulin AspB10 is a human insulin analog polypeptide containing a single amino acid change in the B chain in which the naturally occurring histidine (H) at position 10 in wild-type insulin is replaced by an aspartic acid (D) (e.g., H to D substation). The substitution results in a superactive insulin that is absorbed two times faster than regular insulin (e.g., wild-type human insulin). In some aspects, insulin AspB10 has increased binding affinity to the insulin receptor compared to regular insulin (e.g., wild-type human insulin). The sequence of the A chain of insulin AspB10 is shown in SEQ ID NO:36, and the B chain is shown in SEQ ID NO:41. In some embodiments, cells that make up the stratified epidermis (e.g., basal cells) contain a polynucleotide encoding a proinsulin precursor form of insulin AspB10 containing the A chain shown in SEQ ID NO:36 and the B chain shown in SEQ ID NO:41. In some embodiments, the encoded proinsulin can be processed in keratinocytes into a two-chain form containing an A chain and a B chain linked by, for example, a disulfide bond. In some embodiments, the encoded insulin is processed into a two-chain form containing an A chain as shown in SEQ ID NO:36 and a B chain as shown in SEQ ID NO:41. In some embodiments, cells of the stratified epidermis of the skin substitute produce, e.g., secrete, recombinant insulin. In some embodiments, basal cells of the stratified epidermis of the skin substitute produce, e.g., secrete, recombinant human insulin. In some embodiments, a two-chain recombinant form of insulin containing an A chain as shown in SEQ ID NO:36 and a B chain as shown in SEQ ID NO:41 can be secreted from cells (e.g., basal cells) that make up the stratified epidermis.

[0090] In some embodiments, the recombinant insulin is insulin glargine. Due to the addition of two arginines to the C-terminus of the B chain, the isoelectric point of glargine insulin shifts, making it more soluble at acidic pH. There is an additional amino acid change (N21G) in the A chain to prevent deamidation and dimerization due to acid-sensitive asparagine. The sequence of the A chain of glargine insulin is shown in SEQ ID NO:34, and the B chain is shown in SEQ ID NO:35. In some embodiments, cells that constitute stratified epidermis (e.g., basal cells) contain a polynucleotide that encodes a proinsulin precursor form of insulin glargine, containing the A chain shown in SEQ ID NO:34 and the B chain shown in SEQ ID NO:35. In some embodiments, the encoded proinsulin can be processed in keratinocytes into a two-chain form containing the A chain and the B chain linked by a disulfide bond or the like. In some embodiments, the encoded insulin is processed into a two-chain form containing the A chain shown in SEQ ID NO:34 and the B chain shown in SEQ ID NO:35. In some embodiments, cells of the stratified epidermis of the skin substitute produce, e.g., secrete, recombinant insulin. In some embodiments, basal cells of the stratified epidermis of the skin substitute produce, e.g., secrete, recombinant human insulin. In some embodiments, a two-chain recombinant form of insulin containing the A chain shown in SEQ ID NO:34 and the B chain shown in SEQ ID NO:35 can be secreted from cells (e.g., basal cells) that make up the stratified epidermis.

[0091] In some embodiments, the recombinant insulin is insulin lispro. Human insulin lispro is an insulin polypeptide preparation that contains amino acid changes at positions 28 and 29 of the B chain, such that Pro-Lys at this position in wild-type insulin is inverted to Lys-Pro. These two amino acid inversions result in a polypeptide with a reduced tendency to self-associate, which allows for a more rapid onset of action. Specifically, the sequence inversion in the B chain results in the elimination of two hydrophobic interactions and the weakening of two beta-pleated sheet hydrogen bonds that stabilize the dimer (DeFelippis et al., Insulin Chemistry and Pharmacokinetics. In Ellenberg and Rifkin's Diabetes Mellitus 2002 pp. 481-500, McGraw-Hill Professional). Due to the amino acid modifications, insulin lispro acts more rapidly than regular insulin. The sequence of the A chain of insulin lispro is shown in SEQ ID NO:36, and the B chain is shown in SEQ ID NO:37. In some embodiments, cells constituting the stratified epidermis (e.g., basal cells) contain a polynucleotide encoding a proinsulin precursor form of insulin lispro containing an A chain as set forth in SEQ ID NO:36 and a B chain as set forth in SEQ ID NO:37. In some embodiments, the encoded proinsulin can be processed in keratinocytes into a two-chain form containing an A chain and a B chain linked, such as by a disulfide bond. In some embodiments, the encoded insulin is processed into a two-chain form containing an A chain as set forth in SEQ ID NO:36 and a B chain as set forth in SEQ ID NO:37. In some embodiments, cells of the stratified epidermis of the skin substitute produce, e.g., secrete, recombinant insulin. In some embodiments, basal cells of the stratified epidermis of the skin substitute produce, e.g., secrete, recombinant human insulin. In some embodiments, a two-chain recombinant form of insulin containing the A chain shown in SEQ ID NO:36 and the B chain shown in SEQ ID NO:37 can be secreted from cells that make up the stratified epidermis (e.g., basal cells).

[0092] In some embodiments, the recombinant insulin is insulin aspart. Human insulin aspart is an insulin polypeptide preparation that contains an amino acid substitution of proline to aspartic acid at position 28 of the B chain of human insulin. The modification in insulin aspart imparts a negatively charged side chain carboxyl group, resulting in charge repulsion and destabilizing monomer-monomer interactions. Furthermore, the removal of proline eliminates important hydrophobic interactions between monomers (DeFelippis et al., Insulin Chemistry and Pharmacokinetics. In Ellenberg and Rifkin's Diabetes Mellitus 2002 pp. 481-500, McGraw-Hill Professional). The sequence of the A chain of insulin aspart is shown in SEQ ID NO:36, and the B chain is shown in SEQ ID NO:38. In some embodiments, cells constituting the stratified epidermis (e.g., basal cells) contain a polynucleotide encoding a proinsulin precursor form of insulin aspart containing an A chain as shown in SEQ ID NO:36 and a B chain as shown in SEQ ID NO:38. In some embodiments, the encoded proinsulin can be processed in keratinocytes into a two-chain form containing an A chain and a B chain linked, such as by a disulfide bond. In some embodiments, the encoded insulin is processed into a two-chain form containing an A chain as shown in SEQ ID NO:36 and a B chain as shown in SEQ ID NO:38. In some embodiments, cells of the stratified epidermis of the skin substitute produce, e.g., secrete, recombinant insulin. In some embodiments, basal cells of the stratified epidermis of the skin substitute produce, e.g., secrete, recombinant human insulin. In some embodiments, a two-chain recombinant form of insulin containing the A chain shown in SEQ ID NO:36 and the B chain shown in SEQ ID NO:38 can be secreted from cells that make up the stratified epidermis (e.g., basal cells).

[0093] In some embodiments, the recombinant insulin is insulin glulisine. Human insulin glulisine is an insulin polypeptide preparation that contains an amino acid substitution at position B3 of the B chain from asparagine to lysine and at amino acid B29 from lysine to glutamic acid, compared to the sequence of the B chain of human insulin. This modification makes the polypeptide molecule less prone to self-association compared to human insulin. The sequence of the A chain of insulin glulisine is shown in SEQ ID NO:36, and the B chain is shown in SEQ ID NO:39. In some embodiments, cells that make up the stratified epidermis (e.g., basal cells) contain a polynucleotide that encodes a proinsulin precursor form of insulin glulisine containing the A chain shown in SEQ ID NO:36 and the B chain shown in SEQ ID NO:39. In some embodiments, the encoded proinsulin can be processed in keratinocytes into a two-chain form containing the A chain and the B chain linked by, for example, a disulfide bond. In some embodiments, the encoded insulin is processed into a two-chain form containing the A chain shown in SEQ ID NO:36 and the B chain shown in SEQ ID NO:39. In some embodiments, cells of the stratified epidermis of the skin substitute produce, e.g., secrete, recombinant insulin. In some embodiments, basal cells of the stratified epidermis of the skin substitute produce, e.g., secrete, recombinant human insulin. In some embodiments, a two-chain recombinant form of insulin containing the A chain shown in SEQ ID NO:36 and the B chain shown in SEQ ID NO:39 can be secreted from cells (e.g., basal cells) that make up the stratified epidermis.

[0094] In some embodiments, the proinsulin form of insulin is modified so that proinsulin can be easily cleaved into a two-chain form containing A and B chains. In some cases, human keratinocytes, such as HaCaT cells, lack the enzymes required to efficiently cleave proinsulin for the production of mature insulin. For example, endopeptidases, such as PC-2 and PC-3, are not present or are not present at high enough levels to cleave insulin. Instead, keratinocytes express furin, a calcium-dependent cleavage enzyme that belongs to the subtilisin-like proprotein convertase family of enzymes. In some embodiments, human proinsulin is modified human proinsulin. In some embodiments, modified human proinsulin comprises a sequence that is recognized by an enzyme, such as a protease, expressed in keratinocytes, such as HaCaT cells, allowing the encoded proinsulin to be processed in keratinocytes into a two-chain form containing A and B chains linked by disulfide bonds, etc. In some embodiments, the protease is furin and the modified human proinsulin comprises at least one furin recognition sequence. In some embodiments, the modified human proinsulin comprises two furin recognition sequences introduced in place of the sequence containing the Arg31-Arg32 cleavage site (BC junction) and the Lys64-Arg65 cleavage site (CA junction). In some embodiments, the at least one furin recognition sequence comprises the consensus sequence RXRR, where X is any amino acid (SEQ ID NO:8) or RXKR, where X is any amino acid (SEQ ID NO:9). In some embodiments, the furin cleavage site is RTKR (SEQ ID NO:10). In some embodiments, the furin cleavage site is RQKR (SEQ ID NO:42).

[0095] In some embodiments, the proinsulin is AspB10 insulin containing an A chain as set forth in SEQ ID NO:36 and a B chain as set forth in SEQ ID NO:41, and the proinsulin further contains two furin recognition sequences. In some embodiments, each of the furin recognition sequences comprises the consensus sequence RXRR, where X is any amino acid (SEQ ID NO:8) or RXKR, where X is any amino acid (SEQ ID NO:9). In some embodiments, one of the furin cleavage sites is RTKR (SEQ ID NO:10). In some embodiments, one of the furin cleavage sites is RQKR (SEQ ID NO:42). In some embodiments, the modified human proinsulin comprises a sequence having at least 80% or at least about 80%, at least 85% or at least about 85%, at least 90% or at least about 90%, or at least 95% or at least about 95% sequence identity to the amino acid sequence shown in SEQ ID NO:6, and the proinsulin contains a furin recognition site and an amino acid substitution of Asp at position 10 of the B chain. In some embodiments, the modified human proinsulin comprises the amino acid sequence shown in SEQ ID NO:6. In some embodiments, the modified human proinsulin is shown in SEQ ID NO:6. In some embodiments, the encoded insulin is processed into a two-chain form containing the A chain shown in SEQ ID NO:36 and the B chain shown in SEQ ID NO:41. In some embodiments, the cells of the stratified epidermis of the skin substitute produce, e.g., secrete, recombinant insulin. In some embodiments, the basal cells of the stratified epidermis of the skin substitute produce, e.g., secrete, recombinant human insulin. In some embodiments, a two-chain recombinant form of insulin containing the A chain shown in SEQ ID NO:36 and the B chain shown in SEQ ID NO:41 can be secreted from cells that make up the stratified epidermis (e.g., basal cells).

[0096] In some of any of the provided embodiments, the coding polynucleotide encoding proinsulin is a preproinsulin further containing a signal peptide to facilitate secretion of the growth factor. In some embodiments, the signal peptide is cleaved from the encoded preproinsulin to form a secretable mature proinsulin. In some embodiments, the signal peptide is cleaved upon expression of insulin from cells of the skin substitute. In some embodiments, the mature proinsulin form is further processed into recombinant insulin, which is a two-chain form containing A and B chains as described. In some embodiments, the signal peptide is the endogenous or native signal peptide of insulin. In some embodiments, the signal peptide is a heterologous signal peptide from a different protein. In some embodiments, the sequence is a signal peptide. TIFF2024531827000003.tif4128. In some embodiments, the recombinant insulin is capable of being secreted from the cell. In some embodiments, the secretable recombinant insulin sequence lacks a signal peptide.

[0097] In some embodiments, the recombinant human insulin is encoded by a polynucleotide comprising a sequence having at least 80% or at least about 80%, at least 85% or at least about 85%, at least 90% or at least about 90%, or at least 95% or at least about 95% sequence identity to the sequence shown in SEQ ID NO:2, and the encoded proinsulin contains a furin recognition site and an amino acid substitution of aspartic acid (Asp, D) at position 10 of the B chain. In some embodiments, the recombinant human insulin is encoded by a polynucleotide comprising a sequence shown in SEQ ID NO:2. In some embodiments, the recombinant human insulin is encoded by a polynucleotide shown in SEQ ID NO:2. In some embodiments, the cells of the stratified epidermis of the skin substitute produce, e.g., secrete, recombinant insulin. In some embodiments, the basal cells of the stratified epidermis of the skin substitute produce, e.g., secrete, recombinant human insulin. In some embodiments, a two-chain recombinant form of insulin containing the A chain shown in SEQ ID NO:36 and the B chain shown in SEQ ID NO:41 can be secreted from cells that make up the stratified epidermis (e.g., basal cells).

[0098] C. Exemplary Features of Skin Substitutes In some embodiments, the cells of the stratified epidermis of the skin substitute produce, e.g., secrete, recombinant human growth factors (e.g., any described in Section II.A) and recombinant human insulin (e.g., any described in Section II.B), e.g., a two-chain insulin type containing an A chain and a B chain. In some embodiments, the basal cells of the stratified epidermis of the skin substitute produce, e.g., secrete, recombinant human growth factors (e.g., any described in Section II.A) and recombinant human insulin (e.g., any described in Section II.B), e.g., a two-chain insulin type containing an A chain and a B chain.

[0099] In some embodiments, the recombinant human growth factor is VEGF and is encoded by the polynucleotide set forth in SEQ ID NO:4, and the recombinant human insulin is encoded by the polynucleotide set forth in SEQ ID NO:2. In some embodiments, the recombinant human growth factor is VEGF and is set forth in SEQ ID NO:7, and the recombinant human insulin is set forth in SEQ ID NO:6, or a two-chain form thereof containing an A chain and a B chain linked by, such as, a disulfide bond. In some embodiments, the cells of the stratified epidermis of the skin substitute produce, e.g., secrete, recombinant VEGF and recombinant insulin. In some embodiments, the basal cells of the stratified epidermis of the skin substitute produce, e.g., secrete, recombinant human VEGF and recombinant human insulin. In some embodiments, recombinant human insulin, a two-chain recombinant form of VEGF as shown in SEQ ID NO:7 lacking amino acid residues 1-26 thereof and insulin containing the A chain as shown in SEQ ID NO:36 and the B chain as shown in SEQ ID NO:41, can be secreted by cells constituting the stratified epidermis (e.g., basal cells). In some embodiments, recombinant human insulin, a two-chain recombinant form of VEGF as shown in SEQ ID NO:7 lacking amino acid residues 1-26 thereof and insulin containing the A chain as shown in SEQ ID NO:36 and the B chain as shown in SEQ ID NO:40, can be secreted by cells constituting the stratified epidermis (e.g., basal cells).

[0100] In some aspects, provided herein are skin substitutes that produce, e.g., secrete, recombinant growth factors and recombinant insulin at levels that result in significantly greater improvement in one or more markers of vascularization reconstitution compared to either growth factor or insulin alone, as assessed in a lumen formation assay. In vitro lumen formation assays can provide insight into angiogenesis, the development of new blood vessels from pre-existing vessels (DiCicco-Skinner, J Vis Exp. 2014; (91): 51312). Angiogenesis is a key component of a variety of processes, including organ growth, embryonic development, and wound healing. Markers of angiogenesis or vascularization reconstitution that can be assessed in lumen formation assays include, but are not limited to, the presence or relative increase of nodes, webs, and main segments. A node, also referred to as a union, can be defined as a connection, or a site of attachment, of at least three cords. A web can be defined as a closed circuit surrounded by two or more nodes. A main segment can be defined as a cord that connects two nodes together.

[0101] In some aspects, provided herein is a skin substitute comprising a stratified epidermis, wherein the cells of the stratified epidermis continuously secrete quantifiable levels of recombinant growth factors and recombinant insulin. In some embodiments, the cells of the stratified epidermis continuously secrete recombinant growth factors and recombinant insulin for up to or about 2 days, up to or about 3 days, up to or about 4 days, up to or about 5 days, up to or about 6 days, up to or about 7 days, up to or about 8 days, up to or about 9 days, up to or about 10 days, up to or about 11 days, up to or about 12 days, up to or about 13 days, or up to or about 14 days. In some embodiments, the cells of the stratified epidermis continuously secrete the recombinant growth factors and recombinant insulin for up to or about 1 week, up to or about 2 weeks, up to or about 3 weeks, up to or about 1-2 weeks, or up to or about 2-3 weeks.

[0102] In some embodiments, cells of stratified epidermis secrete quantifiable levels of recombinant growth factors and insulin and / or C-peptide. C-peptide, which is cleaved from proinsulin, is a by-product of insulin production that is also secreted by cells. C-peptide is produced in equimolar amounts to endogenous insulin. C-peptide is widely used as a measure of pancreatic beta cell function and is an indicator for the diagnosis and management of diabetes (Leighton et al., Diabetes Ther. 2017; 8(3):475-487).

[0103] Methods for detecting and quantifying growth factors and insulin and / or C-peptide are known in the art.As an example, in vitro detection of growth factors and insulin and / or C-peptide can include the use of enzyme-linked immunosorbent assay (ELISA).ELISA kits for detecting C-peptide include, for example, KT-881 C-Peptide ELISA Kit from Epitope Diagnostics, Inc. and ab178641 C-Peptide ELISA Kit from Abcam.Alternatively, gene expression can be quantified by PCR, for example, RT-qPCR.Quantifiable level can be defined as any level that exceeds or is equal to the quantification limit of a particular assay.

[0104] In some aspects, the present invention provides a skin substitute that produces, for example, secretes, recombinant growth factors and recombinant insulin at a level that reduces advanced glycation end products (AGEs) in the skin of a subject.In some embodiments, the subject is a human.AGEs are proteins or lipids that are glycated when exposed to sugar.The accumulation of AGEs, for example, in skin, can disrupt cell structure and / or function (Goldin et al., Circulation. 2006;114:597-605).Methods for detecting AGEs are known in the art. In some examples, AGE complexes can be detected using ELISA (see, e.g., abx054078, Advanced Glycation End Product (AGE) ELISA Kit, Abexxa, and STA-817, OxiSelect™ Advanced Glycation End Product (AGE) Competitive ELISA Kit, Cell Biolabs, Inc.), spectrofluorimetry (see, e.g., Villa et al., Metabolism 2017;71:64-69), chromatographic, colorimetric, spectroscopic, mass spectrometry, and serological methods (Perrone et al., Oxidative Medicine and Cellular Longevity 2020; vol. 2020, Article ID 3818196, 18 pages).

[0105] III. Methods of Preparing Skin Substitutes Provided herein is a method for producing a skin substitute comprised of stratified epidermis, where cells of the stratified epidermis produce growth factors and insulin. In some embodiments, the method comprises the steps of: 1) differentiating keratinocytes into stratified epidermis, where the stratified epidermis comprises a basal layer, a spinous layer, a granular layer, and a stratum corneum; and 2) introducing a polynucleotide into the stratified epidermis to produce a skin substitute, where the skin substitute produces and secretes growth factors and insulin. In some embodiments, step 1) of the method comprises differentiating immortalized keratinocytes, and the introduction of the polynucleotide in step 2) comprises transduction with a viral vector.

[0106] In some embodiments, methods for producing skin substitutes may include (1) a system in which cultured keratinocytes are reconstituted into a 3D system to represent human epidermis; (2) a system in which keratinocytes (primary or immortalized) are cultured in 3D on a substrate; (3) a system in which cultured skin cells are reconstituted into a 3D system to represent human skin; and / or (4) a system in which keratinocytes (primary or immortalized) are cultured on a matrix, e.g., a skin matrix.

[0107] In some embodiments, cells differentiate such that cells of the stratified epidermis express tight junction proteins such as occluding or claudins. Epidermal proteins contribute to its role as a permeability barrier. For example, tight junctions and desmosomes contribute to the barrier-like function of the epidermis. Tight junctions are multiprotein networks that form intercellular connections between cells of mammalian epidermis. Strands of tight junctions include occludin and claudins, a multigene family of proteins. Occludin is an integral plasma membrane protein that is localized to tight junctions. Claudins, such as claudin-1, claudin-2, and claudin-4, are integral membrane proteins. Claudins form the backbone of tight junction strands, and occludin is copolymerized into these strands (Furuse et al., J. Cell Biol. 1999;147(4):891-903). Desmosomes are adhesion protein complexes that are localized at cell-cell junctions and are responsible for maintaining the mechanical integrity of tissues. Desmosomal cadherins have been shown to act as attachment receptors for certain adenoviruses. For example, adenovirus serotypes Ad3, Ad7, Ad11, and Ad14, but not Ad2 or Ad5, were found to interact with desmoglein 2 (Wang et al., Nat Med. 2011;17(1): 96-104.).

[0108] Culture conditions affect the localization and abundance of proteins that determine epithelial permeability. For example, it has been shown that growing HaCaT cells to confluence promotes desmosomal superadhesiveness, thereby enhancing cell sheet integrity and decreasing permeability (Kimura et al., J Invest Dermatol 2007;127, 775-781). Regarding calcium levels in culture, it has been shown that increasing calcium levels, for example from 0.1 mM to 2 mM, induces desmosome formation and stratification in human keratinocytes (Watt et al., J Cell Biol. 1984; 99(6):2211-2215). Low or depleted calcium levels, for example 0.03 mM Ca 2+ is associated with dissociation of tight junctions and increased permeability. In contrast, exposure of normal human keratinocytes to high levels of calcium was shown to induce stratification and improve barrier function, i.e., decrease permeability. Elevated calcium levels (1.8 mM Ca 2+ ), increased localization of claudin-1, claudin-4, and occludin is detectable at cell borders and lower epidermal layers, likely contributing to enhanced transepithelial electrical resistance (Yuki et al., Exp Dermatol. 2007;16(4):324-30).

[0109] A. Differentiation and culture of immortalized keratinocytes Examples of immortalized keratinocyte lines include, but are not limited to, HaCaT (Boukamp et al., J Cell Biol. 1988;106:761-771), NM1 (Baden et al., In Vitro Cell Dev Biol. 1987;23:205-213), and NIKS (Allen-Hoffmann et al., J Invest Dermatol. 2000;114:444-455). As described herein, reference to "keratinocytes" includes reference to immortalized keratinocytes. Keratinocytes can differentiate into stratified epidermis, which is composed of four morphologically and biochemically distinct layers: stratum basale, stratum spinosum, stratum granulosum, and stratum corneum. Keratinocyte growth and differentiation can be influenced by a variety of factors, including calcium levels, cell density, and temperature during culture. For example, high levels or concentrations of calcium can induce differentiation in HaCaT cells, as can high cell densities. One major difference between primary keratinocytes and immortalized keratinocytes, such as HaCaT cells, is that differentiated immortalized cells retain their proliferation potential, whereas primary keratinocytes have ceased differentiation. Thus, differentiated immortalized keratinocytes, such as HaCaT cells, can proliferate indefinitely under certain conditions (Wilson, Methods Mol Biol. 2014;1195:33-41).

[0110] 1. Structural characteristics and biochemical components of the stratified epidermis The morphology of keratinocytes can serve as a distinguishing factor between undifferentiated and differentiated cells.Stratified epidermis can be identified and confirmed using microscopy.Stratified epidermis is visually different from simple epithelium, which contains only one cell layer.Stratified epidermis is also visually different from pseudostratified epithelium, which contains a single elongated layer that extends to the basolateral surface of the epithelium.Methods for visually evaluating skin substitutes that contain stratified epidermis are known to those skilled in the art.For example, electron microscopy can be used to visualize stratified epithelium.In some instances, scanning electron microscopy can be used to visualize stratified epithelium.

[0111] Detection of certain proteins, such as transglutaminase, filaggrin and laminin, can also assist in identifying differentiated epidermal layers. Methods for detecting certain proteins that can act as markers of differentiation and / or stratification are known to those skilled in the art and can include, for example, immunofluorescence microscopy (see, for example, Schoop et al., J. Invest Derm 1999;112(3):343-353), RT-PCR (see, for example, Kikkawa et al., Biol Pharm Bull. 2010;33(2):307-10), and RNAseq. Keratinocytes are the main cellular component of the epidermis and comprise approximately 80% of the cells in adult human skin. All epithelia express type I and type II keratins, with molecular weights ranging from 40 kDa to 70 kDa. Different epithelial tissues express special pairs of keratins. The localization and relative amounts of proteins produced by differentiated keratinocytes can be used to distinguish different layers of stratified epidermis. In some examples, transglutaminase, e.g., keratinocyte transglutaminase isoenzyme TGK, can be detected to distinguish layers of stratified epidermis. In some examples, fillagrin, a filament-binding protein that binds to keratin fibers, can be detected to distinguish layers of stratified epidermis. In some examples, laminin, an extracellular matrix glycoprotein, can be detected to distinguish layers of stratified epidermis. In some examples, keratin can be detected to distinguish layers of stratified epidermis. In some examples, involucrin, an extracellular membrane protein, can be detected to distinguish layers of stratified epidermis. In some examples, cadherin adhesion molecules (e.g., N-, E-, and P-cadherin, which play a role in barrier function and formation) can be detected to distinguish layers of stratified epidermis (Allen-Hoffmann, US 2014 / 0127170).

[0112] Basal layer cells are cylindrical in shape and produce keratins K5 and K14. In some instances, basal layer cells reside on a structure called basement membrane, which separates the dermis or dermis equivalent from the epidermis. Laminin can be found in the extracellular matrix of the basement membrane. In structures where there is no dermal-epidermal junction, such as skin substitutes that only have epidermal components, laminin can be found in the invaginations of the basal layer. In some instances, expression of laminin, such as laminin 5, is detected to determine whether a basement membrane can be formed, for example, when a skin substitute is applied to a subject.

[0113] The first suprabasal keratinocyte layer is the spinous layer (stratum spinosum), so named because of the spinous appearance of many of the desmosomal contacts between adjacent cells. Keratinocytes in this layer can no longer produce K5 and K14, but can instead synthesize differentiation-specific keratins K1 and K10. Keratinocytes can begin to produce involucrin and epidermis-specific transglutaminase in the upper spinous layer. Morphologically, spinous cells are larger and flatter than basal cells (Holbrook, 1994).

[0114] As keratinocytes further differentiate, they form the granular theca layer (stratum granulosum). Tight junction proteins have been identified in the stratum granulosum and in deeper layers of the epidermis (Brandner et al., Open Dermatol. J. 2010; 4:14-20). Cells in this layer are characterized by distinct electron-dense keratohyalin granules that contain profilaggrin, the protein precursor of filaggrin (Dale et al., 1994). Granular cells also contain lipid-filled granules that, midway through the transition zone between the granular theca layer and the stratum corneum, fuse with the plasma membrane and release their contents into the extracellular space, thereby conferring hydrophobicity to the epidermal surface. As differentiating keratinocytes transition from the granular layer to the cornified layer, i.e., the stratum corneum or horny layer, profilaggrin is cleaved to generate filaggrin, which is involved in the alignment and aggregation of keratin bundles called macrofibrils via disulfide bonds. Macrofibrils are the basic structural units of the cornified membrane. In normal skin sections, filaggrin is localized in the granular layer and can be found in cornified sheets (Sandilands et al., J Cell Sci. 2009; 122(9):1285-1294). Antibodies against filaggrin detect both profilaggrin and its cleavage products.

[0115] The top epidermal layer is the stratum corneum. Involucrin can be used as a differentiation marker for the stratum corneum. The cells of this layer have completed the differentiation process and have lost their nuclei and all metabolic functions. The cornified membrane is a highly stable, insoluble protein structure formed beneath the plasma membrane, which is resistant to detergents and reducing agents and gives strength and rigidity to the terminally differentiated cells of the top epidermal layer. The cells of the horny layer, also known as corneocytes, are linked together by modified desmosomes and eventually detach in sheets from the surface of the skin. For the stratum corneum or cornified layer, the introduction of an air-liquid interface is necessary for keratinocyte differentiation (Prunieras et al., J Invest Dermatol. 1983 Jul;81(1 Suppl):28s-33s).

[0116] 2. Keratinocyte cell culture Low levels of calcium, for example about 0.3 mM, in serum-free conditions promote the proliferation of keratinocytes in a basal undifferentiated phenotype, and supplementation with bovine pituitary extract (BPE) can also enhance proliferation and cell survival. Switching from low to high calcium conditions (the "calcium switch") can induce markers of differentiation, but other factors may contribute to optimal differentiation into the epidermal layer. In one example, culturing cells in serum-containing medium and lowering the temperature, for example from 37°C to 31°C, combined with the calcium switch was found to induce markers of differentiation on a large scale (Borowiec et al., Plos One 2013: 8(10):e77507). Differentiated keratinocytes can be reverted to their basal state by exposure to low calcium medium. However, high cell densities, for example greater than 75-80% confluency, and temperatures above 37°C can induce differentiation in HaCaT cells, even under low calcium conditions (Wilson, Methods Mol Biol. 2014;1195:33-41).

[0117] In some embodiments, provided herein are methods of culturing keratinocytes in a low calcium medium to culture, e.g., obtain, a basal layer. In some embodiments, provided herein are methods of culturing non-primary keratinocytes in a low calcium medium to culture, e.g., obtain, a basal layer. In some embodiments, provided herein are methods of culturing immortalized keratinocytes in a low calcium medium to culture, e.g., obtain, a basal layer. In some embodiments, provided herein are methods of culturing HaCaT keratinocytes in a low calcium medium to culture, e.g., obtain, a basal layer. In some embodiments, keratinocytes are cultured in a low calcium medium for about 2 to about 6 weeks to form a basal layer. In some embodiments, keratinocytes are cultured in a low calcium medium for about 3 to about 4 weeks to form a basal layer. In some embodiments, keratinocytes are cultured in a low calcium medium for about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, or about 6 weeks to form a basal layer. In some embodiments, the keratinocytes are cultured in low calcium medium for about 4 weeks to form a basal layer.

[0118] In some embodiments, the methods provided herein include a cell culture medium that can be used to support the growth of keratinocytes and / or dermal fibroblasts to form the basal layer of stratified epidermis, also referred to as basal keratinocytes. In some embodiments, a serum-free medium can be used to culture the basal layer of stratified epidermis. In some embodiments, a calcium-free medium can be used to culture the basal layer of stratified epidermis. In some embodiments, a serum-free and calcium-free medium can be used as an initial medium for culturing the basal layer of stratified epidermis. In some embodiments, the culture medium used to culture the basal layer contains a final low calcium level, e.g., about 0.01 mM Ca, to form the basal layer of stratified epidermis. 2+ , approximately 0.02 mM Ca 2+ , about 0.03mM Ca 2+ , about 0.04mM Ca 2+ , about 0.05mM Ca 2+ , about 0.06mM Ca 2+ , about 0.07mM Ca 2+ , about 0.08mM Ca2+ , about 0.09mM Ca 2+ , or about 0.1 mM Ca 2+ can be adjusted to.

[0119] In some examples, the culture medium used to culture the basal layer can further comprise endothelial growth factor (EGF) and / or bovine pituitary extract (BPE). In some embodiments, the low calcium culture medium further comprises about 0.1 ng / ml, about 0.2 ng / ml, about 3 ng / ml, about 4 ng / ml, about 5 ng / ml or about 6 ng / ml EGF, and / or about 10 μg / ml, 20 μg / ml, 30 μg / ml, 40 μg / ml, 50 μg / ml, 60 μg / ml or 70 μg / ml BPE. In some embodiments, the low calcium culture medium for culturing the basal layer further comprises about 0.1 ng / ml, about 0.2 ng / ml or about 0.3 ng / ml EGF, and / or about 20 μg / ml, 30 μg / ml or 40 μg / ml BPE. In some embodiments, the low calcium culture medium for culturing the basal layer further comprises about 0.2 ng / ml EGF and about 30 μg / ml BPE. In some embodiments, the low calcium medium used to culture the basal layer is serum-free. In some embodiments, the low calcium medium used to culture the basal layer is serum-free keratinocyte culture medium.

[0120] In some aspects, provided herein is a method of exposing basal keratinocytes that have been cultured to form a basal layer as described herein to a "calcium switch", which changes the calcium level in the culture medium from a low level to a high level to promote the formation of a stratified epithelium. In some aspects, provided herein is a method of culturing basal keratinocytes in a low calcium medium, followed by culturing the basal keratinocytes in a high calcium medium to form a skin substitute comprising a stratified epidermis. In some aspects, provided herein is a method of culturing basal non-primary keratinocytes in a low calcium medium, followed by culturing the basal non-primary keratinocytes in a high calcium medium to form a skin substitute comprising a stratified epidermis. In some aspects, provided herein is a method of culturing basal immortalized keratinocytes in a low calcium medium, followed by culturing the basal immortalized keratinocytes in a high calcium medium to form a skin substitute comprising a stratified epidermis. In some aspects, provided herein are methods of culturing basal HaCaT keratinocytes in a low calcium medium, followed by culturing the basal HaCaT keratinocytes in a high calcium medium to form a skin substitute comprising a stratified epidermis.

[0121] In some aspects, methods are provided herein that allow basal keratinocytes to be cultured on a substrate in low calcium medium prior to calcium switch (transition from low calcium medium to high calcium medium). The surface that contacts the basal keratinocytes, i.e., the substrate, may include a sidewall and / or be in the form of an insert or cup. In some embodiments, the substrate fits into the opening of a well. In some embodiments, the substrate may include a surface with holes of appropriate size, e.g., diameter and size that supports the growth and differentiation of keratinocytes into stratified epidermis. In some embodiments, the substrate may be composed of a variety of holes and diameter sizes. In some embodiments, the substrate is a mesh or a transwell insert, e.g., a transwell insert with a diameter of at least or about 50 mm, at least or about 75 mm, at least or about 100 mm, or at least or about 125 mm, and a pore size of at least or about 1.0 μm, at least or about 2.0 μm, at least or about 3.0 μm, at least or about 4.0 μm, or at least or about 5.0 μm. In some embodiments, the substrate is a transwell insert or a ring, e.g., a cloning ring. In some embodiments, the substrate may include a mesh, e.g., a wire mesh, and the seeded basal keratinocytes may be positionable on the mesh, e.g., above and / or below the substrate. In some embodiments, the substrate may be made of plastic or metal. In some embodiments, the basal keratinocytes may be seeded onto a liquid permeable substrate, for example, onto and / or underneath a wire mesh or plastic containing holes.

[0122] In some embodiments, the substrate is coated with, for example, a gel. In some embodiments, the gel can be collagen, i.e., gelatinous collagen, and / or a hydrogel. In some embodiments, the surface of the insert can be coated with a neutralized bovine collagen solution that is certified for human use. In some embodiments, a solution, for example, a collagen solution, can be used to coat the substrate, and the substrate coated with the solution can then be incubated for a sufficient time until the solution becomes gelatinous, for example, forms gelatinous collagen. In some embodiments, the substrate or coated substrate is washed, for example, with PBS, before seeding with basal keratinocytes.

[0123] In some embodiments, basal keratinocytes can be seeded on a substrate, for example, a coated substrate, in a serum-free and / or calcium-free medium supplemented to a final low calcium level. In some embodiments, basal keratinocytes can be seeded on a substrate, for example, a coated substrate, in a serum-free and / or calcium-free medium supplemented to a final low calcium level without any additional supplementation. In some embodiments, basal keratinocytes can be seeded on a substrate, for example, a coated substrate, in a serum-free and / or calcium-free medium supplemented to a final low calcium level with additional supplementation. In some embodiments, basal keratinocytes can be seeded on a substrate, for example, a coated substrate, in a serum-free and / or calcium-free medium supplemented to a final low calcium level with supplementation.

[0124] In some embodiments, the low calcium culture medium contains 0.01 mM Ca 2+ Or about 0.01 mM Ca 2+ , 0.02 mM Ca 2+ Or about 0.02 mM Ca 2+ , 0.03 mM Ca 2+ Or about 0.03 mM Ca 2+ , 0.04 mM Ca 2+ Or about 0.04 mM Ca 2+ , 0.05 mM Ca 2+ Or about 0.05 mM Ca2+ , or 0.06 mM Ca 2+ Or about 0.06 mM Ca 2+ Final low Ca 2+ The low calcium culture medium is supplemented to a level of about 0.1 ng / ml, about 0.2 ng / ml, about 3 ng / ml, about 4 ng / ml, about 5 ng / ml or about 6 ng / ml EGF, and / or about 10 μg / ml, 20 μg / ml, 30 μg / ml, 40 μg / ml, 50 μg / ml, 60 μg / ml or 70 μg / ml BPE. In some embodiments, the low calcium culture medium further comprises about 0.1 ng / ml, about 0.2 ng / ml or about 0.3 ng / ml EGF, and / or about 20 μg / ml, 30 μg / ml or 40 μg / ml BPE. In some embodiments, the low calcium culture medium further comprises about 0.2 ng / ml EGF and about 30 μg / ml BPE. In some embodiments, the low calcium medium used to culture keratinocytes on a substrate is serum-free. In some embodiments, the low calcium medium used to culture the keratinocytes on the substrate is a serum-free keratinocyte culture medium.

[0125] In some embodiments, the cells are about 1×10 6 cells / ml, 10 x 10 6 cells / ml, 20 x 10 6 cells / ml, 30 x 10 6 cells / ml, 40 x 10 6 cells / ml, or 50 x 10 6 The cells can be seeded in contact with the substrate at a density of about 1×10 cells / ml. In some embodiments, the cells are seeded under the substrate, on top of the substrate, or under and on top of the substrate. In some embodiments, the cells are seeded at a density of about 1×10 6 cells / ml, 10 x 10 6 cells / ml, 20 x 10 6 cells / ml, 30 x 10 6 cells / ml, 40 x 10 6 cells / ml, or 50 x 10 6The cells can be seeded in contact with the coated substrate at a density of cells / ml. In some embodiments, the cells are seeded under the coated substrate, on top of the coated substrate, or under and on top of the coated substrate.

[0126] In some embodiments, after seeding on the substrate, e.g., seeding on and under the coated substrate, the basal keratinocytes are incubated in low calcium medium for about 2 to about 6 days, about 3 to about 5 days, or about 3 to about 4 days. In some embodiments, after seeding on the substrate, e.g., seeding on and under the coated substrate, the basal keratinocytes are incubated in low calcium medium for about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, or about 7 days. In some embodiments, the low calcium culture medium is changed every day. In some embodiments, the low calcium culture medium is changed every other day. In some embodiments, the low calcium medium is serum-free. In some embodiments, the low calcium medium is a serum-free keratinocyte culture medium.

[0127] In some embodiments, provided herein are methods of culturing keratinocytes, where the introduction of an air-liquid interface and increased calcium levels in culture stimulates keratinocyte differentiation into a stratified epidermis. In some embodiments, the air-liquid interface and increased calcium levels can be introduced on the third, fourth, fifth, sixth, or seventh day of culture in low calcium medium with a substrate. In some embodiments, once the air-liquid interface is introduced, the low calcium keratinocyte culture medium is discarded and replaced with medium adjusted to a relatively high calcium level. In some embodiments, the increased calcium level is 1-5 mM Ca 2+ , 1-4 mM Ca 2+ , 1-3 mM Ca 2+ , 2-4 mM Ca 2+ Or 2-3mM Ca 2+ , or about 1 to 5 mM Ca 2+ , 1-4 mM Ca 2+ , 1-3 mM Ca 2+ , 2-4 mM Ca 2+ Or 2-3mM Ca 2+In some embodiments, the elevated calcium level is 1.5 mM Ca 2+ , 1.6 mM Ca 2+ , 1.7 mM Ca 2+ , 1.8 mM Ca 2+ , 1.9 mM Ca 2+ , 2.0 mM Ca 2+ , 2.1 mM Ca 2+ , 2.2 mM Ca 2+ , 2.3 mM Ca 2+ , 2.4 mM Ca 2+ , 2.5 mM Ca 2+ , 2.6 mM Ca 2+ , 2.7 mM Ca 2+ , 2.8 mM Ca 2+ , 2.9 mM Ca 2+ Or 3mM Ca 2+ , or about 1.5 mM Ca 2+ , 1.6 mM Ca 2+ , 1.7 mM Ca 2+ , 1.8 mM Ca 2+ , 1.9 mM Ca 2+ , 2.0 mM Ca 2+ , 2.1 mM Ca 2+ , 2.2 mM Ca 2+ , 2.3 mM Ca 2+ , 2.4 mM Ca 2+ , 2.5 mM Ca 2+ , 2.6 mM Ca 2+ , 2.7 mM Ca 2+ , 2.8 mM Ca 2+ , 2.9 mM Ca 2+ Or 3mM Ca 2+ It is.

[0128] In some embodiments, the high calcium medium is supplemented with EGF, bovine pituitary extract (BPE), and / or hydrocortisone. In some embodiments, the high calcium medium is supplemented with 0.09ng / ml EGF or about 0.09ng / ml EGF, 0.1ng / ml EGF or about 0.1ng / ml EGF, 0.2ng / ml EGF or about 0.2ng / ml EGF, 0.3ng / ml EGF or about 0.3ng / ml EGF, 0.4ng / ml EGF or about 0.4ng / ml EGF, or 0.5ng / ml EGF or about 0.5ng / ml EGF. In some embodiments, the high calcium medium is supplemented with at or about 10 μg / ml BPE, at or about 20 μg / ml BPE, at or about 30 μg / ml BPE, at or about 40 μg / ml BPE, or at or about 50 μg / ml BPE. In some embodiments, the high calcium medium is supplemented with 0.1 μg / ml or about 0.1 μg / ml, 0.2 μg / ml or about 0.2 μg / ml, 0.3 μg / ml or about 0.3 μg / ml, 0.4 μg / ml or about 0.4 μg / ml, 0.5 μg / ml or about 0.5 μg / ml, 0.6 μg / ml or about 0.6 μg / ml, 0.7 μg / ml or about 0.7 μg / ml, or 0.8 μg / ml or about 0.8 μg / ml of hydrocortisone. In some embodiments, the high calcium medium is supplemented with about 0.2 ng / ml EGF, 30 μg / ml BPE, and 0.4 μg / ml of hydrocortisone. In some embodiments, the high calcium medium is serum-free. In some embodiments, the high calcium medium is a serum-free keratinocyte culture medium.

[0129] In some embodiments, the keratinocytes can be cultured in high calcium medium on a substrate, e.g., on and under a collagen-coated substrate, for about 2-4 weeks, about 2-3 weeks, or about 3 weeks until a stratified epidermis is obtained. In some embodiments, the keratinocytes can be cultured in high calcium medium, e.g., on and under a collagen-coated substrate, for 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, or 28 days until a stratified epidermis is obtained. In some embodiments, the high calcium culture medium can be replaced every day until a stratified epidermis is obtained. In some embodiments, the high calcium culture medium can be replaced every other day until a stratified epidermis is obtained.

[0130] Once an air-liquid interface is introduced, differentiated keratinocytes located on and / or under a substrate, e.g., a collagen-coated substrate, can be positioned so that the top surface of the cells forming the skin substitute is exposed to a gaseous environment rather than tissue culture medium, and / or the cells of the basal layer of the skin substitute are exposed to tissue culture medium rather than a gaseous environment. The introduction of an air-liquid interface can facilitate biphasic culture of the skin substitute, i.e., culture in a gaseous environment and a liquid environment. For example, a skin substitute in a medium can be positioned so that, in use, the stratum corneum is exposed to a gaseous environment rather than tissue culture medium, and / or the basal layer and / or the dermis or dermis equivalent is exposed to a tissue culture medium rather than a gaseous environment. This can be achieved by controlling the height of the liquid interface and / or by positioning the location of the substrate, e.g., a collagen-coated substrate. In some embodiments, the substrate, e.g., a transwell or insert, can be positioned so that the bottom of the transwell touches the liquid, but the liquid does not contact the top of the epidermis / epidermal equivalent.

[0131] The keratinocyte culture medium can be at a temperature of about 33.0-37.5° C., e.g., about 34-37.5° C., about 35-37.5° C., about 36-37.5° C., or about 37° C. The tissue culture medium can also be at a pH of about 6.1-7.9, e.g., about 6.2-7.7, about 6.3-7.7, about 6.4-7.7, about 6.5-7.7, about 6.6-7.7, about 6.7-7.6, about 6.8-7.6, about 6.9-7.6, about 7-7.6, about 7.1-7.6, about 7.1-7.5, or about 7.2-7.4. The tissue culture medium can contain about 2-10%, about 2-8%, about 3-7%, about 4-6%, or about 5% CO2. In some aspects, the top surface of the skin substitute is not exposed to tissue culture medium and the bottom or basal surface of the skin substitute is not exposed to a gaseous environment.

[0132] The skin sample holder may be placed in a laminar flow hood to maintain sterility. An air monitor may be used to monitor conditions in the gas environment. In some embodiments, the gas environment may have a temperature of about 37°C or less than about 37°C, e.g., about 10-36°C, about 12-32°C, about 14-29°C, about 15-25°C, about 18-25°C, about 19-24°C, or about 20-22°C. The gas environment may also have a relative humidity of about 90% or less than about 90%, e.g., about 0-89%, about 0-85%, about 10-80%, about 15-75%, about 20-74%, about 23-70%, about 25-65%, about 30-50%, about 35-50%, about 40-50%, or about 40-45%.

[0133] In some embodiments, the gaseous environment for culturing the skin substitute may include less than 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.045%, or 0.04% CO2. In some embodiments, the gaseous environment may include 0.02-0.05% or 0.035-0.045% CO2. In some embodiments, the gaseous environment may include 18-25%, 18-24%, 18-23%, 19-23%, 19-22%, 20-22%, or about 21% O2. In some embodiments, the gaseous environment may contain about 78% N2, and / or about 1% argon. In some embodiments, the gaseous environment may include atmospheric air, compressed air, and / or medical air. In some aspects, medical air may refer to sterile compressed air, and medical air may have a gas composition similar to atmospheric air (e.g., approximately 78% N2 and 21% O2). In some aspects, the gas environment may mimic a sanitary internal room or physiological conditions.

[0134] B. Engineering Skin Substitutes Containing Stratified Epidermis to Deliver Growth Factors and Insulin Provided herein is a method for introducing a nucleic acid molecule, e.g., a polynucleotide, into a skin substitute comprising cells of a stratified epidermis. Provided herein in some aspects is a method for introducing any desired nucleic acid molecule, any nucleic acid molecule-containing vehicle, construct or complex. In some aspects, cells of the stratified epidermis can translate the introduced nucleic acid into a protein for delivery, e.g., secretion, to a subject. In some aspects, keratinocytes of the stratified epidermis are transduced with a nucleic acid molecule that has a desired function or that encodes a selected polypeptide that has a desired function.

[0135] In some embodiments, the polynucleotides encoding the growth factors or insulins as described can be introduced into the cells of the stratified epidermis by viral or non-viral methods. In some embodiments, non-viral delivery methods include the introduction of DNA (e.g., double-stranded circular or linear), RNA, ribozymes, or aptamers. In some embodiments, the introduction involves using a viral vector containing the polynucleotide encoding the recombinant insulin or growth factor. As an example, an adenoviral vector can be used. In some embodiments, the nucleic acid molecule to be introduced can be provided as a construct containing a heterologous nucleic acid molecule or a transgene.

[0136] There are many constructs known to those skilled in the art for introducing nucleic acid into cells either in vitro or in vivo. In some embodiments, such constructs include viral-based delivery systems (e.g., for transduction) and non-viral-based delivery systems (e.g., transfection). In some embodiments, the polynucleotide to be introduced can be a construct containing a nucleic acid molecule delivered in a vector (e.g., a viral vector or an expression vector), a nanoparticle (e.g., a targeted or radiolabeled nanoparticle), or a plasmid. Such constructs are well known in the art and can be easily adapted for use with the compositions and methods described herein.

[0137] In any of the above-provided aspects, the polynucleotides encoding the recombinant growth factors and recombinant insulins provided herein can be introduced into cells using recombinant DNA and cloning techniques. To this end, recombinant molecules, such as recombinant DNA molecules encoding the recombinant growth factors or recombinant insulins, are prepared. Methods for preparing such DNA molecules are well known in the art. By way of example, the peptide-encoding sequence can be excised from DNA using a suitable restriction enzyme. Alternatively, the DNA molecule can be synthesized using chemical synthesis techniques, such as the phosphoramidite method. A combination of these techniques can also be used. In some cases, recombinant or synthetic nucleic acids can be generated through polymerase chain reaction (PCR). The DNA inserts encoding the recombinant molecules can be cloned into suitable transduction / transfection vectors, which are also known to those skilled in the art. Also provided are expression vectors containing the nucleic acid molecules.

[0138] In some embodiments, the expression vector is capable of expressing recombinant growth factors and recombinant insulin in appropriate cells of differentiated stratified epidermis under suitable conditions for protein expression and secretion. In some aspects, the nucleic acid molecule or expression vector comprises a DNA molecule encoding the recombinant molecule operably linked to an appropriate expression control sequence. Methods for achieving this functional linkage either before or after the DNA molecule is inserted into the vector are well known. Expression control sequences include promoters, activators, enhancers, operators, ribosome binding sites, start signals, stop signals, cap signals, polyadenylation signals, and other signals involved in the control of transcription or translation.

[0139] In some embodiments, expression of a recombinant molecule is controlled by a promoter or enhancer to control or regulate expression. The promoter is operably linked to a portion of the nucleic acid molecule encoding the recombinant molecule.

[0140] The resulting recombinant expression vector carrying the DNA molecule thereon is used to transform a suitable host. This transformation can be performed using methods well known in the art. In some embodiments, the resulting expression vector carrying the DNA molecule thereon is used to transform, e.g., transduce, a suitable cell. The introduction can be performed using methods well known in the art. Exemplary methods include methods for the transfer of nucleic acid, including via viruses, e.g., adenovirus, transduction, transposon, and electroporation. In some embodiments, the expression vector is a viral vector. In some embodiments, the nucleic acid is transferred to the cell by adenovirus transduction.

[0141] In some aspects, provided herein is a polynucleotide encoding a growth factor and a precursor of human insulin for introduction into keratinocytes of a skin substitute comprising a stratified epidermis. In some embodiments, the introducing step comprises contacting cells of a skin substitute comprising a stratified epidermis with the polynucleotide (e.g., present in a viral vector) for up to or about 10 minutes, up to or about 20 minutes, up to or about 30 minutes, up to or about 45 minutes, up to or about 60 minutes, up to or about 75 minutes, up to or about 90 minutes, or up to or about 120 minutes. In some embodiments, the introducing step comprises contacting a layer of the stratified epidermis, e.g., the basal layer, with the polynucleotide (e.g., present in a viral vector) for up to or about 10 minutes, up to or about 20 minutes, up to or about 30 minutes, up to or about 45 minutes, up to or about 60 minutes, up to or about 75 minutes, up to or about 90 minutes, or up to or about 120 minutes.

[0142] 1. Polynucleotides and Secreted Polypeptides Encoded Thereby for Delivery to Skin Substitutes In some aspects, methods are provided herein that include introducing polynucleotides into skin substitutes that include stratified epidermis.In some embodiments, the particular polynucleotides delivered or introduced into the skin substitutes are or include nucleic acid molecules whose expression results in useful activities or properties when present in a localized target area and / or secreted into the bloodstream.In some embodiments, introducing polynucleotides into skin substitutes that include stratified epidermis results in the production, e.g., secretion, of one or more encoded polypeptides that have a desired effect or therapeutic effect.In some embodiments, the delivered or introduced nucleic acid molecules can be translated by cells of stratified epidermis to produce and / or secrete one or more proteins that provide a desired effect, e.g., wound closure in wound healing situations.

[0143] In some embodiments, the nucleic acid molecule can be delivered or introduced as a vehicle, for example, as part of a viral vector, as a complex or construct, or as naked DNA.In some embodiments, the nucleic acid molecule can comprise a vector or plasmid that contains the nucleic acid molecule, for example, a viral vector or a non-viral vector.In some embodiments, the nucleic acid molecule can be encapsulated in a liposome.In some embodiments, the nucleic acid molecule can be complexed with other agents, for example, targeting ligands or other moieties, and delivered as nanoparticles.

[0144] In some embodiments, the polynucleotide introduced into the cells of the skin substitute is or includes a nucleic acid molecule encoding one or more desired polypeptides, such as growth hormone and insulin or variants thereof. In some embodiments, the encoded polypeptides can be secreted or released from the cells of the skin substitute, including the stratified epidermis. In some embodiments, the polynucleotide introduced into the cells of the skin substitute can encode a growth factor, such as VEGF or any isoform thereof, and a hormone protein, such as proinsulin and / or insulin, that regulates cell growth, cell differentiation, or cell metabolism.

[0145] a. Recombinant growth factors Provided herein are methods of introducing a polynucleotide encoding a recombinant growth factor into keratinocytes of a skin substitute. In some aspects, the methods provided herein comprise transducing cells of a skin substitute comprised of stratified epidermis with a polynucleotide encoding a recombinant growth factor. In some aspects, the polynucleotide molecule can encode a polypeptide that is a growth factor or portion thereof that binds to a receptor or a growth factor receptor or portion thereof that binds to a ligand.

[0146] In some embodiments, a nucleic acid molecule encoding a growth factor is introduced into the keratinocytes of the stratified epidermis. In some embodiments, the keratinocytes of the stratified epidermis are transduced with a polynucleotide encoding a growth factor selected from epidermal growth factor (EGF), erythropoietin (EPO), fibroblast growth factor (FGF), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), hepatocyte growth factor (HGF), insulin-like growth factor (IGF), platelet-derived growth factor (PDGF), transforming growth factor alpha and beta, vascular endothelial growth factor (VEGF), and any isoforms or alternative splice variants thereof. In some embodiments, cells of the stratified epidermis are transduced with polynucleotides encoding other members of the PDGF / VEGF family of proteins that can be used in the present invention, such as vascular endothelial growth factor B (VEGF-B) polypeptides, platelet-derived growth factor A (PDGF-A) polypeptides, platelet-derived growth factor B (PDGF-B) polypeptides, c-fos-induced growth factor (FIGF) polypeptides, or placenta growth factor (P1GF) polypeptides.

[0147] In some embodiments, the polynucleotide encodes a growth factor sequence that contains a signal peptide that facilitates secretion of the growth factor. In some embodiments, the signal peptide is present in the precursor growth factor sequence and is cleaved to form the mature secretable growth factor. In some embodiments, the signal peptide is the endogenous or native signal peptide of the growth factor. In some embodiments, the signal peptide is a heterologous signal peptide from a different protein. In some embodiments, the signal peptide is cleaved upon expression of the growth factor from the cells of the skin substitute. In some embodiments, the secretable growth factor sequence lacks a signal peptide. In some embodiments, the growth factor is secretable from the cells. In some embodiments, the recombinant growth factor is secretable from the stratified epidermis. In some embodiments, the cells of the stratified epidermis secrete the recombinant growth factor.

[0148] In some embodiments, the growth factor is VEGF-A or its isoform or alternatively spliced ​​variant. VEGF-A is an important mediator of angiogenesis and signals through the class IV tyrosine kinase receptor family of VEGF receptors (VEGFR). VEGF-A ligands bind to both VEGFR1 and VEGFR2, but they primarily signal through VEGFR2, leading to endothelial cell proliferation, survival, migration and vascular permeability. Separate VEGF-A isoforms result from alternative splicing. Any isoform or alternatively spliced ​​variant of VEGF-A that retains the ability to bind to VEGF-R (e.g., VEGFR2) is contemplated in the provided skin substitute. Usually, VEGF-A isoforms vary in length and are named VEGF xxx (where xxx represents the number of amino acids present in the final protein sequence).

[0149] Exemplary VEGF-A isoforms include the vascular endothelial growth factor A (VEGF-A) polypeptide VEGF 206 (SEQ ID NO:11), VEGF-A isoform VEGF 189 (SEQ ID NO:19), VEGF-A isoform VEGF 183 (SEQ ID NO:20), VEGF-A isoform VEGF 148 (SEQ ID NO:21), VEGF-A isoform VEGF 145 (SEQ ID NO:22), VEGF-A isoform VEGF 165B (SEQ ID NO:23), VEGF-A isoform VEGF 121 (SEQ ID NO:24), VEGF-A isoform VEGF111 (SEQ ID NO:25), VEGF-A isoform VEGF 165 (SEQ ID NO:7), VEGF-A isoform L-VEGF165 (SEQ ID NO:8), VEGF-A isoform L-VEGF 121 (SEQ ID NO:27), VEGF-A isoform L-VEGF 189 (SEQ ID NO:28), VEGF-A isoform L-VEGF 206 (SEQ ID NO:29), VEGF-A isoform 15 (SEQ ID NO:30), VEGF-A isoform 16 (SEQ ID NO:31), VEGF-A isoform 17 (SEQ ID NO:32), or VEGF-A isoform 18 (SEQ ID NO:33). It is also understood that the mature sequence thereof, which is cleaved and lacks the signal peptide when expressed and produced from a cell, is also included.

[0150] In some embodiments, the polynucleotide encodes a recombinant human VEGF-A isoform having at least or at least about 80%, at least 85% or at least about 85%, at least 90% or at least about 90%, or at least 95% or at least about 95% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:7, 11, and 19-33, and that retains binding to a VEGFR (e.g., VEGFR-2). In some embodiments, the polynucleotide encodes a recombinant human VEGF-A isoform set forth in any one of SEQ ID NOs:7, 11, and 19-33. In some embodiments, the polynucleotide encodes a protein that contains a signal peptide, which is proteolytically cleaved and removed such that the protein lacking the signal peptide is secreted, such as via a constitutive secretory pathway. In some embodiments, the polynucleotide encodes a VEGF-A isoform having an amino acid sequence having at least or at least about 80%, at least or at least about 85%, at least or at least about 90%, or at least or at least about 95% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:7, 11, and 19-33, and that retains binding to a VEGFR (e.g., VEGFR-2). In some embodiments, the polynucleotide encodes a recombinant VEGF-A isoform set forth in any one of SEQ ID NOs:7, 11, and 19-33. In some embodiments, the polynucleotide encodes a protein that lacks a signal peptide that is proteolytically cleaved and removed, e.g., the encoded protein lacks the signal peptide set forth in any one of SEQ ID NOs:7, 11, and 19-33 (e.g., lacks amino acid residues 1-26).In some embodiments, the polynucleotide comprises a sequence having at least 80% or at least about 80%, at least 85% or at least about 85%, at least 90% or at least about 90%, or at least 95% or at least about 95% sequence identity to the sequence shown in SEQ ID NO:4. In some embodiments, the polynucleotide comprises a sequence shown in SEQ ID NO:4. In some embodiments, the polynucleotide is shown in SEQ ID NO:4. In some embodiments, the polynucleotide encodes a recombinant human VEGF-A comprising a sequence having at least 80% or at least about 80%, at least 85% or at least about 85%, at least 90% or at least about 90%, or at least 95% or at least about 95% sequence identity to the sequence shown in SEQ ID NO:7, or a sequence thereof lacking a signal peptide. In some embodiments, the polynucleotide encodes a recombinant human VEGF-A comprising a sequence shown in SEQ ID NO:7, or a sequence thereof lacking a signal peptide. In some embodiments, the polynucleotide encodes recombinant human VEGF-A as set forth in SEQ ID NO:7, or a sequence thereof lacking the signal peptide.

[0151] In some embodiments, the polynucleotide encoding the growth factor sequence contains a signal peptide that facilitates secretion of VEGF. In some embodiments, the signal peptide is present in a precursor growth factor sequence and is cleaved to form a mature secretable growth factor. In some embodiments, the signal peptide is the endogenous or native signal peptide of the growth factor. In some embodiments, the signal peptide is a heterologous signal peptide from a different protein. In some embodiments, the signal peptide is The sequence is shown as TIFF2024531827000004.tif4128. In some embodiments, the signal peptide is cleaved upon expression of VEGF from cells of the skin substitute.

[0152] In some embodiments, the growth factor is a member of the PDGF / VEGF family of proteins. In some embodiments, the polynucleotide encodes a growth factor that is a vascular endothelial growth factor B (VEGF-B) polypeptide (e.g., SEQ ID NO:12), a c-fos-induced growth factor (FIGF) polypeptide (also called VEGF-D) (e.g., SEQ ID NO:13), a platelet-derived growth factor A (PDGF-A) polypeptide (e.g., SEQ ID NO:14), a platelet-derived growth factor B (PDGF-B) polypeptide (e.g., SEQ ID NO:15), or a placenta growth factor (PLGF) polypeptide (e.g., SEQ ID NO:16), and any isoform or alternatively spliced ​​variant thereof.

[0153] In some embodiments, the polynucleotide encodes a recombinant human growth factor having an amino acid sequence having at least 80% or at least about 80%, at least 85% or at least about 85%, at least 90% or at least about 90%, or at least 95% or at least about 95% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs: 12-16, or an alternatively spliced ​​form or isoform thereof. In some embodiments, the polynucleotide encodes a recombinant human growth factor set forth in any one of SEQ ID NOs: 12-16, or an alternatively spliced ​​form or isoform thereof. In some embodiments, the polynucleotide encodes a recombinant human growth factor set forth in any one of SEQ ID NOs: 12-16. In some embodiments, the encoded protein lacks a signal peptide, which is proteolytically cleaved and removed such that the encoded protein lacks the signal peptide set forth in any one of SEQ ID NOs: 12-16 (see, e.g., the sequence listing).

[0154] In some embodiments, the keratinocytes of the skin substitute comprising a polynucleotide encoding a growth factor secrete or release a growth factor. In some embodiments, the keratinocytes of the skin substitute comprising a polynucleotide encoding a growth factor secrete or release a mature growth factor. In some embodiments, the keratinocytes of the skin substitute comprising a polynucleotide molecule encoding a growth factor secrete or release a mature growth factor, wherein the growth factor comprises VEGF or any isoform thereof. In some embodiments, the polynucleotide encoding the secreted VEGF comprises a sequence having at least 80% or at least about 80%, at least 85% or at least about 85%, at least 90% or at least about 90%, or at least 95% or at least about 95% sequence identity to the sequence shown in SEQ ID NO:4. In some embodiments, the polynucleotide encoding VEGF comprises a sequence shown in SEQ ID NO:4. In some embodiments, the VEGF isoform comprises a sequence having at least or at least about 80%, at least 85% or at least about 85%, at least 90% or at least about 90%, or at least 95% or at least about 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 7. In some embodiments, the VEGF isoform comprises the amino acid sequence set forth in SEQ ID NO: 7. In some embodiments, cells of the basal layer of the stratified epidermis secrete recombinant growth factors.

[0155] b. Recombinant insulin Insulin is a polypeptide composed of 51 amino acid residues with a molecular weight of 5808 daltons. Insulin is produced in the beta cells of the islets of Langerhans in the pancreas. Exemplary human insulin is translated as a 110 amino acid precursor polypeptide containing a 24 amino acid signal peptide, and in the ER, the signal sequence is cleaved, resulting in proinsulin. The proinsulin molecule is then converted to mature insulin by the action of prohormone convertases (e.g., PC1 / 3) and by the action of the exoprotease carboxypeptidase E (CPE) (Ramzy et al, Diabetes 2020; 69(7): 1451-1462). This cleavage results in the removal of four basic amino acid residues and the remaining 31 amino acid C-peptide or linking chain (corresponding to amino acid residues 57-87 of the preproinsulin polypeptide). The resulting insulin contains a 21 amino acid A chain (corresponding to amino acid residues 66-86 of the proinsulin polypeptide) and a 30 amino acid B chain (corresponding to amino acid residues 1-30 of the proinsulin polypeptide) that are cross-linked by disulfide bonds. Mature insulin normally contains three disulfide bridges: one between position 7 of the A chain and position 7 of the B chain, a second between position 20 of the A chain and position 19 of the B chain, and a third between positions 6 and 11 of the A chain.

[0156] In some aspects, the methods provided herein comprise introducing a polynucleotide encoding a precursor of recombinant human insulin into the keratinocytes of the skin substitute. In some aspects, the methods provided herein comprise introducing a polynucleotide encoding proinsulin into the keratinocytes of the skin substitute.

[0157] In some embodiments, the polynucleotide encodes a recombinant regular insulin that is a natural or wild-type insulin polypeptide. These include recombinant human insulin, as well as insulin from bovine, porcine, and other species. In some embodiments, the recombinant insulin is a recombinant insulin of regular human insulin sold as Humulin® R, Novolin® R, and Velosulin®. In some embodiments, the recombinant insulin is a recombinant insulin of regular porcine insulin sold as Iletin II®.

[0158] In some embodiments, the polynucleotide encodes recombinant human insulin. In some embodiments, the polynucleotide encodes a proinsulin precursor form of insulin. In some embodiments, the precursor of human insulin is human proinsulin. In some embodiments, the polynucleotide encodes a human proinsulin amino acid sequence having at least 80% or at least about 80%, at least 85% or at least about 85%, at least 90% or at least about 90%, or at least 95% or at least about 95% sequence identity to the amino acid sequence shown in SEQ ID NO:5. In some embodiments, the polynucleotide encodes human proinsulin shown in SEQ ID NO:5.

[0159] In some embodiments, the polynucleotide encodes a recombinant insulin that is a variant of human insulin, such as a functional variant or a species or allelic variant, or is a truncated form of active human insulin. In some embodiments, such polynucleotides encoding variants of insulin, including allelic and species variants, variants encoded by splice variants, and other functional variants, such as insulin analogs or other derivatives or modifications, encode insulin that has at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the sequence of human insulin shown in SEQ ID NO:5 or to its processed insulin containing A and B chains, so long as the insulin binds to the human insulin receptor and initiates a signaling cascade that leads to increased glucose uptake and storage and / or reduced endogenous glucose production.

[0160] In some embodiments, the polynucleotide encodes a recombinant insulin that is a species variant of human insulin. These include, but are not limited to, insulins derived from bovine and porcine. Bovine insulin differs from human insulin at amino acids 8 and 10 of the A chain and amino acid 30 of the B chain (SEQ ID NO:17). Porcine insulin differs from human insulin only at amino acid 30 of the B chain, where, like the bovine sequence, there is an alanine substitution for threonine (SEQ ID NO:18). In some embodiments, the polynucleotide encodes a proinsulin precursor form of bovine or porcine insulin, such as the proinsulin form of SEQ ID NO:17 (e.g., amino acids 25-105 of SEQ ID NO:17) or the proinsulin form of SEQ ID NO:18 (e.g., amino acids 25-105 of SEQ ID NO:18), or a sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the insulin shown in SEQ ID NO:17 or SEQ ID NO:18 or to the processed insulin thereof containing the A and B chains, so long as the insulin binds to the human insulin receptor and initiates a signaling cascade that leads to increased glucose uptake and storage and / or decreased endogenous glucose production.

[0161] In some embodiments, the polynucleotide encodes a variant of human insulin that contains one or more amino acid modifications compared to human insulin. Exemplary insulin analogs (A and B chains) include fast-acting and slow-acting analog forms or superactive insulin (see, e.g., Vajo et al. 2001 Endocrine Reviews 22:706-717). Fast-acting insulin analogs are modified forms of insulin that usually contain one or more amino acid changes. The analogs are designed to reduce the self-association of the insulin molecule, with the aim of increasing the absorption rate and onset of action compared to regular insulin. For example, insulin analogs include, but are not limited to, glulisine (LysB3, GluB29), HMR-1 153 (LysB3, IleB28), HMR-1423 (GlyA21, HisB32), insulin aspart (AspB28), insulin lispro (LysB28, ProB29) and AspB10. In all the above examples, the nomenclature of the analogues is based on describing the amino acid substitution at a particular position on the A or B chain of insulin, numbering from the N-terminus of the chain, with the remainder of the sequence being that of native human insulin.

[0162] In some embodiments, the polynucleotide encodes a recombinant insulin that is insulin AspB10. Insulin AspB10 is a human insulin analog polypeptide that contains a single amino acid change in the B chain in which the naturally occurring histidine (H) at position 10 in wild-type insulin is replaced by aspartic acid (D) (e.g., H to D substation). The substitution results in a superactive insulin that is absorbed two times faster than regular insulin (e.g., wild-type human insulin). In some aspects, insulin AspB10 has increased binding affinity to the insulin receptor compared to regular insulin (e.g., wild-type human insulin). In some embodiments, the polynucleotide encodes a proinsulin precursor form of insulin AspB10 that contains an A chain as shown in SEQ ID NO:36 and a B chain as shown in SEQ ID NO:41.

[0163] In some embodiments, the polynucleotide encodes a recombinant insulin that is insulin glargine. The addition of two arginines to the C-terminus of the B chain shifts the isoelectric point of glargine insulin, making it more soluble at acidic pH. There is an additional amino acid change (N21G) in the A chain to prevent deamidation and dimerization due to acid-sensitive asparagine. In some embodiments, the polynucleotide encodes the proinsulin precursor form of insulin glargine, containing the A chain shown in SEQ ID NO:34 and the B chain shown in SEQ ID NO:35.

[0164] In some embodiments, the polynucleotide encodes a recombinant insulin that is insulin lispro. Human insulin lispro is an insulin polypeptide preparation that contains amino acid changes at positions 28 and 29 of the B chain, such that Pro-Lys at this position in wild-type insulin is inverted to Lys-Pro. These two amino acid inversions result in a polypeptide with a reduced tendency to self-associate, which allows for a more rapid onset of action. Specifically, the sequence inversion in the B chain results in the elimination of two hydrophobic interactions and the weakening of two beta-pleated sheet hydrogen bonds that stabilize the dimer (DeFelippis et al., Insulin Chemistry and Pharmacokinetics. In Ellenberg and Rifkin's Diabetes Mellitus 2002 pp. 481-500, McGraw-Hill Professional). The amino acid modifications make insulin lispro act more rapidly than regular insulin. In some embodiments, the polynucleotide encodes a proinsulin precursor form of insulin lispro containing an A chain as shown in SEQ ID NO:36 and a B chain as shown in SEQ ID NO:37.

[0165] In some embodiments, the polynucleotide encodes a recombinant insulin that is insulin aspart. Human insulin aspart is an insulin polypeptide preparation that contains an amino acid substitution of proline to aspartic acid at position 28 of the B chain of human insulin. The modification in insulin aspart imparts a negatively charged side chain carboxyl group, resulting in charge repulsion and destabilizing monomer-monomer interactions. Furthermore, the removal of proline eliminates important hydrophobic interactions between monomers (DeFelippis et al., Insulin Chemistry and Pharmacokinetics. In Ellenberg and Rifkin's Diabetes Mellitus 2002 pp. 481-500, McGraw-Hill Professional). In some embodiments, the polynucleotide encodes a proinsulin precursor form of insulin aspart that contains the A chain shown in SEQ ID NO:36 and the B chain shown in SEQ ID NO:38.

[0166] In some embodiments, the polynucleotide encodes a recombinant insulin that is insulin glulisine. Human insulin glulisine is an insulin polypeptide preparation that contains an amino acid substitution at position B3 of the B chain from asparagine to lysine and at amino acid B29 from lysine to glutamic acid, compared to the sequence of the B chain of human insulin. This modification makes the polypeptide molecule less prone to self-association compared to human insulin. In some embodiments, the polynucleotide encodes a proinsulin precursor form of insulin glulisine that contains an A chain as shown in SEQ ID NO:36 and a B chain as shown in SEQ ID NO:39.

[0167] In some embodiments, the polynucleotide encodes a proinsulin form of insulin that is modified so that proinsulin can be easily cleaved into a two-chain form that contains A and B chains. In some cases, human keratinocytes, such as HaCaT cells, lack the enzymes required to efficiently cleave proinsulin for the production of mature insulin. For example, endopeptidases, such as PC-2 and PC-3, are not present or are not present at high enough levels to cleave insulin. Instead, keratinocytes express furin, a calcium-dependent cleavage enzyme that belongs to the subtilisin-like proprotein convertase family of enzymes. In some embodiments, the polynucleotide encodes a human proinsulin that is a modified human proinsulin. In some embodiments, the encoded modified human proinsulin comprises a sequence that is recognized by an enzyme, such as a protease, that is expressed in keratinocytes, such as HaCaT cells, allowing the encoded proinsulin to be processed in keratinocytes into a two-chain form that contains A and B chains linked by disulfide bonds or the like. In some embodiments, the protease is furin and the modified human proinsulin comprises at least one furin recognition sequence. In some embodiments, the encoded modified human proinsulin comprises two furin recognition sequences introduced in place of a sequence containing an Arg31-Arg32 cleavage site (BC junction) and a Lys64-Arg65 cleavage site (CA junction). In some embodiments, the at least one furin recognition sequence comprises the consensus sequence RXRR, where X is any amino acid (SEQ ID NO:8), or RXKR, where X is any amino acid (SEQ ID NO:9). In some embodiments, the furin cleavage site is RTKR (SEQ ID NO:10). In some embodiments, the furin cleavage site is RQKR (SEQ ID NO:42).

[0168] In some embodiments, the polynucleotide encodes a proinsulin that is AspB10 insulin containing an A chain as set forth in SEQ ID NO:36 and a B chain as set forth in SEQ ID NO:41, and the proinsulin further contains two furin recognition sequences. In some embodiments, each of the furin recognition sequences comprises the consensus sequence RXRR, where X is any amino acid (SEQ ID NO:8), or RXKR, where X is any amino acid (SEQ ID NO:9). In some embodiments, one of the furin cleavage sites is RTKR (SEQ ID NO:10). In some embodiments, one of the furin cleavage sites is RQKR (SEQ ID NO:42). In some embodiments, the polynucleotide encodes a modified human proinsulin comprising a sequence having at least 80% or at least about 80%, at least 85% or at least about 85%, at least 90% or at least about 90%, or at least 95% or at least about 95% sequence identity to the amino acid sequence shown in SEQ ID NO:6, wherein the proinsulin contains a furin recognition site and an amino acid substitution of Asp at position 10 of the B chain. In some embodiments, the polynucleotide encodes a modified human proinsulin comprising the amino acid sequence shown in SEQ ID NO:6. In some embodiments, the polynucleotide encodes a modified human proinsulin shown in SEQ ID NO:6.

[0169] In some of any of the provided embodiments, the polynucleotide is a preproinsulin further containing a signal peptide that facilitates secretion of the growth factor. In some embodiments, the signal peptide is cleaved from the encoded preproinsulin to form a secretable mature proinsulin. In some embodiments, the signal peptide is cleaved upon expression of insulin from cells of the skin substitute. In some embodiments, the mature proinsulin form is further processed into recombinant insulin, which is a two-chain form containing A and B chains as described. In some embodiments, the signal peptide is the endogenous or native signal peptide of insulin. In some embodiments, the signal peptide is a heterologous signal peptide from a different protein. In some embodiments, the sequence is a signal peptide. Encode TIFF2024531827000005.tif4128.

[0170] In some embodiments, the polynucleotide comprises a sequence having at least 80% or at least about 80%, at least 85% or at least about 85%, at least 90% or at least about 90%, or at least 95% or at least about 95% sequence identity to the sequence shown in SEQ ID NO:2, and the encoded proinsulin contains a furin recognition site and an amino acid substitution of aspartic acid (Asp, D) at position 10 of the B chain. In some embodiments, the polynucleotide comprises the sequence shown in SEQ ID NO:2. In some embodiments, the polynucleotide is shown in SEQ ID NO:2.

[0171] 2. Expression constructs and regulatory elements In some aspects, expression constructs and regulatory elements are provided herein that facilitate expression of recombinant growth factors and recombinant insulin in cells of a skin substitute, including stratified epidermis. In some embodiments, cells of the stratified epidermis, such as cells of the basal layer, are transduced with an expression construct that includes the regulatory elements described herein. In some embodiments, the expression cassette is a bicistronic expression cassette, in which the polynucleotide encoding the growth factor and the polynucleotide encoding insulin are separated in the expression cassette by a bicistronic element.

[0172] Promoter The polynucleotides described herein may be driven by promoters or enhancers to control or regulate their expression. In some embodiments, the promoter is operably linked to the coding region of the nucleic acid for which relatively high expression is desired. In some embodiments, the promoter is operably linked to the coding region of the nucleic acid that requires post-translational modification after translation. Non-limiting examples of promoters include cytomegalovirus (CMV), Simian Virus 40 (SV40), phosphoglycerate kinase 1 (PGK1), ubiquitin C (Ubc), human beta actin, CAG, TRE, UAS, Ac5, polyhedron, CaMKIIa, GALl, GAL 10, TEF1, GDS, ADH1, CaMV35S, Ubi, HI, U6, SSFV, MNDU3, and EFl-a (also known as Efla).

[0173] In some embodiments, the promoter can be tissue specific. Tissue specific promoters allow the production of proteins in certain cell populations that have the appropriate transcription factors to activate the promoter. Numerous promoters are commercially available and widely known in the art; exemplary sequences can be found in Entrez Gene ID 1915. In some embodiments, the promoter is selected from the group of cytomegalovirus immediate early promoter (CMV), Simian Virus 40 early promoter (SV40), or Rous Sarcoma Virus LTR promoter (RSV).

[0174] Any strong promoter known to those skilled in the art can be used to drive the expression of DNA.The promoter can be a constitutive promoter, such as a CMV promoter, a tissue-specific promoter, an inducible or regulatable promoter.In some embodiments, the polynucleotide to be introduced into the cell contains an inducible promoter operably linked to the coding region, so that the expression of the nucleic acid can be controlled by controlling the presence or absence of a suitable inducer of transcription.

[0175] In some embodiments, the promoter is a regulated promoter and transcription factor expression system, such as the published tetracycline regulated system or other regulatable systems (see, e.g., WO01 / 30843), that allows regulated expression of the encoded polypeptide. Exemplary other promoters are tissue-selective promoters, such as those described in U.S. Pat. No. 5,998,205, including, e.g., fetoprotein, DF3, tyrosinase, CEA, surfactant protein and ErbB2 promoters. Exemplary regulatable promoter systems are, e.g., the Tet-On (and Tet-Off) system available from Clontech (Palo Alto, Calif.). This promoter system allows regulated expression of the transgene controlled by tetracycline or a tetracycline derivative (e.g., doxycycline). Other regulatable promoter systems are known (see, e.g., U.S. Patent Publication No. 2002-0168714, entitled "Regulation of Gene Expression Using Single-Chain, Monomeric, Ligand Dependent Polypeptide Switches," which describes gene switches containing ligand binding domains and transcriptional regulatory domains, such as those derived from hormone receptors).

[0176] In some embodiments, the promoter is a constitutive promoter. Exemplary promoters include, but are not limited to, the CMV promoter, the truncated CMV promoter, the human serum albumin promoter, or the C-1-antitrypsin promoter. In some embodiments, the promoter is a truncated CMV promoter that lacks binding sites for known transcriptional repressors. The CMV-derived promoter can be of human or monkey origin. In some embodiments, the promoter is an inducible promoter. For example, the promoter is an inducible ecdysone promoter. Other examples of promoters include steroid promoters, such as estrogen and androgen promoters, and metallothionein promoters. In some embodiments, the enhancer can be a tissue-specific or non-specific enhancer. For example, the enhancer is a liver-specific enhancer element. Exemplary enhancer elements include, but are not limited to, the human serum albumin (HSA) enhancer, the human prothrombin (HPrT) enhancer, the C-1-microglobulin enhancer, the intronic aldolase enhancer, and the apolipoprotein E hepatic control region.

[0177] In some embodiments, promoters such as, but not limited to, animal virus-derived promoters, mammalian cell-derived promoters, or hybrid promoters of both promoters can be used. In many cases, it is desirable to express genes, including therapeutic genes, at a relatively high level. Examples of high expression promoters include CMV promoters (Foecking MK et al., Gene 1986;45:101-105) and CAG promoters (Niwa H. et al., Gene 1991;108:193-200). The CMV promoter consists of the enhancer and promoter of the immediate early (IE) gene of cytomegalovirus (CMV), and the CAG promoter consists of the IE enhancer of CMV, chicken β-actin promoter, splice acceptor, and rabbit β-globin poly(A) sequence. Thus, both the CMV promoter and the CAG promoter contain the enhancer of the IE gene of CMV (Boshart M. et al., Cell 1985;41:521-530). As used herein, this enhancer of the CMV IE gene may be simply referred to as the "CMV enhancer."

[0178] Examples of constitutive promoters include CAG promoter, CMV promoter, EF-1α promoter, SRα promoter, SV40 promoter, RSV promoter, adenovirus major late promoter (MLP), etc. Examples of inducible promoters include metallothionein gene promoter, mouse mammary tumor virus (MMTV) promoter, etc. In addition, a system in which the expression of a constitutive promoter is induced by tetracycline or ecdysone can be used. Expression vectors and expression induction systems having such promoters are commercially available or available from public institutions. If available, commercial products can be purchased from Invitrogen Inc., Clontech Inc., etc.

[0179] In some embodiments, in addition to the above promoters containing the CMV enhancer, promoters derived from viruses such as the SV40 promoter and the Rous sarcoma virus (RSV) promoter (Takebe Y. et al., Mol. Cell. Biol. 1988;8:466-472) can also be used.

[0180] In some aspects, the present invention provides a viral vector with a CAG promoter that expresses genes such as growth factors and insulin or variants thereof. In some embodiments, the nucleic acid sequence that encodes the protein that undergoes most post-translational modifications is in front of any other nucleotides downstream of the promoter, e.g., the CAG promoter. In some embodiments, the nucleic acid sequence that encodes the growth factor is upstream of the nucleic acid sequence that encodes insulin or variants thereof downstream of the promoter. These aspects should not be interpreted as limiting, since other specific or promiscuous promoters may be used with different transgenes that encode other proteins. The gene to be inserted into the adenoviral vector of the present invention is not particularly limited, and genes that encode proteins such as growth factors and hormones (e.g., insulin) may be used.

[0181] In some embodiments, the nucleotide sequence of the CAG promoter can be replaced with a nucleotide sequence that comprises a nucleotide sequence having at least 60% sequence identity or similarity to SEQ ID NO: 1. In some embodiments, preferred nucleotide sequences are at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% similar to SEQ ID NO: 1.

[0182] In some embodiments, the coding sequences encoding each of the different polypeptide chains can be operably linked to promoters that can be the same or different, hi some embodiments, the nucleic acid molecule can contain a promoter that drives expression of a polynucleotide encoding a growth factor and a polynucleotide encoding insulin.

[0183] b. Bicistronic elements In some embodiments, the expression cassette containing the coding polynucleotide can be multicistronic (bicistronic or tricistronic, see, e.g., U.S. Pat. No. 6,060,273). In some embodiments, the transcription unit can be engineered as a bicistronic unit containing a bicistronic element, allowing for co-expression of gene products with messages from a single promoter. In some embodiments, the bicistronic element is an IRES (internal ribosome entry site). In some embodiments, the bicistronic element can be a self-cleaving sequence, such as a 2A sequence (e.g., P2A, FTA, or T2A).

[0184] An internal ribosome entry site (IRES) is a sequence that initiates translation from an internal start codon (usually AUG) within a bi- or multicistronic RNA transcript followed by multiple protein coding regions. IRES have been characterized in encephalomyocarditis virus and related picornaviruses (e.g., Jackson et al., RNA 1995;1:985-1000 and Herman, Trends in Biochemical Sciences 1989;14(6):219-222). IRES sequences are also detected in mRNAs from other viruses, such as cardioviruses, rhinoviruses, aphthoviruses, hepatitis C virus (HCV), Friend murine leukemia virus (FrMLV) and Moloney murine leukemia virus (MoMLV). The presence of IRES in cellular RNA has also been described. Examples of cellular mRNAs that contain IRES include those encoding immunoglobulin heavy chain binding protein (BiP), vascular endothelial growth factor (VEGF), fibroblast growth factor 2, insulin-like growth factor, translation initiation factor eIF4G, and the yeast transcription factors TFIID and HAP4 (e.g., Macejak et al., Nature 1991;353:90-94; Oh et al., Genes Dev. 1992;6:1643-1653; Vagner et al., Mol. Cell. Biol. 1995;15:35-44; He et al., PNAS 1996;93:7274-7278; He et al., Gene 1996;175:121-125; Tomanin et al., Gene 1997;193:129-140; Gambotto et al., Cancer Gene Therapy 1999;6:45-53; Qiao et al., Cancer Gene Therapy 1999;6:373-379). Expression vectors containing IRES elements have been described. See, e.g., PCT / US98 / 03699 and PCT / EP98 / 07380.

[0185] In some embodiments, the viral vector described herein comprises one or more transgenes.In one example, the vector encodes two transgenes, for example, a transgene encoding a growth factor and a transgene encoding insulin, or variants thereof.In some embodiments, the same regulatory element exerts transcriptional control on the first and second transgenes, and optionally, one transgene is under the translational control of an internal ribosome entry site.In some embodiments, different elements regulate the transcription of each of the two transgenes, and optionally, one transgene is under the translational control of an IRES.

[0186] c. 3' untranslated region (UTR) 3'-untranslated region (3'-UTR) is a part of mRNA that is usually located between the protein coding region (i.e., open reading frame) and poly(A) sequence of mRNA. 3'-UTR of mRNA is not translated into amino acid sequence. 3'-UTR sequence is generally encoded by a gene that is transcribed into each mRNA during gene expression process. Genomic sequence is first transcribed into premature mRNA that contains any intron. Premature mRNA is then further processed into mature mRNA in maturation process. This maturation process includes steps such as 5'-capping, splicing of premature mRNA that excises any intron and modification of 3' end, such as polyadenylation of 3' end of premature mRNA, and cleavage by any endo- or exonuclease. The 3'-UTR may correspond to a sequence of a mature mRNA that is located 3' to the stop codon of the protein coding region, preferably immediately 3' to the stop codon of the protein coding region, and extends to the nucleotides 5' to the poly(A) sequence, preferably immediately 5' to the poly(A) sequence. The term "corresponding to" indicates that the 3'-UTR sequence may be an RNA sequence, such as an RNA sequence in the mRNA sequence used to define the 3'-UTR sequence, or a DNA sequence that corresponds to such an RNA sequence. The term "3'-UTR of a gene", for example "3'-UTR of the insulin gene", is a sequence that corresponds to the 3'-UTR of the mature mRNA derived from this gene, i.e., the mRNA obtained by transcription of the gene and maturation of the immature mRNA. The term "3'-UTR of a gene" encompasses the DNA and RNA sequences of the 3'-UTR.

[0187] In some embodiments, a suitable 3'-UTR sequence can be operably linked to a nucleotide sequence encoding one or more desired transgenes. In some embodiments, a 3'-UTR sequence can be operably linked to a nucleotide sequence encoding growth factors and insulin or variants thereof. In some embodiments, a suitable 3'-UTR region can be the one naturally associated with the nucleotide sequence or can be derived from a different gene, such as the bovine growth hormone 3'-UTR region (bGH polyadenylation signal, SV40 polyadenylation signal, SV40 polyadenylation signal and enhancer sequence). In the context of the present invention, when referring to "SV40", reference is made to the SV40 polyadenylation signal. When referring to "SV40 enhancer sequence", reference is made to the SV40 polyadenylation signal and enhancer sequence.

[0188] In some embodiments, the 3'-UTR sequence comprises a poly-A tail, also referred to as a 3'-poly(A) tail or poly(A) sequence. A poly-A tail is a long sequence of adenosine nucleotides added to the 3' end of an RNA molecule. Polyadenylation is the addition of a poly(A) sequence to a nucleic acid molecule, such as an RNA molecule, for example to a premature mRNA. Polyadenylation can be induced by a polyadenylation signal. This signal is preferably located within consecutive nucleotides at the 3' end of the nucleic acid molecule, such as an RNA molecule, to be polyadenylated. A polyadenylation signal usually comprises a hexamer of adenine and uracil / thymine nucleotides, preferably the hexamer sequence AAUAAA. Other sequences, preferably hexamer sequences, are also envisaged. Polyadenylation usually occurs during the processing of pre-mRNA (also referred to as premature mRNA). Usually, maturation of RNA (from pre-mRNA to mature mRNA) comprises a step of polyadenylation.

[0189] In some embodiments, an expression construct described herein can include a 3'-UTR region including a poly-A tail having up to about 400 adenosine nucleotides, e.g., about 25 to about 400, about 50 to about 400, about 50 to about 300, about 50 to about 250, about 60 to about 250 adenosine nucleotides, about 70 to about 250 adenosine nucleotides, about 80 to about 250 adenosine nucleotides, about 90 to about 250 adenosine nucleotides, about 100 to about 250 adenosine nucleotides, about 100 to about 200 adenosine nucleotides, or about 100 to about 150 adenosine nucleotides.

[0190] 3. Viral vectors for transduction Viruses can be used as gene delivery vehicles when exogenous nucleic acid sequences are inserted into viral vectors.Provided herein is a viral vector that contains a polynucleotide that encodes a recombinant growth factor (e.g., any of those described herein, e.g., VEGF) and a polynucleotide that encodes a recombinant insulin, e.g., recombinant human insulin (e.g., any of those described herein).Also provided herein is a viral vector that contains any of the expression cassettes, e.g., bicistronic expression cassettes, as described herein.

[0191] Viruses are useful in in vivo delivery of nucleic acid molecules, e.g., polynucleotides, because they are efficient at transferring viral DNA into host cells. They can infect and be incorporated into specific target cells depending on the viral attachment proteins (e.g., capsid or glycoproteins), and they can be engineered to remove non-essential genes and add heterologous nucleic acid molecules. Many viral vectors are known to those skilled in the art. Examples of viruses that can be used in the methods herein include, but are not limited to, adenoviruses, adeno-associated viruses, alphaviruses, baculoviruses, hepadenaviruses, poxviruses, herpesviruses, retroviruses, lentiviruses, orthomyxoviruses, papovaviruses, paramyxoviruses, and parvoviruses. The choice of virus is within the skill of the skilled artisan and depends on a number of factors, such as the requirement for viral DNA replication or integration, the tropism of the virus, and / or the immunogenicity of the virus.

[0192] Such viruses and their derivatives are well known and available to those skilled in the art. For example, many are available from the American Type Culture Collection (ATCC, Rockville, Md.) or from commercial suppliers (e.g., Vector Biolabs, Philadelphia, Pa.; Applied Biological Materials, Inc., Richmond, British Columbia, Canada). Viral vectors for use in making recombinant viruses include replication-competent and replication-defective viruses. In replication-defective viruses, the virus usually lacks one or more genes associated with viral replication, and in some cases cannot replicate beyond the first cycle of infection. To produce replication-defective viruses, transfer vectors, packaging vectors, or helper viruses may be required. For example, packaging vectors can be provided as cosmids or in cell lines that provide viral structural proteins for packaging of defective vectors. Viral vectors can also contain expression cassettes that include regulatory elements such as promoters and enhancers operably linked to a transgene of choice. Any suitable promoter can be used. Suitable promoters and enhancers are widely available in the art for use in the viral vector of choice.

[0193] a. Adenovirus vector Adenovirus vectors have several advantages for use as gene delivery vehicles, including tropism for both dividing and non-dividing cells, minimal pathogenicity, the ability to replicate to high titers for preparation of vector stocks, and the potential to carry large inserts (see, e.g., Berkner Curr. Top. Micro. Immunol. 1992;158:39-66; Jolly et al. Cancer Gene Therapy 1994;1:51-64). Adenoviruses are nuclear DNA viruses with a genome of approximately 36 kb and have been well characterized through studies in classical genetics and molecular biology (Horwitz, MS, "Adenoviridae and Their Replication in Virology, 2nd edition, Fields, BN, et al., eds., Raven Press, New York, 1990). The genome is divided into early (known as E1-E4) and late (known as L1-L5) transcription units, referring to the production of two temporal classes of viral proteins. The dividing line between these events is viral DNA replication. Adenoviruses show a natural tropism for epithelial cells of the respiratory and digestive tracts. Adenoviruses can also infect liver cells, such as hepatocytes and endothelial cells, which can occur when the virus is excreted in the liver after systemic administration. The penton base and fiber proteins on the viral surface are responsible for viral tropism. Multiple interactions between the adenoviral particle and the host cell are required to facilitate efficient cell entry (Nemerow, Virology 2000;274:1-4).

[0194] For subgroup C adenoviruses, such as adenovirus 2 and 5 (Ad2 or Ad5), the viral entry pathway has been well characterized and is thought to involve two separate cell surface events. First, a high affinity interaction between the adenovirus fiber knob and the Coxsackie-adenovirus receptor (CAR) mediates attachment of the adenovirus particle to the cell surface. Subsequent binding of the penton and cell surface integrin α v Beta3 and Alphav Engagement of β5 (acting as a coreceptor) enhances viral internalization. CAR, which is expressed in many human tissues, including lung epithelial cells (Bergelson et al., Science 1997;275:1320-1323), appears to function as a cellular receptor for most adenovirus subgroups, except for subgroup B (Bergelson et al., Science 1997;275:1320-1323; Roelvink et al., J. Virol. 1998;72:7909-7915). In some embodiments, the adenovirus used to transduce cells of the skin substitute comprising stratified epidermis is a type 5 adenovirus. In some embodiments, the adenovirus used to transduce basal cells of the skin substitute comprising stratified epidermis is a type 5 adenovirus.

[0195] Adenoviruses include over 50 serotypes classified into six distinct subgroups, A through F. Any of these adenovirus serotypes available from the American Type Culture Collection (ATCC, Rockville, Md.) and other commercial and non-commercial vendors can be used in the methods herein or can be used as a source of further modification as known in the art. Also, any other serotype of adenovirus available from any other source can be used or further modified. By way of example, the adenovirus can be of subgroup A (e.g., serotypes 12, 18, 31), subgroup B (e.g., serotypes 3, 7, 11a, 11p. 14, 16, 21, 34, 35, 50), subgroup C (e.g., serotypes 1, 2, 5, 6), subgroup D (e.g., serotypes 8, 9, 10, 13, 15, 17, 19, 19p. 20, 22-30, 32, 33, 36-39, 42-49, 51), subgroup E (e.g., serotype 4), subgroup F (e.g., serotypes 40, 41), or any other adenovirus serotype. In certain embodiments, the adenovirus is a subgroup C adenovirus or is derived from a subgroup C adenovirus. In a preferred example, the adenovirus is a type 5 adenovirus of subgroup C.Adenovirus vectors are available in the art (e.g., from the American Type Culture Collection (ATCC, Rockville, Md.)), and the sequences of wild-type adenovirus proteins from many different adenovirus serotypes are well known in the art (e.g., Roberts et al. J. Biol. Chem. 1984;259:13968-13975; Chroboczek et al. Virology 1992;186:280-285; Sprengel et al. J. Virol. 1994;68:379-389; Chillon et al. J. Virol. 1999;73:2537-2540; Davison et al. J. Mol. Biol. 1993;234:1308-1316; www.binfgmu.edu / wiki / index.php / Human_Adenovirus_Genome_Sequences_2002 ... and Annotations). Adenovirus vectors are widely available to those of skill in the art, for example, from the American Type Culture Collection (ATCC) or other commercial or non-commercial vendors. From the ATCC, adenoviruses are available under ATCC numbers VR-1 through VR-1616. For example, wild-type adenovirus type 5 is available as VR-5 and VR-1082. Any of a number of recombinant or modified adenoviruses derived from any of the above serotypes can be made as described in the art and herein, or by any suitable method known to one of skill in the art.

[0196] Adenoviral vectors for use in the methods described herein can include defective adenoviral vectors that contain at least one deletion in the immediate early gene region (E1-E4). Modifications to adenoviral vectors include deletions known in the art. Such deletions can be made in one or more of the E1, E2a, E2b, E3, or E4 coding regions. For example, adenoviral vectors for gene therapy can be prepared by substituting a heterologous nucleic acid molecule in place of the E1, E2a, E2b, E3, and / or E4 genes. Deletions can be achieved using restriction endonucleases. For example, the E1a region can be deleted using a convenient restriction endonuclease site in the E1a region. Often, a portion of E3 is also deleted by restriction endonuclease addition to allow for the insertion of a large piece of foreign DNA while still meeting the size constraints required for packaging into new viral particles. With the deletion of these regions, the cloning capacity of the adenoviral vector can be approximately 8 kb. Such adenoviral vectors are usually referred to as replication-deficient adenoviruses due to at least one deletion in the first viral early gene region, such as E1, including the E1a and E1b regions.

[0197] In some embodiments, the adenovirus used to transduce cells of a skin substitute comprising stratified epidermis is a replication-deficient type 5 adenovirus. In some embodiments, the adenovirus used to transduce cells of a skin substitute comprising stratified epidermis is a replication-deficient type 5 adenovirus with a deletion in region E1. In some embodiments, the adenovirus used to transduce cells of a skin substitute comprising stratified epidermis is a replication-deficient type 5 adenovirus with a deletion in region E3. In some embodiments, the adenovirus used to transduce cells of a skin substitute comprising stratified epidermis is a replication-deficient type 5 adenovirus with a deletion in regions E1 and E3. In some embodiments, the adenovirus used to transduce basal cells of a skin substitute comprising stratified epidermis is a replication-deficient type 5 adenovirus with a deletion in regions E1 and / or E3.

[0198] The deletion of early genes, such as viral regions E1 and E3, makes recombinant adenoviruses replication-deficient and subsequently unable to produce infectious viral particles in infected target cells. Therefore, a complementation system is required to provide the missing gene products to enable genome replication of early gene-deleted adenoviruses, such as genome replication of E1-deleted adenoviruses, and to produce viral particles. For example, E1 complementation is usually provided by a cell line that expresses E1, such as a human embryonic kidney packaging cell line called 293, an epithelial cell line (deposited with ATCC under registration number CRL-1573). Cell line 293 contains the E1 region of adenovirus, which provides the E1 gene region product that supports the growth of E1-deleted viruses in cell lines (see, for example, Graham et al., J. Gen. Virol. 36:59-71, 1977). In addition, cell lines have been reported that can be used to produce defective adenoviruses with a portion of the adenovirus E4 region (see, for example, International Publication No. WO 96 / 22378). E3 can also be deleted from vectors, but since it is not required for vector production, it can be omitted from complementing producer cells. Complementing producer cell lines and methods for making complementing producer cell lines are known in the art (see, for example, Morris et al., BMC Biotechnology 2010;10(92)).

[0199] The advantage of using replication-defective viruses as vectors is that they can replicate within the initially infected cell but cannot form new infectious viral particles, limiting the extent to which they can spread to other cell types. Several defective adenovirus vectors and complementing cell lines have also been described (see, e.g., International PCT Publication No. WO 95 / 34671, U.S. Patent No. 5,994,106). The construction of replication-defective adenoviruses has been described (Berkner et al., J. Virol. 1987;61:1213-20;Massie et al., Mol. Cell. Biol. 1986;6:2872-83;Haj-Ahmad et al., J. Virol. 1986;57:267-74;Davidson et al., J. Virol. 1987;61:1226-39;Zhang et al., BioTechniques 1993;15:868-72;Berkner Nuc. Acids Res. 1983;11:6003:Ghosh-Choudhury Biochem. Biophys. Res. Commun., 1987;147:964;Gilardi et al., FEBS 1990;267:60;Mittal Virus Res. 1993;28:67; Yang PNAS 1993;90:4601; and international publication PCT WO1995 / 026411).

[0200] Adenoviral vectors also include "gutless" or "gutted" vectors, in which all viral genes have been removed, leaving only the inverted terminal repeats (ITRs) necessary for vector propagation. Such adenoviral vectors are named pseudoadenoviral vectors (PAV) because they are derived from the genome of adenovirus, which contains the minimal cis-acting nucleotide sequences necessary for replication and packaging of the vector genome. PAV vectors contain 5' ITR and 3' ITR nucleotide sequences containing the origin of replication, as well as the cis-acting nucleotide sequences necessary for packaging of the PAV genome. They can be modified to contain more than one transgene with appropriate regulatory elements (e.g., promoters or enhancers). PAVs are up to 36 kb in size, with a carrying capacity of much more than 8 kb, due to the deletion of most of the viral coding sequences (see, e.g., U.S. Pat. Nos. 5,882,887 or 5,670,488; PCT Publication Nos. WO 96 / 40955, WO 97 / 25466, WO 95 / 29993, WO 97 / 00326; Morral et al. Hum. Gene Ther. 1998;10:2709-2716; Kochanek et al. PNAS 1996;93:5731-5736; Parks et al. PNAS 1996;93:13565-13570; Lieber et al. J. Virol. 1996;70:8944-8960; Fisher et al. J. Virol. 1996;217:11-22).

[0201] PAV grows by co-infection of producer cells with a "helper" virus (e.g., using an E1-deleted adenovirus vector), where the packaging cells express the E1 gene product. The helper virus trans-complements the missing adenovirus functions, including the production of viral structural proteins required for particle assembly. For example, a helper adenovirus vector genome and a gutless adenovirus vector genome are delivered to the packaging cells. The cells are maintained under standard cell maintenance or growth conditions, whereby the helper vector genome and the packaging cells together provide the complementing proteins for packaging of the adenovirus vector particles. Such gutless adenovirus vector particles are recovered by standard techniques. The helper vector genome can be delivered by standard transfection techniques in the form of a plasmid or similar construct, or can be delivered through infection by a viral particle containing the genome. Such viral particles are generally referred to as helper viruses. Similarly, gutless adenovirus vector genomes can also be delivered to cells by transfection or viral infection.

[0202] Adenoviruses also include replication-conditional adenoviruses, which are viruses that replicate in certain cell or tissue types but not in others as a result of placing an adenoviral gene essential for replication under the control of a heterologous promoter (discussed above; see also U.S. Pat. No. 5,998.205, U.S. Pat. No. 5,801,029, and U.S. Patent Application No. 10 / 081,969, published as US 2003 / 0104625 and corresponding International PCT Publication No. WO 2002 / 067861).

[0203] Adenovirus also includes adenoviruses that are modified to contain targeting ligands, for example, to change the tropism of the virus, thereby enhancing infection of specific target cells that express receptors (proteins, lipids, carbohydrates, or their parts) for the targeting ligands.Although adenovirus vectors and the like have many potential therapeutic applications, their usefulness is limited by the wide tissue distribution of CAR, which limits the delivery of adenovirus vectors to specific cell types.In addition, the absence of CAR and / or C, integrin receptors on certain cells in vivo limits the cell or tissue types that can be targeted by adenovirus vectors. Thus, adenoviruses also include adenoviruses that have been modified to incorporate targeting ligands for desired cellular or tissue-specific receptors by reducing or eliminating binding to the native receptor and / or by manipulating the capsid proteins, such as the HI loop, the C-terminus of the fiber, the L1 loop of the hexon or the RGD loop of the penton base, or capsid protein IX (see, e.g., Krasnykh et al., Mol. Ther, 2000; 1(5):P391-405 and Wickham, Gene Ther. 2000; 7:110-4). The capsid proteins can be modified, for example, by the addition of targeting ligands or by replacement of the fiber with other types of adenoviral fibers. The targeting ligand can be any protein or portion thereof that binds to a moiety in or on a cell, such as a cell surface protein, lipid, carbohydrate or other moiety. For example, targeting ligands include, but are not limited to, growth factors, adhesion molecules, cytokines, protein hormones, neuropeptides (neurotransmitters) and single-chain antibodies, or suitable portions thereof.In other examples, adenoviral vectors can be conjugated to adapter molecules, such as antibodies and fusion proteins or the extracellular domain of a CAR, containing anti-Ad single chain antibodies (sclv) bearing a targeting ligand, or chemically modified with polymers, such as polyethylene glycol (PEG) moieties, containing a targeting ligand (see, e.g., Mizuguchi et al. (2004) Hum. Gene Ther. 15:1034-44; Eto et al. (2008) Int. J. Pharm., 354:3-8).

[0204] Any of the above adenoviruses, or any known in the art, can be modified to contain desired heterologous nucleic acid molecules for use as delivery agents herein. Adenoviruses containing desired heterologous nucleic acid sequences can be prepared by any technique known to those skilled in the art (Levrero et al., Gene 1991;101(2):195-201, EP185 573; Graham, EMBO.J. 3 (1984) 2917; WO95 / 26411). In particular, such viruses can be prepared by homologous recombination between an adenoviral vector and a plasmid carrying a heterologous DNA sequence. Homologous recombination can occur after co-transfection of an adenoviral vector and a plasmid into a suitable cell line. The cell line used is generally one that can be transformed. Transfection can be performed in the presence of a reagent that induces the entry of adenoviral particles into a production cell. Such reagents include, but are not limited to, polycations and bifunctional reagents.

[0205] In some embodiments, when the adenovirus is a defective adenovirus (due to deletion of early genes, e.g., E1 and / or E3, or fiber protein), the cell line in which the adenovirus is packaged or grown contains sequences capable of complementing the defective adenoviral genome portion, e.g., in an integrated form to avoid the risk of recombination. Examples of complementing cell lines include, but are not limited to, the human embryonic kidney line 293 (HEK293), which contains the left hand portion of the Ad5 adenovirus genome (Graham et al., J Gen Virol. 1977; 36(1):59-74). Complementing cells also include, for example, cells of the PER.C6 cell line, which contains the adenovirus E1 gene (PER.C6 is available, for example, from Crucell, the Netherlands; deposited under ECACC accession number 96022940; see also Fallaux et al. Hum Gene Ther. 1998; 9(13):1909-17; U.S. Pat. No. 5,994,128). Another example of a complementing cell line is the A549-derived cell line AE1-2a (see, for example, Gorziglia et al. J Virol. 1996; 70(6):4173-4178 and Von Seggern et al. (1998) J. Gen. Virol. 1998; 79, 1461-1468). In some embodiments, adenovirus propagated in a complementing cell or cell line is recovered and purified according to conventional molecular biology techniques.

[0206] References illustrating the use of adenoviruses in gene therapy include, but are not limited to, Vorburger and Hunt, The Oncologist 2002;7:46-59 and St. George, Gene Therapy 2003;10:1135-1141.

[0207] b. Adeno-associated virus (AAV) Viral vectors for use as delivery agents include adeno-associated viruses (AAV). AAV is a single-stranded human DNA parvovirus with a genome size of 4.6 kb. The AAV genome contains two major genes: the rep gene and the cap gene. The rep gene codes for the rep proteins (Rep. 76, Rep. 68, Rep. 52 and Rep 40). The cap gene codes for AAV replication, rescue, transcription and integration, while the cap protein forms the AAV virus particle. AAV is so named because it depends on adenovirus or other helper viruses (e.g., herpesvirus) to provide essential gene products that allow AAV to productively infect (i.e., produce itself in the host cell). In the absence of helper virus, AAV integrates into the host cell chromosome as a provirus until rescued by superinfection of the host cell with a helper virus, usually an adenovirus (Muzyczka, Curr. Top. Micro. Immunol. 1992;158:97-129).

[0208] AAV virus can integrate into cellular genome. The integration mechanism is mediated by the presence of inverted terminal repeats (ITRs) at both ends of the AAV genome, which contain cis-acting nucleotide sequences necessary for viral replication, rescue, packaging and integration. The integration function of ITRs, mediated in trans by rep protein, allows AAV genome to integrate into cellular chromosomes after infection in the absence of helper virus. The site of AAV integration is well established and is localized to chromosome 19 in humans (Kotin et al., PNAS 1990;87:2211-2215). Knowledge of the integration site reduces the risk of random insertion events into the cellular genome that may activate or inactivate host genes or interrupt coding sequences. AAV is also useful for gene therapy applications because of its broad host range and tropism for many cell types. AAV can also infect both non-dividing and dividing cells.

[0209] AAV vectors can be derived from any naturally occurring AAV serotype, including AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8 or AAV-9. Such viruses are well known and available to those of skill in the art (see, e.g., Grimm et al. (2003) Current Gene Therapy, 3:28.1-304; Muramatsu et al. (1996) Virol., 221:208-217; Chiorini et al. (1997) J. Virol., 71:6823-6833; Chiorini (1999) J. Virol., 73: 1309-1319; Rutledge et al. (1998) J. Virol, 72:309-319; Xiao et al. (1999). Virol., 73:3994-4003; Gao et al. (2002) Proc Natl. Acad. Sci., 99:1 1854-11859; Kotin (1994) Human Gene. Therapy, 5:793-801). Other serotypes are known and available, including AAV-8 through AAV-12. For example, many AAV vectors are available from the American Type Culture Collection (ATCC, Rockville, Md.; see, e.g., VR-197, VR-645, VR-646, VR-680, VR-681, VR-1449, VR-1523, VR-1616). Compatible host cells and helper viruses are also available. AAV vectors also include "pseudotyped" AAV vectors in which the AAV-2 vector genome is cross-packaged into the capsid of other AAV serotypes (Burger et al., Mol Ther. 2004; 10(2):302-17 and U.S. Pat. No. 7,094,604). Such pseudotyped AAV vectors overcome the limitations of AAV-2 derived serotypes, e.g., their inefficiency in transducing some cells, such as liver or muscle cells.

[0210] Many AAV vectors show broad transduction across multiple tissues, such as skeletal and cardiac muscles, following delivery methods that achieve systemic expression. These include, for example, AAV serotypes 6, 8 and 9. In particular, AAV vectors include adenovirus-associated serotype 9 (AAV-9; GenBank accession number AY530629.1; Gao et al. J. Virol., 2004; 78:6381-6388). AAV-9 is a vector that can bypass the blood-brain barrier and target the central nervous system (CNS) (see, for example, Foust et al., Nature Biotechnology, 2009; 27:59-65; Duque et al. Mol. Ther:, 2009; 17:1187-1196). Thus, in the examples of neurodegenerative diseases or other diseases herein that affect or involve the brain or CNS, AAV-9 can be used as a delivery agent that encodes a protein of interest for systemic delivery (e.g., delivery to the liver or portions thereof for expression in the blood).

[0211] AAV vectors include recombinant AAV vectors that contain heterologous nucleic acid of interest. Procedures for producing such vectors are known to those skilled in the art. For example, the standard approach of AAV vector production requires transfection of AAV vector genomes that contain nucleic acid molecules of interest flanked by AAV ITR sequences into host cells, transfection of host cells with a plasmid that encodes the AAV rep and cap protein genes required in trans, and infection of transfected cells with a helper virus that provides the non-AAV helper functions required in trans (Muzyczka Curr. Top. Micro. Immunol., 1992; 158:97-129 and U.S. Patent No. 5,139,941). The helper virus can be adenovirus or other helper viruses. The helper virus protein activates the transcription of AAV rep gene, and then the rep protein activates the transcription of AAV cap gene. The cap protein then utilizes the ITR sequences to package the AAV genome into viral particles.

[0212] Alternatively, a plasmid containing helper function genes can be used in combination with infection by one of the well-known helper viruses that can be used as a source of replication function to help the recombination of AAV virions (see, for example, U.S. Patent Nos. 5,622,856 and 5,139,941).Similarly, those skilled in the art can utilize a plasmid containing accessory function genes in combination with infection by wild-type AAV to provide the necessary replication function.Also, a triple transfection method can be used to produce recombinant virions (rAAV), which is a method that does not require a helper virus (see, for example, U.S. Patent No. 6,001,650).This is achieved by using three vectors for rAAV virion production: an AAV helper function vector, an accessory function vector and a rAAV vector.

[0213] References illustrating the use of AAV viruses in gene therapy include, but are not limited to, Sheridan, Nature Biotechnology 2011; 29:121-128.

[0214] c. Retroviral vectors Viral vectors for use as delivery agents include retroviral vectors. Retroviral vectors are well suited for the delivery of nucleic acids to cells because of their ability to deliver unrearranged single copy genes to a wide range of rodent, primate and human somatic cells. Retroviral vectors are integrated into the genome of host cells. Unlike other viral vectors, they only infect dividing cells. Retroviruses are RNA viruses, and therefore the viral genome is RNA. When a host cell is infected with a retrovirus, the genomic RNA is reverse transcribed into a DNA intermediate, which is integrated very efficiently into the chromosomal DNA of the infected cell. This integrated DNA intermediate is called a provirus. The transcription and assembly of the provirus into an infectious virus occurs in the presence of a suitable helper virus or in a cell line that contains the appropriate sequences that allow encapsidation without the co-production of a contaminating helper virus. If the sequences for encapsidation are provided by co-transfection with a suitable vector, a helper virus is not required for the production of recombinant retrovirus.

[0215] Retroviral genomes and proviral DNA have three genes: gag, pol and env, flanked by two long terminal repeat (LTR) sequences. The gag gene codes for the internal structural (matrix, capsid and nucleocapsid) proteins, and the env gene codes for the viral envelope glycoproteins. The pol gene codes for products including the RNA-dependent DNA polymerase reverse transcriptase, which transcribes viral RNA into double-stranded DNA, integrase, which integrates the DNA produced by reverse transcriptase into the host chromosomal DNA, and protease, which acts to process the encoded gag and pol genes. The 5' and 3' LTRs serve to facilitate transcription and polyadenylation of virion RNA. The LTRs contain all other cis-acting sequences necessary for viral replication.

[0216] Retroviral vectors are described in Coffin et al., Retroviruses, Cold Spring Harbor Laboratory Press (1997). Exemplary retroviruses are Moloney murine leukemia virus (MMLV) or murine stem cell virus (MSCV). Retroviral vectors can be replication-competent or replication-defective. Typically, retroviral vectors are replication-defective, with the coding regions of genes required for further virion replication and packaging rounds deleted or replaced by other genes. As a result, the virus cannot continue its normal lytic pathway once the initial target cell is infected. Such retroviral vectors and the necessary agents (e.g., packaging cell lines) to produce such viruses are commercially available (see, for example, retroviral vectors and systems available from Clontech, e.g., catalog numbers 634401, 631503, 631501, etc., Clontech, Mountain View, Calif.).

[0217] Such retroviral vectors can be produced as delivery agents by replacing the viral genes required for replication with the nucleic acid molecule to be delivered. The resulting genome contains an LTR at each end with the desired gene or genes in between. Methods for producing retroviruses are known to those skilled in the art (see, for example, WO1995 / 26411). Retroviral vectors can be produced in packaging cell lines containing one or more helper plasmids. The packaging cell lines provide the viral proteins required for vector capsid production and virion maturation (e.g., gag, pol and env genes). Typically, at least two separate helper plasmids (containing gag and pol genes separately; and env gene separately) are used to prevent recombination between vector plasmids. For example, the retroviral vector can be transferred into the packaging cell line using standard transfection methods, such as calcium phosphate-mediated transfection. Packaging cell lines are well known to those skilled in the art and are commercially available. An exemplary packaging cell line is the GP2-293 packaging cell line (Catalog No. 631505, 631507, 631512, Clontech). After sufficient time for virion production, the virus is harvested. If desired, the harvested virus can be used to infect a second packaging cell line, for example, to produce viruses with different host tropisms. The end result is a replication-incompetent recombinant retrovirus that contains the nucleic acid of interest but lacks other structural genes so that new viruses cannot be formed in the host cell.

[0218] References illustrating the use of retroviral vectors in gene therapy include Clowes et al., Clin. Invest. 1994;93:644-651; Kiem et al., Blood 1994;83:1467-1473; Salmons and Gunzberg, Human Gene Therapy 1993;4:129 141; Grossman and Wilson, Curr. Opin. in Genetics and Devel. 1993;3:110-114; Sheridan, Nature Biotechnology 2011;29:121; Cassani et al., Blood 2009;114:3546-3556.

[0219] d. Lentiviral vectors Lentiviruses are a subclass of retroviruses. Exemplary lentiviruses are human immunodeficiency virus (HIV), simian immunodeficiency virus (SIV), and feline immunodeficiency virus (FIV). Unlike other retroviruses, lentiviruses are capable of integrating into the genome of non-dividing cells. Thus, for example, lentivirus vectors have been reported to efficiently and permanently deliver genes to primary liver cells and integrate into the genome of non-dividing primary liver cells (Lewis and Emerman, J. Virol. 1994;68:510-6). Lentivirus vectors also do not suffer from the same transcriptional silencing mechanism as MMLV retroviral vectors. Lentiviruses differ from other retroviruses in that they have karyophilic determinants contained in some virion proteins, such as matrix or VPR, which interact with the nuclear import machinery and mediate the active transport of viral preintegration complexes through the nucleopore. Therefore, lentivirus integration into the genome of host cells is not dependent on cell division.

[0220] Like other retroviruses, lentiviruses contain the gag, pol and env genes, which are the main genes that code for viral proteins. In addition, there are other accessory genes (e.g., Tat and Rev in HIV) that are involved in the regulation of synthesis, processing of viral RNA and other replication functions. These are flanked by two long terminal repeat (LTR) sequences. The replication cycle is initiated by the binding of viral glycoproteins to the host cell receptor, membrane fusion and viral entry into the cell. Upon entry, the virus uncoats and reverse transcription occurs, resulting in the formation of the preintegration complex (PIC). Other accessory genes play a role in the formation of the PIC and in the ability of lentiviruses to infect non-dividing cells by actively entering the nucleus of the cell through the nuclear envelope via the PIC. Once the provirus enters the nuclear envelope, it integrates itself into the host genome.

[0221] Exemplary lentiviral vectors are based on HIV-1, HIV-2, SIV or FIV. To make a safe lentiviral vector, a packaging cell line is made that contains several plasmid vectors, such as a four-plasmid vector system. For example, the first plasmid contains accessory proteins (e.g., tat, brf, vpr and nef) that are deleted to contain only promoter, gag and pol and the Psi packaging sequence that allows transcribed viral RNA to be incorporated into the assembly of new viruses, the second plasmid contains reverse transcriptase, the third plasmid contains the env gene replaced by vesicular stomatitis virus envelope protein (VSV-G), and the fourth plasmid is the vector of interest in which the viral genes required for replication are replaced by the nucleic acid molecule to be delivered.

[0222] Such lentiviral vectors, as well as systems and methods for producing lentiviruses, are known in the art (see, for example, Buchshacher and Wong-Staal, Blood 2000; 95:2499-2504; Blomer et al., J. Virol. 1997; 71:6641-9; Choi et al., Stem Cells 2001; 19:236-46; U.S. Patent No. 6,218,186). Lentiviral vectors are replication-deficient and do not contain genes required for replication. To produce lentivirus, several packaging plasmids are transfected into packaging cell lines, generally derivatives of HEK 293 or other similar cell lines (e.g., 293FT cells, catalog number R700-07, Invitrogen, Life Technologies, Carlsbad, Calif.); 293LTV cell line, catalog number LTV-100, Cell Biolabs, Inc., San Diego, Calif.; Lenti Pac 293Ta cell line, catalog number CLV-PK-01, GeneCo poeia, Rockville, Md.). The packaging plasmids separately code for virion proteins (e.g., capsid and reverse transcriptase) and the nucleic acid molecule delivered by the vector (which can be transfected into the packaging cell line). The single-stranded RNA viral genome is transcribed and packaged into virions. Methods for making lentiviral vectors are well known to those skilled in the art (see, for example, Naldine et al., Science 1996;272:263-267). Lentiviral vectors and virus production systems are commercially available (see, for example, lentiviral expression vectors, such as the pSMPUW lentiviral vector and its derivatives available from Cell Biolabs, Inc., and lentiviral expression and packaging systems).

[0223] Lentiviral vectors have been used for gene therapy applications (see, e.g., Manilla et al., Human Gene Therapy 2005;16:17-25; Sheridan, Nature Biotechnology 2011;29:121). In particular, lentiviral vectors have been used to deliver small interfering RNA (siRNA) (Sachdeva et al., Journal of Medical Virology 2007;79:118-26).

[0224] C. Cryopreservation and Storage In some aspects, provided herein is a method for producing a skin substitute, comprising: 1) differentiating keratinocytes into stratified epidermis, the stratified epidermis comprising basal layer, spinous layer, granular layer, and stratum corneum; and 2) introducing a polynucleotide into cells of the stratified epidermis to produce a skin substitute, the skin substitute comprising growth factors and insulin. In some aspects, provided herein is a method for producing a skin substitute, comprising: 1) differentiating keratinocytes into stratified epidermis, the stratified epidermis comprising basal layer, spinous layer, granular layer, and stratum corneum; and 2) transducing cells of the stratified epidermis with a viral vector comprising a polynucleotide to produce a skin substitute, the skin substitute comprising growth factors and insulin. In some embodiments, the methods provided herein further comprise a method for cryopreserving and storing a skin substitute comprising a stratified epidermis, the cells of which produce, e.g., secrete, growth factors and insulin. In some aspects, the methods provided herein further include performing a quality control assessment on the skin substitute prior to cryopreservation and storage.

[0225] In some embodiments, the skin substitute comprising a stratified epidermis is cryopreserved. In some embodiments, the skin substitute comprising a stratified epidermis is formulated with a cryoprotectant prior to cryopreservation. In some embodiments, the cryoprotectant comprises albumin and a monosaccharide. In some embodiments, the cryoprotectant comprises human albumin and glucose, e.g., D-glucose. In some embodiments, the cryoprotectant does not comprise DMSO.

[0226] In some embodiments, quality control evaluation is performed before the skin substitute comprising stratified epidermis is formulated with cryoprotectant.Quality control evaluation can include, but is not limited to, identification and / or detection of certain proteins, such as markers of epidermal differentiation, evaluation of potency, and evaluation of purity.Quality control evaluation can also include evaluation of sterility and safety.

[0227] In some embodiments, the quality control assessment comprises identifying and / or detecting genes associated with the viral genome of the vector used for transduction. In some embodiments, the quality control assessment comprises identifying and / or detecting genes associated with the adenoviral vector. In some embodiments, adenoviral genes are identified and / or detected to distinguish between replication-incompetent and replication-competent viruses. In some embodiments, the quality control assessment comprises identifying and / or detecting the levels of adenoviral genes E1, E4. In some embodiments, the quality control assessment comprises identifying and / or detecting the levels of molecules associated with the skin substitute, such as recombinant growth factors, recombinant insulin, filaggrin, laminin and transglutaminase, using methods known to those skilled in the art, such as PCR, e.g., qPCR, and / or by immunohistochemical staining.

[0228] In some embodiments, the quality control assessment includes detecting molecules secreted from the skin substitute, such as recombinant growth factors and C-peptide, such as by using ELISA. In some embodiments, the quality control assessment includes evaluating the potency of recombinant growth factors and recombinant insulin secreted from the skin substitute, such as by using an angiogenesis assay, such as an endothelial tube formation assay. In some embodiments, the quality control assessment includes evaluating the purity, such as the purity of bovine collagen, using methods known to those skilled in the art. In some embodiments, the quality control assessment includes evaluating sterility, such as by methods known to those skilled in the art, such as by detecting endotoxin using PCR. In some embodiments, all components of the skin substitute as provided herein are screened for infectious agents. In some embodiments, keratinocytes, such as HaCaT, master cell banks are screened for tumorigenicity and chromosomal abnormalities.

[0229] In some embodiments, packaging or storing the cryopreserved skin substitute includes the use of an absorbent material such as a dressing, e.g., absorbent gauze, where the cryopreserved skin substitute is placed on the dressing. In some embodiments, the size of the cryopreserved skin substitute placed on the dressing, e.g., gauze, is about 30-55 cm. 2 , about 30~50cm 2 , about 35~45cm 2 , about 40~50cm 2 , about 40~45cm 2 , or about 45-50cm 2 In some embodiments, the size of the cryopreserved skin substitute placed on the dressing, e.g., gauze, is 30 cm 2 Or about 30cm 2 , 31cm 2 Or about 31 cm 2 , 32cm 2 Or about 32cm 2 , 33cm 2 Or about 33cm 2 , 34cm 2 Or about 34cm 2 , 35cm 2Or about 35cm 2 , 36cm 2 Or about 36cm 2 , 37cm 2 Or about 37cm 2 , 38cm 2 Or about 38cm 2 , 39cm 2 Or about 39cm 2 , 40cm 2 Or about 40cm 2 , 41cm 2 Or about 41cm 2 , 42cm 2 Or about 42cm 2 , 43cm 2 Or about 43cm 2 , 44cm 2 Or about 44cm 2 , 45cm 2 Or about 45cm 2 , 46cm 2 Or about 46cm 2 , 47cm 2 Or about 47cm 2 , 48cm 2 Or about 48cm 2 , 49cm 2 Or about 49cm 2 , or 50cm 2 Or about 50cm 2 It is.

[0230] In some embodiments, packaging or storing the cryopreserved skin substitute includes the use of absorbent gauze, e.g., where the cryopreserved skin substitute is placed on the absorbent gauze. In some embodiments, the dressing includes absorbent gauze. In some embodiments, the absorbent gauze is Vaseline petrolatum gauze. In some embodiments, the dressing, e.g., gauze, on which the skin substitute is placed is about 40-60 cm in size. 2 , about 45~60cm 2 , about 45~55cm 2 , or about 50-60cm 2 In some embodiments, the size of the dressing, e.g., gauze, on which the cryopreserved skin substitute is placed is 40 cm 2Or about 40cm 2 , 41cm 2 Or about 41cm 2 , 42cm 2 Or about 42cm 2 , 43cm 2 Or about 43cm 2 , 44cm 2 Or about 44cm 2 , 45cm 2 Or about 45cm 2 , 46cm 2 Or about 46cm 2 , 47cm 2 Or about 47cm 2 , 48cm 2 Or about 48cm 2 , 49cm 2 Or about 49cm 2 , 50cm 2 Or about 50cm 2 , 51cm 2 Or about 51cm 2 , 52cm 2 Or about 52cm 2 , 53cm 2 Or about 53cm 2 , 54cm 2 Or about 54cm 2 , 55cm 2 Or about 55cm 2 , 56cm 2 Or about 56cm 2 , 57cm 2 Or about 57cm 2 , 58cm 2 Or about 58cm 2 , 59cm 2 Or about 59cm 2 , or 60cm 2 Or about 60cm 2 It is.

[0231] In some embodiments, the ratio of the size of the dressing, e.g., absorbent gauze, to the size of the cryopreserved skin substitute placed on the dressing is about 1:1 to about 1.5: 1. In some embodiments, the ratio of the size of the dressing, e.g., absorbent gauze, to the size of the cryopreserved skin substitute placed on the dressing is about 1:1, about 1.1:1, about 1.2:1, about 1.3:1, about 1.4:1, or about 1.5:1.

[0232] In some embodiments, the cryopreserved skin substitute is packaged or stored in a container. In some embodiments, the cryopreserved skin substitute, placed on a sterile dressing, is packaged or stored in a container. In some embodiments, the container is sterile. In some embodiments, the container is sealed using heat, e.g., heat sealable or heat sealed. In some embodiments, the container is sterile and heat sealable or heat sealed. In some embodiments, the container is transparent. In some embodiments, the container comprises a polyester resin. In some embodiments, the container is a bag. In some embodiments, the skin substitute packaged or stored in the container is contained in a package. In some embodiments, one or both of the container and the package are sterile and / or heat sealable. In some embodiments, the cryopreserved skin substitute, optionally stored in a container, can be stored at about -20°C, -30°C, -40°C, -50°C, -60°C, -70°C, or -80°C.

[0233] In some embodiments, the cryopreserved skin substitute can be stored for up to 6 months. In some embodiments, the cryopreserved skin substitute can be stored for up to 6 months and retain the function of providing, e.g., secreting, an effective amount of recombinant growth factors and recombinant insulin. In some embodiments, the cryopreserved skin substitute can be stored for up to 5 months and retain the function of providing, e.g., secreting, an effective amount of recombinant growth factors and recombinant insulin. In some embodiments, the cryopreserved skin substitute can be stored for up to 4 months and retain the function of providing, e.g., secreting, an effective amount of recombinant growth factors and recombinant insulin. In some embodiments, the cryopreserved skin substitute can be stored for up to 3 months and retain the function of providing, e.g., secreting, an effective amount of recombinant growth factors and recombinant insulin.

[0234] IV. Methods of Using Skin Substitutes In some aspects, provided herein is a method of using a skin substitute comprising stratified epidermis, the cells of which produce, e.g., secrete, growth hormone and insulin or variants thereof. In some embodiments, the skin substitute provided herein can be applied to a subject, e.g., a human subject, to improve wound conditions and / or promote wound healing. In some embodiments, the skin substitute provided herein can be applied to a diabetic subject to promote wound healing and / or prevent microbial infection of the wound.

[0235] A. Advanced glycation end products In some embodiments, the skin substitute as provided herein can be used to improve the condition associated with advanced glycation end products (AGEs), such as impaired or prolonged wound healing.The elevated concentration of reducing sugars (e.g., glucose) in the blood and intracellular environment of animals, i.e., humans, usually leads to the non-enzymatic formation of glycation and dehydration condensation complexes AGEs.AGE complex products are formed on free amino groups, on proteins, on lipids and on DNA (Bucala and Cerami, Adv Pharmacol 1992;23:1-34; Bucala et al., Proc Natl Acad Sci 1993;90:6434-6438; Bucala et al., Proc Natl. Acad Sci 1984;81:105-109). In one example, AGE levels in diabetic patients are significantly increased as a result of persistently high blood glucose levels, frequently leading to tissue damage through a variety of mechanisms, including alteration of tissue protein structure and function, stimulation of cellular responses through AGE-specific receptors, and / or generation of reactive oxygen species (ROS) (Boel et al., J Diabetes Complications 1995;9:104-29). AGEs have been shown to cause complications in patients with diabetes mellitus who experience wounds, such as micronicks, cuts, burns, sores, ulcers, abscesses, and / or any other form of bodily injury. In some embodiments, the growth factors and insulin secretable from the skin substitute as provided herein can promote angiogenesis and reduce the amount of AGEs in the skin of a subject without affecting systemic glucose levels.

[0236] Accumulation of AGEs may lead to poor skin function, especially as it relates to wound healing in diabetic patients (Putte et al., Scars Burn Heal. 2016; 5(2):1-14). AGEs are involved in delayed or incomplete wound healing of diabetic subjects' skin (Peppa et al., Diabetes 2003; 52(11):2805-2813) and even bone (Santana et al., Diabetes 2003;52(6):1502-10). In delayed or prolonged wound healing, the time it takes for the wound to heal, as indicated by wound closure, in some cases exceeds the time to heal observed in healthy or non-diabetic subjects. In some embodiments, the skin substitutes as provided herein can reduce wound healing time (e.g., time to wound closure) in diabetic subjects to a comparable time required to achieve the same effect in non-diabetic subjects.

[0237] In some cases, defective wound healing refers to abnormalities in epithelial organization during the wound healing process, which can include a reduced ability to form scars. Scar formation is a major part of wound healing. Scars are areas of fibrous tissue that form during the wound healing process in place of the normal skin that existed before wound formation. Scars exhibit altered extracellular matrix and have reduced elastin fiber levels compared to normal skin. In healthy skin, nearly all wounds result in some degree of scarring. In some aspects, the skin substitutes as provided herein can induce scar formation, thereby facilitating the wound healing process.

[0238] Wounds can be described as acute or chronic. Acute wounds are usually the result of injury to the skin that occurs suddenly rather than over time (e.g., surgical or traumatic wounds). In normal subjects, acute wounds usually heal at a predictable and expected rate according to the normal wound healing process. In contrast, chronic wounds are wounds in which the stages of wound healing do not progress in an orderly and timely manner (e.g., do not show substantial progress toward healing at 30 days). In some embodiments, the skin substitutes as provided herein can be applied to acute wounds. In some embodiments, the skin substitutes as provided herein can be applied to chronic wounds. Non-limiting examples of chronic wounds include venous stasis ulcers, diabetic foot ulcers, and decubitus ulcers. Non-healing wounds are a significant health care burden. Non-healing wounds can lead to prolonged hospital stays, reduced quality of life, increased risk of mortality, need for amputation, and increased likelihood of discharge to long-term care facilities.

[0239] B. Diabetic Foot Wounds and Infections Diabetic foot wounds, including ulcers, erosions, lesions and / or abscesses, result from peripheral neuropathy and vascular insufficiency, two common diabetic complications. Diabetic foot, hereafter referred to as diabetic foot, is a condition with high morbidity and adversely affects patients' quality of life, in part due to frequent hospitalizations, prolonged hospital stays and amputations (Alosaimi et al., Journal of Foot and Ankle Research 2019; 12:57). It is estimated that one in ten people diagnosed with type 2 diabetes (diabetes mellitus) have risk factors for foot disease (Boulton et al., Lancet 2005; 366(9498):1719-24).

[0240] Approximately 15%-25% of all diabetic patients will develop foot or leg ulcers during the course of their disease (Boulton et al., Lancet 2005; 366(9498):1719-24, and Pinzur and Dart, Foot Ankle Clin. 2001;6(2):205-142005). Risk factors include long duration of diabetes mellitus (>10 years), age (>50 years), history of ulcer or amputation, presence of neuropathy, arthropathy or vascular disease, presence of other diabetic complications, low socioeconomic status and / or social isolation of the patient, poor diet, inadequate education in foot care, as well as other risk factors associated with vascular disease (Nongmaithem et al., J Family Med Prim Care 2016;5(2):399-403).

[0241] The diagnosis of diabetic foot conveys the risk of injury and / or amputation. Wounds or lesions pose a threat, especially to high-risk patients, including smokers and those with a history of vascular complications of the lower extremities. Early detection of diabetic foot is performed through foot examination, including the use of monofilaments, palpation, visual and sensory testing. If preventive measures fail and injury occurs or a high-risk foot is identified, multidisciplinary management should be implemented, especially in cases with a history of ulcers or amputations in other extremities.

[0242] The Wagner classification (grading lesions on a scale of 0 to 5, with 5 indicating the most severe disease) is widely used to determine management strategies for neuropathic diabetic foot. The primary goal of treatment is wound closure. Initial low-severity management may include rest, foot elevation, and oral antibiotic treatment as necessary. If no response to these measures is observed, patients should be referred for more aggressive interventions. For subjects with lesions that do not respond to initial treatment and / or present with severe (severe) wounds and / or infection, surgical intervention and / or intravenous antibiotic treatment are recommended (Frykberg, Am Fam Physician. 2002;66(9):1655-1663).

[0243] Table 1. Wagner's Diabetic Foot Ulcer Grading System TIFF2024531827000006.tif41165 * Quoted from Frykberg, Am Fam Physician. 2002;66(9):1655-1663

[0244] Currently available skin substitutes or skin equivalents are of a variety of different compositions, such as compositions that contain keratinocyte stem cells, normal immortalized keratinocytes (NIKS) and / or human fibroblasts, and structures, such as structures in the form of epidermal layers or structures in the form of dermis and epidermal layers. These skin substitutes can secrete a wide range of different molecules, such as growth factors, collagen and / or extracellular matrix proteins. For example, Dermagraft® and Apligraf® are skin substitutes that have been approved by the FDA to treat wounds in diabetic patients, such as diabetic foot ulcers and / or venous leg ulcers. However, currently available skin substitutes have many limitations, including the need for multiple applications with high costs. In some embodiments, the skin substitutes as provided herein can reduce wound healing time in diabetic subjects to the equivalent time required to achieve the same effect in non-diabetic subjects with only one application. In some embodiments, the skin substitutes as provided herein can reduce wound healing time in diabetic subjects to the equivalent time required to achieve the same effect in non-diabetic subjects with only two applications. In some embodiments, the skin substitutes as provided herein can reduce wound healing time in diabetic subjects to the equivalent time required to achieve the same effect in non-diabetic subjects with only three applications. In some embodiments, the skin substitutes as provided herein can reduce wound healing time in diabetic subjects to the equivalent time required to achieve the same effect in non-diabetic subjects with only four applications.

[0245] In some embodiments, the skin substitute as provided herein is applied to the skin or wound of a subject. In some embodiments, the skin substitute as provided herein is placed on an absorbent dressing, such as gauze, and is applied to the skin or wound of a subject. In some embodiments, the skin substitute as provided herein can be replaced once every 10 days, once every 11 days, once every 12 days, once every 13 days, once every 14 days, once every 15 days, once every 16 days, once every 17 days, once every 18 days, once every 19 days, once every 20 days, or once every 21 days. In some embodiments, the skin substitute as provided herein can be left applied to a subject without replacement for at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days, at least 16 days, at least 17 days, at least 18 days, at least 19 days, at least 20 days, or at least 21 days.

[0246] In some embodiments, the skin substitutes as provided herein can provide sustained release of recombinant growth factors and recombinant insulin. In some embodiments, the sustained release of recombinant growth factors and recombinant insulin from the skin substitutes as provided herein lasts for up to or about 2 days, up to or about 3 days, up to or about 4 days, up to or about 5 days, up to or about 6 days, up to or about 7 days, up to or about 8 days, up to or about 9 days, up to or about 10 days, up to or about 11 days, up to or about 12 days, up to or about 13 days, or up to or about 14 days. In some embodiments, the sustained release of recombinant growth factors and recombinant insulin from the skin substitutes as provided herein lasts for up to or about 1 week, up to or about 2 weeks, up to or about 3 weeks, up to or about 1-2 weeks, or up to or about 2-3 weeks.

[0247] The microbiota of the skin is composed of various viruses, bacteria and fungi, and wounds of diabetic subjects are particularly susceptible to infection. The presence of microorganisms alone is not an indicator of infection. Infection must be clinically diagnosed by the presence of systemic signs (e.g., fever, chills and leukocytosis), pus or secretions, or local inflammatory symptoms (e.g., heat, redness, pain or tenderness, and induration).

[0248] In addition to complicating the diagnosis of infection, chronic wounds may further exhibit delayed healing, abnormal coloration, fragility or foul odor. Infection should be suspected at the first appearance of foot problems and when systemic infection or metabolic disturbances become evident. Vascular abnormalities, including peripheral neuropathy or ischemia, may mask or mimic inflammation (Lipsky, Clinical Infectious Diseases 2004;39:S104-14). Interestingly, signs of systemic toxicity, such as sepsis, systemic inflammatory response syndrome (SIRS) or multiple organ dysfunction syndrome (MODS), have been reported only rarely in diabetic foot infections. However, when SIRS is evident, for example, diabetic foot infections are likely to become life-threatening as well as lower limb threatening (Lin et al., J. Clin. Med. 2019;8(10):1538). Any suspected infection should be aggressively pursued, especially considering how quickly the severity can spread, sometimes within hours.

[0249] In one example, diabetic foot wounds can result in a wide range of infections, from superficial to severe, spreading to the deeper layers of the skin and / or bone. Infection is a risk factor for surgical intervention, which can include minor foot-sparing surgery or major surgery such as amputation. By some estimates, 60% of amputations follow the development of infected foot ulcers (Lipsky, Clinical Infectious Diseases 2004; 39:S104-14). Methods for preventing infection are of paramount importance given the serious impact on patients. Furthermore, prevention avoids the need for antibiotic treatment, which is expensive and can lead to off-target effects that are harmful to patients.

[0250] In some embodiments, a skin substitute as provided herein can be used to prevent microbial infection. In some embodiments, a skin substitute as provided herein can be used to prevent bacterial infection. In some embodiments, a skin substitute as provided herein can be used to prevent viral infection. In some embodiments, a skin substitute as provided herein can be used to prevent microbial infection of one or more wounds of a subject. In some embodiments, a skin substitute as provided herein can be used to prevent bacterial infection of one or more wounds of a subject. In some embodiments, a skin substitute as provided herein can be used to prevent viral infection of one or more wounds of a subject. In some embodiments, a skin substitute as provided herein can be used to prevent microbial infection of one or more wounds of a diabetic subject. In some embodiments, a skin substitute as provided herein can be used to prevent bacterial infection of one or more wounds of a diabetic subject. In some embodiments, a skin substitute as provided herein can be used to prevent viral infection of one or more wounds of a diabetic subject.

[0251] V. Illustrative Embodiments Aspects provided include: 1. A skin substitute comprising a stratified epidermis including a stratum basale, a stratum spinosum, a stratum granulosum, and a stratum corneum, the cells of the stratified epidermis express recombinant growth factors and recombinant insulin; The skin substitute. 2. The skin substitute of embodiment 1, wherein recombinant growth factors and recombinant insulin are secretable from cells of the stratified epidermis. 3. The skin substitute of embodiment 1 or embodiment 2, wherein the stratified epidermis has a thickness of 100 to 200 μm. 4. The skin substitute of any of aspects 1-3, wherein the cells of the stratified epidermis that express recombinant growth factors and recombinant insulin comprise cells of the stratum basale. 5. The skin substitute of any of aspects 1-4, wherein the recombinant insulin is or comprises recombinant human insulin. 6. Recombinant insulin is (i) the amino acid sequence shown in SEQ ID NO:5; (ii) a functional variant having an amino acid sequence having at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO:5; or (iii) a two-chain form of (i) or (ii) comprising an A chain and a B chain having Optionally, the A chain and the B chain are linked by a disulfide bond. The skin substitute of any one of embodiments 1 to 5. 7. Recombinant insulin is (i) the amino acid sequence set forth in SEQ ID NO:5; (ii) a functional variant having an amino acid sequence that has at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO:5. The skin substitute of any of embodiments 1 to 6, wherein the skin substitute is encoded by a polynucleotide encoding the 8. The skin substitute of any of aspects 1-7, wherein the recombinant insulin is an AspB10 insulin analog that includes a histidine to aspartic acid mutation at position 10 in the B chain of modified human proinsulin compared to the wild-type insulin shown in SEQ ID NO:5. 9. The skin substitute of any of aspects 1-8, comprising a polynucleotide encoding a proinsulin that includes at least one furin recognition sequence in place of the endopeptidase Arg31-Arg32 cleavage site or the endopeptidase Lys64-Arg65 cleavage site. 10. The skin substitute of embodiment 9, wherein at least one furin recognition sequence is in place of the endopeptidase Arg31-Arg32 cleavage site and the endopeptidase Lys64-Arg65 cleavage site. 11. At least one furin recognition sequence has the consensus sequence: RXRR (SEQ ID NO:8), where X is any amino acid; or RXKR (SEQ ID NO:9), where X is any amino acid. 11. The skin substitute of embodiment 9 or embodiment 10, comprising: 12. The skin substitute of any of aspects 9-11, wherein at least one furin cleavage site is RTKR (SEQ ID NO:10) or RQKR (SEQ ID NO:42). 13. Recombinant insulin is (i) the amino acid sequence shown in SEQ ID NO:6; (ii) a functional variant having an amino acid sequence having at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO:6; or (iii) a two-chain form of (i) or (ii) comprising an A chain and a B chain having Optionally, the A chain and the B chain are linked by a disulfide bond. The skin substitute of any one of embodiments 1 to 12. 14. The skin substitute of any of aspects 1-13, wherein the recombinant insulin comprises the sequence shown in SEQ ID NO:6, or a two-chain form of (i) or (ii) comprising an A chain and a B chain, optionally wherein the A chain and the B chain are linked by a disulfide bond. 15. The skin substitute of any of aspects 1-14, wherein the recombinant insulin comprises an A chain as shown in SEQ ID NO:36 and a B chain as shown in SEQ ID NO:41, and optionally, the A chain and the B chain are linked by a disulfide bond. 16. The skin substitute of any of aspects 1-15, wherein the recombinant insulin is encoded by a polynucleotide comprising a sequence having at least 85%, or at least about 85%, at least 90%, or at least about 90%, or at least 95%, or at least about 95% sequence identity to the sequence set forth in SEQ ID NO:2. 17. The skin substitute of any of aspects 1-16, wherein the recombinant insulin comprises the sequence shown in SEQ ID NO:2. 18. The skin substitute of any of aspects 1-17, wherein the recombinant growth factor is selected from the group consisting of epidermal growth factor (EGF), erythropoietin (EPO), fibroblast growth factor (FGF), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), hepatocyte growth factor (HGF), insulin-like growth factor (IGF), platelet-derived growth factor (PDGF), transforming growth factors alpha and beta, vascular endothelial growth factor (VEGF), epidermal growth factor receptor (EGFR), fibroblast growth factor receptor (FGFR), and any isoforms or alternatively spliced ​​variants thereof. 19. The skin substitute of any of aspects 1-5, wherein the recombinant growth factor is VEGF or an isoform or alternatively spliced ​​variant thereof. 20. The skin substitute of embodiment 19, wherein the VEGF is encoded by a polynucleotide sequence having at least 85% or at least about 85%, at least 90% or at least about 90%, or at least 95% or at least about 95% sequence identity to the sequence shown in SEQ ID NO:4. 21. The skin substitute of embodiment 19 or embodiment 20, wherein VEGF is encoded by a polynucleotide sequence comprising the sequence shown in SEQ ID NO:4. 22. VEGF, A sequence having at least 85%, or at least about 85%, at least 90%, or at least about 90%, or at least 95%, or at least about 95% sequence identity to the sequence shown in SEQ ID NO:7; or Its sequence lacks the signal peptide 22. The skin substitute of any of embodiments 19 to 21, comprising: 23. VEGF, The sequence shown in SEQ ID NO:7, or Its sequence lacks the signal peptide 23. The skin substitute of any of embodiments 19 to 22, comprising: 24. The skin substitute of any of aspects 19-23, wherein the VEGF comprises a sequence having at least 85% or at least about 85%, at least 90% or at least about 90%, or at least 95% or at least about 95% sequence identity to the sequence shown in SEQ ID NO:44. 25. The skin substitute of any of aspects 19-24, wherein the VEGF comprises the sequence shown in SEQ ID NO:44. 26. The skin substitute of any of aspects 1-25, wherein the recombinant growth factor and recombinant insulin are encoded by a bicistronic expression cassette comprising a polynucleotide encoding the recombinant growth factor and a polynucleotide encoding recombinant insulin separated by a bicistronic element. 27. The skin substitute of embodiment 26, wherein the bicistronic element is an IRES. 28. The skin substitute of any of aspects 1-27, wherein the polynucleotide encoding the recombinant growth factor and the polynucleotide encoding recombinant insulin are operably linked to a promoter. 29. The skin substitute of embodiment 28, wherein the promoter is a constitutive promoter or an inducible promoter. 30. The skin substitute of embodiment 28 or embodiment 29, wherein the promoter is a CAG promoter. 31. The skin substitute of any of aspects 26-30, wherein the polynucleotide encoding the recombinant growth factor is upstream of the polynucleotide encoding recombinant insulin in a bicistronic expression cassette. 32. The skin substitute of any of aspects 1-31, wherein cells of the stratified epidermis secrete recombinant growth factors and recombinant insulin at levels that result in greater improvement in one or more markers of vascularization reconstitution compared to skin substitutes comprising either the recombinant growth factors or recombinant insulin alone, optionally as assessed in a tube formation assay. 33. The skin substitute of embodiment 32, wherein the marker of vascularization remodeling is an increase in the number of nodes or unions, defined as attachment sites of at least three chords. 34. The skin substitute of embodiment 32, wherein a marker of vascularization remodeling is an increase in the number of webs, defined as closed circuits surrounded by two or more nodes. 35. The skin substitute of embodiment 32, wherein a marker of vascularization remodeling is an increase in the number of main segments, defined as a cord connecting two nodes together. 36. The skin substitute of any of aspects 1-35, wherein the cells of the stratified epidermis continuously secrete quantifiable levels of recombinant growth factors and recombinant insulin. 37. The skin substitute of any of embodiments 1-36, wherein the cells of the stratified epidermis secrete recombinant growth factors and recombinant insulin continuously for up to or about 2 days, up to or about 3 days, up to or about 4 days, up to or about 5 days, up to or about 6 days, up to or about 7 days, up to or about 8 days, up to or about 9 days, up to or about 10 days, up to or about 11 days, up to or about 12 days, up to or about 13 days, or up to or about 14 days. 38. The skin substitute of any of aspects 1-37, wherein the cells of the stratified epidermis continuously secrete recombinant growth factors and recombinant insulin for up to or about 1 week, up to or about 2 weeks, up to or about 3 weeks, up to or about 1-2 weeks, or up to or about 2-3 weeks. 39. The skin substitute of any of embodiments 1-38, wherein the cells of the stratified epidermis secrete quantifiable levels of recombinant growth factors and C-peptide that are detectable for up to or about 2 days, up to or about 3 days, up to or about 4 days, up to or about 5 days, up to or about 6 days, up to or about 7 days, up to or about 8 days, up to or about 9 days, up to or about 10 days, up to or about 11 days, up to or about 12 days, up to or about 13 days, or up to or about 14 days. 40. The skin substitute of any of aspects 1-39, wherein the cells of the stratified epidermis secrete quantifiable levels of recombinant growth factors and C-peptide that are detectable for up to or about 1 week, up to or about 2 weeks, up to or about 3 weeks, up to or about 1-2 weeks, or up to or about 2-3 weeks. 41. The skin substitute of any of aspects 1-40, wherein the cells of the stratified epidermis secrete levels of recombinant growth factors and recombinant insulin that reduce levels of advanced glycation end products (AGEs) in the subject's skin. 42. The skin substitute of any of aspects 1 to 41, wherein the cells of the stratified epidermis are cells differentiated from keratinocytes. 43. The skin substitute of embodiment 42, wherein the keratinocytes are human keratinocytes. 44. The skin substitute of embodiment 42 or embodiment 43, wherein the keratinocytes are HaCaT keratinocyte cells. 45. A bicistronic expression cassette comprising a polynucleotide encoding a recombinant human growth factor and recombinant insulin. 46. ​​The bicistronic expression cassette of embodiment 45, wherein the recombinant insulin is or comprises recombinant human insulin. 47. The recombinant insulin encoded by: (i) having the amino acid sequence set forth in SEQ ID NO:5, or (ii) a functional variant having an amino acid sequence having at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO:5; The bicistronic expression cassette of embodiment 45 or embodiment 46. 48. A polynucleotide encoding recombinant insulin comprising: (i) comprising the amino acid sequence set forth in SEQ ID NO:5; or (ii) a functional variant having an amino acid sequence having at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO:5; 48. The bicistronic expression cassette of any of embodiments 45 to 47. 49. The bicistronic expression cassette of any of aspects 45-48, wherein the encoded recombinant insulin is an AspB10 insulin analogue comprising a histidine to aspartic acid mutation at position 10 in the B chain of modified human proinsulin compared to the wild-type insulin shown in SEQ ID NO:5. 50. The bicistronic expression cassette of any of aspects 45-49, wherein the polynucleotide encoding recombinant insulin encodes a proinsulin comprising at least one furin recognition sequence in place of the endopeptidase Arg31-Arg32 cleavage site or the endopeptidase Lys64-Arg65 cleavage site. 51. The bicistronic expression cassette of embodiment 50, wherein at least one furin recognition sequence is in place of the endopeptidase Arg31-Arg32 cleavage site and the endopeptidase Lys64-Arg65 cleavage site. 52. At least one furin recognition sequence has the consensus sequence: RXRR (SEQ ID NO:8), where X is any amino acid; or RXKR (SEQ ID NO:9), where X is any amino acid. 52. The bicistronic expression cassette of embodiment 50 or embodiment 51, comprising: 53. The bicistronic expression cassette of any of aspects 50-52, wherein at least one furin cleavage site is RTKR (SEQ ID NO:10) or RQKR (SEQ ID NO:42). 54. The recombinant insulin encoded by: (i) having the amino acid sequence set forth in SEQ ID NO:6, or (ii) a functional variant having an amino acid sequence having at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO:6; 54. The bicistronic expression cassette of any of embodiments 45 to 53. 55. The bicistronic expression cassette of any of aspects 45-54, wherein the encoded recombinant insulin comprises the sequence shown in SEQ ID NO:6. 56. The bicistronic expression cassette of any of aspects 45-55, wherein the polynucleotide encoding recombinant insulin comprises a sequence having at least 85%, or at least about 85%, at least 90%, or at least about 90%, or at least 95%, or at least about 95% sequence identity to the sequence shown in SEQ ID NO:2. 57. The bicistronic expression cassette of any of aspects 45-56, wherein the polynucleotide encoding recombinant insulin comprises the sequence shown in SEQ ID NO:2. 58. The bicistronic expression cassette of any of embodiments 45-57, wherein the encoded recombinant growth factor is selected from the group consisting of epidermal growth factor (EGF), erythropoietin (EPO), fibroblast growth factor (FGF), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), hepatocyte growth factor (HGF), insulin-like growth factor (IGF), platelet-derived growth factor (PDGF), transforming growth factors alpha and beta, vascular endothelial growth factor (VEGF), epidermal growth factor receptor (EGFR), fibroblast growth factor receptor (FGFR), and any isoforms or alternatively spliced ​​variants thereof. 59. The bicistronic expression cassette of any of aspects 45-58, wherein the recombinant growth factor is VEGF or an isoform or alternatively spliced ​​variant thereof. 60. The bicistronic expression cassette of embodiment 59, wherein the polynucleotide encoding the growth factor comprises a sequence having at least 85% or at least about 85%, at least 90% or at least about 90%, or at least 95% or at least about 95% sequence identity to the sequence shown in SEQ ID NO:4. 61. The bicistronic expression cassette of embodiment 59 or embodiment 60, wherein the polynucleotide encoding the growth factor comprises the sequence shown in SEQ ID NO:4. 62. The bicistronic expression cassette of any of embodiments 59-61, wherein the encoded VEGF comprises a sequence having at least 85%, or at least about 85%, at least 90%, or at least about 90%, or at least 95%, or at least about 95% sequence identity to the sequence shown in SEQ ID NO:7, or the sequence thereof lacking the signal peptide. 63. The bicistronic expression cassette of any of embodiments 59-62, wherein the encoded VEGF comprises the sequence shown in SEQ ID NO:7, or a sequence thereof lacking the signal peptide. 64. The bicistronic expression cassette of any of embodiments 59-63, wherein the encoded VEGF comprises a sequence having at least 85%, or at least about 85%, at least 90%, or at least about 90%, or at least 95%, or at least about 95% sequence identity to the sequence shown in SEQ ID NO:44. 65. The bicistronic expression cassette of any of embodiments 59-64, wherein the encoded VEGF comprises the sequence shown in SEQ ID NO:44. 66. The bicistronic expression cassette of any of aspects 45 to 65, wherein the polynucleotide encoding the recombinant growth factor and the polynucleotide encoding recombinant insulin are separated by a bicistronic element. 67. The bicistronic expression cassette of embodiment 66, wherein the bicistronic element is an IRES. 68. The bicistronic expression cassette of any of aspects 45 to 67, wherein the polynucleotide encoding the recombinant growth factor and the polynucleotide encoding recombinant insulin are operably linked to a promoter. 69. The bicistronic expression cassette of embodiment 68, wherein the promoters are the same. 70. The bicistronic expression cassette of embodiment 68 or embodiment 69, wherein the promoter is a constitutive promoter or an inducible promoter. 71. The bicistronic expression cassette of any of aspects 68-70, wherein the promoter is a CAG promoter. 72. The bicistronic expression cassette of any of aspects 68-71, wherein a polynucleotide encoding a recombinant growth factor is upstream of the polynucleotide encoding recombinant insulin in the bicistronic expression cassette. 73. A vector comprising the bicistronic expression cassette of any of aspects 45 to 72. 74. The vector of embodiment 73, which is a viral vector. 75. The vector of embodiment 74, wherein the viral vector is an adenoviral vector. 76. The vector of any of aspects 73 to 75, which is a non-replicating type 5 adenovirus. 77. The vector of any of aspects 73 to 76, wherein the non-replicating adenovirus lacks or is deleted in the E1 and E3 regions. 78. The vector of any of aspects 73 to 77, wherein a bicistronic expression cassette is inserted into the E1 region. 79. (1) Differentiating keratinocytes into stratified epidermis, the stratified epidermis including a basal layer, a spinous layer, a granular layer, and a stratum corneum; and (2) introducing into cells of said stratified epidermis a bicistronic expression cassette according to any one of embodiments 45 to 72 or a vector according to any one of embodiments 73 to 78 to produce a skin substitute, said skin substitute comprising a recombinant growth factor and recombinant insulin. A method for producing a skin substitute comprising: 80. The method of embodiment 79, wherein the introducing step is by transduction with a viral vector of any of embodiments 73 to 78. 81. (1) Differentiating keratinocytes into stratified epidermis, the stratified epidermis including a basal layer, a spinous layer, a granular layer, and a stratum corneum; and (2) transducing cells of the stratified epidermis with the viral vector of any of embodiments 73 to 78 to produce a skin substitute, the skin substitute comprising growth factors and insulin. A method for producing a skin substitute comprising: 82. (1) Differentiating keratinocytes into stratified epidermis, the stratified epidermis including a basal layer, a spinous layer, a granular layer, and a stratum corneum; and (2) transducing cells of the stratified epidermis with an adenoviral vector encoding modified proinsulin and growth factors to produce a skin substitute, wherein the skin substitute comprises the growth factors and insulin. A method for producing a skin substitute comprising: 83. The method of any of embodiments 79-81, wherein at the time of introducing or transducing, cells of the stratified epidermis express occludin and claudins. 84. The method of any of aspects 79 to 83, wherein the cells of the basal layer are transduced or transduced. 85. The method of any of aspects 79-84, comprising culturing the keratinocytes in low calcium medium for 2 weeks, 3 weeks, 4 weeks, 5 weeks, or 6 weeks, optionally for 4 weeks or about 4 weeks, prior to differentiation in step (1). 86. The method of embodiment 85, wherein the low calcium medium comprises a calcium concentration of 0.01 to 0.1 mM at the time of seeding the cells or during the culturing step. 87. The method of any of embodiment 85 or embodiment 86, wherein the low calcium medium comprises a calcium concentration of up to or about 0.05 mM at the time of seeding said cells or during said culturing step. 88. The method of any of aspects 85-87, wherein the low calcium medium comprises a calcium concentration that is about 0.03 mM at the time of seeding said cells or during said culturing step. 89. The method of any of aspects 85-88, wherein the low calcium medium further contains epidermal growth factor (EGF) and bovine pituitary extract (BPE). 90. The method of embodiment 89, wherein the low calcium medium comprises 0.05 ng / mL to 1 ng / ml of EGF and 1 μg / ml to 100 μg / ml of BPE at the time of seeding the cells or during the culturing step. 91. The method of embodiment 89 or embodiment 90, wherein the low calcium medium comprises 0.2 ng / ml or about 0.2 ng / ml EGF and 30 μg / ml or about 30 μg / ml BPE at the time of seeding said cells or during said culturing step. 92. The method of any of aspects 79 to 91, wherein the keratinocytes are human keratinocytes. 93. The method of any of aspects 79 to 92, wherein the keratinocytes are HaCaT keratinocyte cells. 94. The method of any of aspects 79-93, wherein step (1) comprises culturing keratinocytes on an extracellular matrix substrate. 95. The method of embodiment 94, wherein the extracellular matrix substrate is collagen. 96. The method of embodiment 94 or embodiment 95, wherein the extracellular matrix substrate is certified for human use. 97. Keratinocytes grow in a density of 5×10 on an extracellular matrix substrate. 6 / ml to 50 x 10 6 97. The method of any of embodiments 94 to 96, wherein the cells are seeded at a cell density of between 100 and 150 cells / ml. 98. The cell density is 10 x 10 6 pieces / ml, 20×10 6 pieces / ml, 30×10 6 / ml or 40 x 10 6 / ml, or approximately 10 x 10 6 pieces / ml, 20×10 6 pieces / ml, 30×10 6 / ml or 40 x 10 6 98. The method of embodiment 97, wherein the cell count is 100 / ml, 100 / ml, or any value therebetween. 99. The cell density is 20 x 10 6 / ml or approximately 20 x 10 6 The method of embodiment 97 or embodiment 98, wherein the number of cells / ml. 100. The method of any of embodiments 94-99, wherein the extracellular matrix substrate is coated onto a Transwell insert. 101. The method of any of aspects 94 to 99, wherein the culture in step (1) is for about 23 to 28 days. 102. The method of any of embodiments 94 to 100, wherein the culturing in step (1) comprises a first incubation in a low calcium medium and a second incubation in a high calcium medium. 103. The method of embodiment 101, wherein the first incubation in the low calcium medium is for about 3-5 days, and the second incubation in the high calcium medium is for about 20-23 days. 104. The method of embodiment 102 or embodiment 103, wherein the low calcium medium contains 0.01 to 0.1 mM calcium. 105. The method of any one of aspects 102 to 104, wherein the high calcium medium comprises 1.0 to 3.0 mM. 106. The method of any of aspects 102-105, wherein the low calcium medium comprises 0.03 mM calcium and the high calcium medium comprises 2.4 mM calcium. 107. The method of any of embodiments 101-106, wherein the low calcium medium and the high calcium medium further comprise EGF and BPE. 108. The method of embodiment 107, wherein the low calcium medium and the high calcium medium contain 0.05 ng / mL to 1 ng / ml of EGF and 1 μg / ml to 100 μg / ml of BPE. 109. The method of any of embodiment 107 or embodiment 108, wherein the low calcium medium and the high calcium medium comprise 0.2 ng / ml or about 0.2 ng / ml EGF and 30 μg / ml or about 30 μg / ml BPE. 110. The method of any of aspects 102-109, wherein the high calcium medium further contains hydrocortisone. 111. The method of embodiment 110, wherein the high calcium medium contains 0.1 to 1.0 μg / ml of hydrocortisone. 112. The method of embodiment 110 or embodiment 111, wherein the high calcium medium contains 0.4 μg / ml or about 0.4 μg / ml of hydrocortisone. 113. The method of any one of aspects 85 to 112, wherein the low-calcium medium is a serum-free medium. 114. The method of any one of aspects 102 to 113, wherein the high-calcium medium is a serum-free medium. 115. The method of any of aspects 102-114, wherein an air-liquid interface is introduced when culturing the keratinocytes in high calcium medium during the second incubation, such that the cells of the basal layer are exposed to the high culture medium but not to the gaseous environment. 116. The method of any of embodiments 102-114, wherein the low calcium medium is changed daily during the first incubation. 117. The method of any of embodiments 102-116, wherein the high calcium medium is changed daily during the second incubation. 118. The method of any of embodiments 79-117, further comprising, after step (2), formulating the skin substitute with a cryoprotectant. 119. The method of embodiment 118, wherein the cryoprotectant comprises human albumin and glucose. 120. The method of any of embodiments 79-119, further comprising freezing the skin substitute after step (2). 121. The method of any of aspects 79-120, further comprising the step of subjecting the skin substitute to a quality control evaluation, optionally prior to formulating the skin substitute with a cryoprotectant. 122. The method of embodiment 121, wherein up to or about 24 hours elapse between the completion of step (2) and the quality control step. 123. The method of embodiment 121 or embodiment 122, wherein the quality control step comprises detecting one or more polypeptides selected from the group consisting of proinsulin, modified proinsulin, insulin, insulin variants, growth factors, and variants thereof. 124. The method of any of embodiments 79-123, further comprising placing a skin substitute over the absorbent gauze. 125. The method of any of embodiments 79-124, wherein the keratinocytes comprise immortalized keratinocytes. 126. The method of any of embodiments 79-125, wherein the keratinocytes comprise cells from, and / or cells derived from, the HaCaT, NM1, or NIKS cell line. 127. A skin substitute produced by any of the methods of any of embodiments 79 to 126. 128. A cryopreserved skin substitute comprising the skin substitute of any of embodiments 1 to 44 or embodiment 127 and a cryoprotectant. 129. The cryopreserved skin substitute of embodiment 128, wherein the cryoprotectant comprises human albumin (0.02 g / mL) and D-glucose (0.09 g / mL). 130. A skin substitute dressing comprising a skin substitute of any of embodiments 1 to 44 or embodiment 127 or a cryopreserved skin substitute of embodiment 128 or embodiment 129, and absorbent gauze, wherein the cryopreserved skin substitute is placed on the absorbent gauze. 131. The substitute skin dressing of embodiment 130, wherein the absorbent gauze is Vaseline Petrolatum gauze. 132. The size of the frozen skin substitute is about 40-50 cm. 2 , about 40~45cm 2 , or about 45-50cm 2 The size of the absorbent gauze is about 40 to 60 cm. 2 , about 45~60cm 2 , about 45~55cm 2 132. The substitute skin dressing of embodiment 130 or embodiment 131, 133. The size of the cryopreserved skin substitute is 41 cm 2 or about 41cm 2 , 42cm 2 Or about 42cm 2 , 43cm 2 Or about 43cm 2 , 44cm 2 Or about 44cm 2 , 45cm 2 Or about 45cm 2 , 46cm 2 Or about 46cm 2 , 47cm 2 Or about 47cm 2 The size of the absorbent gauze is about 47 cm 2 Or about 47cm 2 , 48cm 2Or about 48cm 2 , 49cm 2 Or about 49cm 2 , 50cm 2 Or about 50cm 2 , 51cm 2 or about 51cm 2 , 52cm 2 or about 52cm 2 , 53cm 2 or about 53cm 2 132. The substitute skin dressing of embodiment 130 or embodiment 131, 134. The skin substitute of any of embodiments 1 to 44 or 127, the cryopreserved skin substitute of embodiment 128 or embodiment 129, or the skin substitute dressing of any of embodiments 130 to 133, which is sterile. 135. A container comprising a skin substitute of any of embodiments 1-44 or 127, a cryopreserved skin substitute of embodiment 127 or embodiment 128, or a skin substitute dressing of any of embodiments 130-134. 136. The container of embodiment 135, which is a bag. 137. The container of embodiment 135 or 136, which is sterile and / or heat sealed. 138. A package including a container of any one of embodiments 135 to 137, the container being a bag. 139. The package of embodiment 138, which is sterile and / or heat sealed. 140. A method for preparing a skin substitute dressing, comprising placing a skin substitute of any of embodiments 1-44 or 127 or a cryopreserved skin substitute of embodiment 128 or 129 onto absorbent gauze. 141. The method of embodiment 140, wherein the absorbent gauze is Vaseline petrolatum gauze. 142. The size of the frozen skin substitute is about 40-50 cm. 2 , about 40~45cm 2 , or about 45-50cm 2 The size of the absorbent gauze is about 40 to 60 cm. 2 , about 45~60cm 2 , about 45~55cm 2The method of embodiment 140 or embodiment 141, 143. The size of the frozen skin substitute is 41 cm 2 or about 41cm 2 , 42cm 2 Or about 42cm 2 , 43cm 2 Or about 43cm 2 , 44cm 2 Or about 44cm 2 , 45cm 2 Or about 45cm 2 , 46cm 2 Or about 46cm 2 , 47cm 2 Or about 47cm 2 The size of the absorbent gauze is about 47 cm 2 Or about 47cm 2 , 48cm 2 Or about 48cm 2 , 49cm 2 Or about 49cm 2 , 50cm 2 Or about 50cm 2 , 51cm 2 or about 51cm 2 , 52cm 2 or about 52cm 2 , 53cm 2 or about 53cm 2 The method of embodiment 140 or embodiment 141, 144. A method of promoting wound healing comprising applying to a wound a skin substitute of any of embodiments 1-44 or embodiment 127, a cryopreserved skin substitute of embodiment 128 or embodiment 129, or a skin substitute dressing of any of embodiments 130-133. 145. The method of embodiment 144, wherein the skin substitute prevents microbial infection. 146. The method of embodiment 144 or embodiment 145, wherein the skin substitute is applied to an acute wound and / or a chronic wound. 147. The method of any of embodiments 144-146, wherein the wound is selected from the group consisting of a sore, an open wound, an ulcer, and an abscess. 148. The method of any of embodiments 144-147, wherein the skin substitute is applied to a wound of a diabetic patient. 149. The method of any of embodiments 144-148, wherein the wound is a diabetic ulcer. 150. The method of any of aspects 144-149, wherein the wound is a diabetic foot ulcer. 151. The method of any of aspects 144-150, wherein the wound is a venous leg ulcer. EXAMPLES

[0252] VI. Working Examples The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.

[0253] Example 1: Methods for keratinocyte differentiation into epidermis and adenoviral transduction This example describes the generation of a skin substitute by culturing human keratinocytes from the HaCaT cell line (human keratinocytes immortalized by spontaneous mutation) and their differentiation until all epithelial layers are obtained (basal layer, spinous layer, granular layer and stratum corneum), followed by transduction with an adenoviral vector for transgene expression from the differentiated epithelial layers.

[0254] Prior to differentiation, HaCaT cells were maintained in low calcium (0.03 mM) medium for 4 weeks to alter cell properties to obtain a basal layer suitable for transduction of adenovirus type 5. For differentiation, HaCaT cells were cultured at 75 mm 2 They were seeded in culture flasks and then subcultured at a ratio of 1:4 when they reached approximately 80% confluency. Cells were cultured in serum-free and calcium-free Keratinocyte Medium (ThermoFisher, Cat. No. 37010022) modified by adding calcium to a final concentration of 0.03 mM and supplemented with 0.2 ng / mL endothelial growth factor (EGF) and 30 μg / mL bovine pituitary extract (BPE).

[0255] Transwell® polyester inserts (pore size 3 μM and diameter 75 mm) were covered with 23.5 mL of neutralized bovine collagen solution containing bovine collagen (2.5 mg / mL) certified for human use, neutralized with 1 M NaOH to reach pH 7.4. The inserts covered with neutralized bovine collagen solution were incubated at 37°C for 45 minutes. Once the collagen had gelled, they were washed twice with 1×PBS. Serum-free and calcium-free keratinocyte medium supplemented with 0.03 mM calcium, 0.2 ng / mL EGF and 30 μg / ml BPE was added to the top and bottom of the Transwell inserts for cell seeding. Cultured HaCaT cells (20×10 6 The cells were seeded in low calcium medium, and the medium was changed daily for 4 days.

[0256] On day 5, the medium on the inserts was discarded and the low calcium medium was replaced with supplemented serum-free keratinocyte medium with 0.2 ng / mL EGF, 30 μg / mL bovine pituitary extract, 0.4 μg / mL hydrocortisone and 2.4 mM calcium. An air-liquid interface was introduced so that only the basal zone of the cells was in contact with the medium and the upper part of the cell surface was exposed to the air. The cells were incubated at 37°C and 5% CO2, and the medium was changed daily for 21 days. After this period, a fully stratified epidermis 150 μM thick had developed, as shown by the eosin-hematoxylin staining at day 25 in Figure 1B.

[0257] Increasing calcium concentration in the medium (from 0.01-0.1 mM to 2.4 mM) increases the presence of occludin and claudin proteins. Expression of occludin and claudins, the major transmembrane proteins of tight junctions that affect diffusion across epithelia, is necessary for proper basal-lateral communication. From days 5 to 21, skin substitutes were maintained at high calcium concentrations.

[0258] On day 26, skin substitutes were obtained by peeling the skin from the insert with forceps and removing it from the insert before exposing the basal layer to non-replicating type 5 adenovirus. As a model for transgene expression, skin substitutes were transduced with non-replicating type 5 adenovirus expressing GFP (Ad-CMV-GFP). Transduction of skin substitutes was performed in serum-free medium after washing with 1x PBS. Adenovirus was prepared in keratinocyte medium 1 hour before incubation with skin substitutes at 37°C and 5% CO2.

[0259] Fluorescence microscopy demonstrated that the basal cells were permeable to Ad-CMV-GFP transduction and that GFP expression was colocalized with the basal cells of the skin substitute (data not shown). These results indicate that the basal cells of the skin substitute were transduced with adenovirus and that the epidermis of the skin substitute was capable of producing the protein encoded by the transduced adenovirus vector.

[0260] 24 hours after transduction, the skin substitutes were formulated for packaging by adding cryoprotective medium, placing the skin substitutes on petrolatum gauze, and placing the gauze pack into a container. The cryoprotective medium consisted of human albumin (0.02 g / mL) and D-glucose (0.09 g / mL) added to serum-free keratinocyte medium. The containers can then be stored at -20°C for up to 6 months. The process for generating and transducing the skin substitutes is depicted in Figure 1A.

[0261] Example 2: Construction of adenoviral expression vectors encoding insulin and VEGF This example describes the construction of adenoviral vectors used to facilitate protein expression in engineered skin substitutes. The skin substitutes described herein were transduced to express and release insulin and VEGF.

[0262] A type 5 adenovirus, designated Ad-CAG-VEGF-INS, was generated with nucleotides encoding human modified proinsulin (SEQ ID NO:2) and human VEGF (SEQ ID NO:4) separated by an IRES (SEQ ID NO:3) for bicistronic expression of the transgene under the control of the CAG promoter (SEQ ID NO:1). The encoded human VEGF corresponds to isoform 165 (VEGF165; SEQ ID NO:7). The encoded human modified proinsulin sequence (SEQ ID NO:6, mature sequence without signal peptide) contains mutations compared to the precursor sequence encoding wild-type proinsulin shown in SEQ ID NO:5 (encoded by amino acids 25-110 of wild-type human insulin NP_000198.1 and nucleotides 60-389 of wild-type human insulin NM_000207.3). The mutations were incorporated so that human insulin can be properly secreted and functional after release from the skin substitute described herein, which is composed of human keratinocytes immortalized by spontaneous mutation (HaCaT cells). Mature insulin is processed by pancreatic β cells through cleavage between the C and B chains (Arg-Arg dibasic site) and between the CA chains (Lys-Arg dibasic site). These enzymatic cleavages are performed by endopeptidases PC3 and PC2, respectively, which are not present in the HaCaT cells that form the skin substitute. Therefore, these modifications were achieved by creating furin cleavage sites at the desired positions for processing by HaCaT endogenous enzymes. Specifically, a furin consensus sequence (e.g., RX-[R / K]-R) (SEQ ID NO:8 or SEQ ID NO:9, e.g., SEQ ID NO:10) was introduced at the AC and BC junctions, and position 10 of the B chain was modified from a histidine (H) residue to an aspartic acid (D).

[0263] The adenovirus was made replication-deficient by deleting the E1 and E3 regions, and an expression cassette containing the CAG-VEGF-INS sequence was substituted into the E1 region. Specifically, the expression cassette was subcloned into the Dual-Basic adenovirus shuttle vector and recombined with the Ad5(DE1 / DE3) vector (Vector Biolabs, Philadelphia, Pa.). The adenovirus was packaged in HEK293 cells, purified by cesium chloride ultracentrifugation, and titered using a conventional HEK293 plaque assay. Figure 2 shows the structure of the adenovirus vector and expression cassette.

[0264] Example 3: In vitro evaluation of transduced epidermal cells reveals continued expression and release of insulin and VEGF This example describes the creation of a skin substitute composed of genetically modified basal keratinocytes that continuously release VEGF-A and insulin. The skin substitute was created by differentiating human keratinocytes of the cell line HaCaT by increasing the calcium level in the culture medium until a fully stratified epidermis was formed, as described in Example 1, and then transducing the cells of the stratified epidermis with the Ad-CAG-VEGF-INS expression construct described in Example 2.

[0265] Insulin and VEGF release from the skin substitutes was evaluated in vitro over a period of one week to determine the protein expression and release profile of the transduced epidermal cells. The level and duration of protein expression are important factors, especially when determining frequency of application.

[0266] The adenoviral vectors described in Example 2 were used to transduce basal cells of skin substitutes (n=3) at a multiplicity of infection (MOI) of 12 using the method described in Example 1. The transduced skin substitutes were then cryopreserved and stored in a -20°C freezer. The transduced skin substitutes were thawed before performing the experiment. A time course experiment was performed by withdrawing 100 μL of culture medium every 24 hours for 7 days. Expression of human insulin and VEGF was determined by ELISA according to the manufacturer's instructions (Invitrogen and Cloud-Clone, respectively). Experiments were performed in triplicate. Specifically, insulin was measured by detecting C-peptide, a peptide consisting of 31 amino acids released during the cleavage process of insulin from proinsulin (corresponding to residues 33-63 of SEQ ID NOs: 5 and 6).

[0267] Figures 3A and 3B show the average detection levels of C-peptide and VEGF, respectively, over a 7-day period. Both proteins remained detectable throughout the experiment, with VEGF levels below 4.9 ng / mL and insulin levels below 0.49 IU (based on approximately 0.0011 pmol / L, IU = approximately 2.247 ng / mL insulin). These results indicate that insulin and VEGF are released from basal cells and that protein expression is sustained for at least one week. Furthermore, the detected levels are below levels that induce systemic side effects or side effects, which may also be considered individually subtherapeutic.

[0268] Example 4: In vitro evaluation of synergy between VEGF and insulin The synergistic effect between VEGF and insulin was evaluated at the protein level within the ranges measured in Example 3. This study was performed to determine whether the combination of insulin and VEGF would produce a greater therapeutic benefit than either protein alone.

[0269] To evaluate the effects of VEGF and / or insulin on angiogenesis, an endothelial cell tube formation assay was performed. In this assay, the culture of endothelial cells on a basement membrane matrix (Matrigel) results in the formation of tubular structures resembling capillaries, characteristic of an angiogenic phenotype. The activity of 2ng / mL VEGF in combination with 0.1UI insulin was evaluated according to the range of proteins detected in Example 3. To prepare for the experiment, 96-well plates were covered with 30μL of matrigel (Corning Cat.) diluted in a 1:1 ratio. The plates were incubated at 37°C for 20 minutes and, once gelled, washed with serum-free DMEM-F12. Approximately 42,000 mouse hemangioendothelioma endothelial cells (EOMA cells) were seeded in 60μL of DMEM-F12 containing 0.5% simulated body fluid (SBF). EOMA cells were incubated for 20 min at 37° C., 5% CO 2 , after which 40 μL of medium was discarded without disturbing the adherent cells.

[0270] Stimulation or control solutions (60 μL) were then added to the cells. Stimuli included supernatants from NIH3T3 cells transfected with adenovirus encoding human VEGF 165 (SEQ ID NO:7) at MOI=12, NIH3T3 cells transfected with adenovirus encoding proinsulin (SEQ ID NO:6) modified as described in Example 2 at MOI=24, and NIH3T3 cells transfected with a combination of proinsulin and VEGF (24 MOI and 12 MOI, respectively). The negative control was supernatant from untransfected NIH3T3 cells, and the positive control was DMEM-F12 with 5% SBF. The cells were incubated at 37° C. and 5% CO2 for 1.5 hours, at which point an additional 60 μL of each supernatant stimulus was added. Experiments were performed in triplicate. At least four pictures were taken per sample in each group a total of 3 hours after the initial stimulus exposure. Endothelial tube formation was quantified using the Angiogenesis Analyzer for Image J.

[0271] Figures 4A-4C show the various wound healing features observed in each experimental group. Figure 4A shows the number of webs, defined as closed circuits surrounded by two or more nodes. Figure 4B shows the number of nodes, also referred to as unions, defined as the attachment sites of at least three cords. Figure 4C shows the number of main segments, which are cords that connect two nodes together. In all cases, the number of wound healing features observed in the combination groups greatly exceeded those observed in the single agent treatment groups (VEGF alone or insulin alone). The results showed that individual expression of VEGF at a concentration of 2 ng / mL or insulin at 0.1 UI did not induce the formation of segments, nodes or vascularized networks for wound healing. Compared to insulin alone, insulin in combination with VEGF showed significantly greater wound healing features in all assessments (webs, nodes and main segments). Compared to VEGF alone, the combination consistently resulted in more wound healing features throughout all groups, reaching a level of significance in the assessment of main segments.

[0272] Collectively, these results indicate that the therapeutic benefits of insulin and VEGF can be synergistically enhanced when delivered in combination. The study further supports that the therapeutic effect can be maximized at low levels of each protein, amounts that are unlikely to induce systemic effects or toxicity.

[0273] Example 5: Insulin and VEGF releasing skin substitutes improve wound healing in diabetic rats and hyperglycemic pigs Two animal models, diabetic rats and diabetic pigs, were used to evaluate the effect of the VEGF / insulin skin substitute on wound healing.

[0274] A. Rats Thirty Wistar rats (4 weeks old) weighing 200g-250g were kept in a temperature-controlled room (22°C) with a set humidity of 53%. The rats were housed in a micro-insulator with a 12-h light / dark cycle. The rats were allowed to acclimate to this environment for 1 week before starting the experiment.

[0275] To induce diabetes, 60 mg / kg streptozocin (also known as streptozotocin) (Sigma-Aldrich) was administered to rats via the intraperitoneal (IP) route. Streptozocin was prepared in 0.01 M sodium citrate buffer (pH 4.5) immediately prior to IP injection. Rats with glucose concentrations of 350 mg / dl (15 mM) or greater at 72 hours post-injection were diagnosed as diabetic. Blood glucose levels were determined using reactive strips using an Accu-Chek® Instant glucometer (Roche Corp).

[0276] Diabetic rats that had been maintained in a hyperglycemic state for 2 months were divided into two groups: a diabetic control group (without skin substitute) and a diabetic treatment group (with skin substitute). Healthy rats were included in the third group in this study. All rats in each group were given a 1 cm 2 Wounds were created by making a puncture. Wounds in the control diabetic and healthy groups were covered with petrolatum gauze. Wounds in the treated diabetic rats were covered with the VEGF / insulin skin substitute. Gauze dressings were changed every 3 days throughout the duration of the study, whereas the skin substitute was applied only once. Wounds were measured every 3 days until complete wound closure was observed.

[0277] Figure 5A shows the percentage of open wound area over 21 days among the three experimental groups. The time from wound initiation to complete wound closure was 21 days for both the healthy control group and the diabetic group treated with skin substitutes. In comparison, the diabetic group treated with gauze dressings had an increased time to healing by more than one week (time to wound closure = 30 days). Representative images of wounds on days 1 and 21 are shown in Figure 5B.

[0278] As a further evaluation of wound healing activity, histological examination was performed to determine the effect of skin substitute on scar formation. For each experimental group, cross-sections of scarred areas were taken for histopathological examination on the 21st day after wound initiation. As shown in Figure 6, the structure and remodeling of both dermis and epidermis were comparable between healthy rats and diabetic rats treated with skin substitute. In contrast, examination of diabetic samples revealed tissue inflammation, cell infiltration and thickening. Taken together, these results provide evidence of the wound healing activity of skin substitute in vivo and are consistent with the findings described in Examples 3 and 4.

[0279] B. Pig A surgical approach was employed to induce hyperglycemia in pigs. Using aseptic technique, the left jugular vein on the ventral fascia of the neck was exposed, and then an 18G x 30mm catheter was inserted into the left jugular vein to withdraw 15mL of blood. After sample collection, the catheter was secured to the vessel with a suture, and a venous perfusion device was connected to maintain vascular access. One blood sample was taken from each ear to obtain a baseline blood glucose measurement. One single dose of 124mg / kg streptozocin was administered through the vascular access using an infusion pump for 15 minutes. This was followed by administration of a 5% glucose solution (200mL) over a 30 minute period. Once the streptozocin and glucose were administered, a new glucose measurement was taken from each ear to confirm the rise in blood glucose levels.

[0280] Pigs showing normal behavior and blood glucose levels below 180 mg / dL during the first 48 hours after injection were included in the control group. Glucose measurements were performed in the pigs' fasting state and 1 hour after breakfast. If glucose levels above 350 mg / dL were measured, insulin aspart (NovoMix®), insulin isophane (Aurax®) and insulin glargine (Lantus®) were administered to maintain hyperglycemic levels of approximately 250 mg / dL. The pigs were maintained in a hyperglycemic state for 2 months to induce glycation in the skin. There were a total of 3 pigs in each group.

[0281] After 2 months, three 4x3 cm wounds were made on the back of anesthetized pigs using the spine as a reference. The wounds were located 2 cm from the midline with 5 cm between them. The wounds were identified using location-based designations, i.e., wound #1 was located on the left side of the skull, wound #2 was located on the left side of the tail, and wound #3 was located on the right side of the center.

[0282] Once the effects of the anesthetic had worn off, 0.01-0.03 mg / kg buprenorphine (Brospine®) was administered via intramuscular injection to alleviate pain. For the first 3 days after wound initiation (days 1-3), one dose of buprenorphine was administered every 8 hours. From day 3 onwards (days 4-6), one dose was administered per hour. From days 7-9, one dose was given every 24 hours. At the discretion of the treating physician, antimicrobial treatment was administered in the event of infection with either 3.0 mg / kg enrofloxacin (every 24 hours) or 15 mg / kg 3 Sulfas® (sulfamethazine, sulfamerazine and sulfadiazine, in equal portions) (every 12 hours) administered via the intramuscular (IM) route for 8 days.

[0283] Photographs of the wounds were taken on a standard centimeter scale at 0, 1, 4, 7, 11, 18, 21, 25 and 28 days after wound initiation. During the first 7 days, the wounds were cleaned with saline solution and new sterile gauze and dressings were provided. For wounds with a skin substitute, cleaning was performed only around the wound periphery to avoid wetting or slipping of the treatment tools. After the first 7 days, gauze and dressings were changed every other day, including the day the photographs were taken. As a comparison, the VEGF / insulin skin substitute was applied only once. Surgical debridement was performed under sedation for wounds that showed signs of infection. The area (cm) was measured using ImageI software. 2 Wound closure was assessed by measuring the mean mean closure time (min) and mean mean mean closure time (min).

[0284] FIG. 7A shows the wound area (cm2) between healthy pigs, diabetic pigs (without skin substitute) and diabetic pigs treated with VEGF / insulin skin substitute. 2) are shown. Similar to the results of the rat study, complete wound closure was observed in the healthy control group and the group treated with VEGF / insulin skin substitute over the same time period. Complete wound closure was observed on day 25 in the healthy control and skin substitute-treated pigs, while observable wound areas persisted until day 28 in the diabetic pigs (without skin substitute). Given that open wounds persisted for the diabetic group, observations were extended until complete wound closure. As shown in FIG. 7B, complete wound healing was not achieved until day 52 in the diabetic group, while healing was observed after nearly 4 weeks in the skin substitute-treated group. FIG. 8 shows representative images of wound healing over time among the three pig groups. The photographed wound in the skin substitute group began to close on day 14, and no signs of infection were observed throughout the study. As shown in the top and bottom panels, wounds in the healthy control and diabetic subjects treated with VEGF / insulin skin substitute, respectively, closed on day 23. On this same day, the wound in the diabetic pig was still open and infection was present (middle panel).

[0285] Figure 9 shows wounds from diabetic pigs treated with wound cleansing and gauze dressing and VEGF / insulin skin substitute on days 1 and 7 after wounding. Infection was noted in the diabetic pig wounds (without skin substitute) from day 3. In comparison, the diabetic pigs treated with VEGF / insulin did not show signs of infection and therefore did not require antibiotic treatment. Treatment of infection in the diabetic pigs (without skin substitute) consisted of oral, e.g., 3 Sulfas®, and topical antibiotics, e.g., topical silver sulfadiazine, for 45 days.

[0286] Glucose levels were measured from one week before wound initiation (day -7) until day 11 after wounding to evaluate the systemic effect of the epidermis releasing insulin and VEGF. Figure 10 shows the detected blood glucose levels (mg / dL) among all experimental groups. The levels were maintained at relatively low levels (about 100mg / dL) in the healthy group. For both diabetic groups, there was a large fluctuation in blood glucose of about 100mg / dL throughout the course of the study. Compared to diabetic controls, treatment with VEGF / insulin skin substitutes did not dramatically change blood glucose levels. These results are consistent with the observation that enhanced synergy between molecules not only allows faster and more orderly healing and scarring, but also ensures the lack of toxicity and safety of the product.

[0287] Taken together, these in vivo studies support the potent wound-healing activity and safety of VEGF / insulin skin substitutes for wound treatment in diabetic mice. In both rats and pigs, diabetic skin treated with VEGF / insulin skin substitutes healed on a timeline comparable to that of healthy skin. Furthermore, treatment with VEGF / insulin skin substitutes was not accompanied by microbial infections and did not dramatically alter systemic blood glucose levels, indicating that insulin and VEGF are not present in amounts that significantly affect blood glucose. In addition to the therapeutic benefit, this study demonstrates that repeated replacement of the skin substitute is unnecessary, an advantage that provides positive implications for patient compliance.

[0288] Example 6: Sustained release of insulin and VEGF from VEGF / insulin skin substitutes reduces the presence of advanced glycation end products (AGEs) in porcine skin Hyperglycemia causes structural changes in proteins and lipids in cells and tissues, such as glycation, which occurs as a result of the covalent attachment of sugar molecules (glucose or fructose) to proteins or lipids. Glycated proteins or lipids are referred to as AGEs. High AGE concentrations are found in the skin of diabetic patients and act to inhibit the healing process. The porcine skin of Example 5 was examined to determine the effect of the skin substitute on advanced glycation end products (AGE) concentrations.

[0289] Skin biopsies were performed on the experimental groups described in the pig study in Example 5: healthy pigs, diabetic pigs (without skin substitute), and diabetic pigs treated with VEGF / insulin skin substitute. Pigs in each diabetic group maintained blood glucose levels >350 mg / dL for 2 months as described in Example 5. For pigs treated with VEGF / insulin skin substitute, dermal and epidermal samples were taken at wound initiation and 2 days after wound healing was evident (at or about day 25). For healthy and diabetic controls, skin sample biopsies were taken at the moment of wounding and when the wound had healed after 21 days (healthy pigs) or 60 days (diabetic pigs).

[0290] For each biopsy, a portion of the collected sample was divided and weighed using an analytical balance. Approximately 20 mg of tissue was disrupted in 500 μL of 1× PBS containing the protease inhibitor Complete (Sigma) using a TissueRuptor. Samples were then centrifuged at 4,000 rpm for 5 min to pellet the disrupted tissue and the supernatant was removed. Protein concentration was adjusted to 50 μg / mL using the Bradford method of protein estimation (Sigma). AGEs were quantified using a 1:10 dilution of the sample using an ELISA kit. ELISA was performed according to the manufacturer's instructions. Briefly, 50 μL of diluted sample and 50 μL of diluted biotinylated antibody (1:100) were incubated for 45 min. After this initial incubation, three washes were completed with approximately 359 μL of 1× wash solution. 100 μL of secondary antibody with HRP (1:100) was then added and incubated for 30 min at 37°C. At the end of this incubation, five washes were performed with 350 μL of 1× wash solution. After removal of the wash solution, 90 μL of 3,3′,5,5′-tetramethylbenzidine (TMB) was added and incubated at 37° C. for 15 min. The reaction was stopped by adding 50 μL of stop solution. The absorbance was read at 450 nm and a second-order polynomial regression equation (R 2 = 0.99) to obtain the AGE concentration.

[0291] The concentration in ng / mg protein was calculated from the concentration obtained with the AGE ELISA kit (data in ng / mL). Figure 11 shows the amount of AGE (in ng / mg protein) per experimental group before and after wound healing as calculated from ELISA. The results showed that in the healthy group, the level of AGE increased from wound initiation to healing, while in the diabetic (no skin substitute) group, AGE remained at a relatively high level. Treatment with VEGF / insulin skin substitute reduced the number of AGE from wound initiation to healing. Of note, at the time of wound initiation, AGE in the VEGF / insulin skin substitute treatment group was similar to that of the diabetic group, but treatment with VEGF / insulin skin substitute reduced AGE to the level observed in healthy skin.

[0292] The reduction of AGEs is associated with improved angiogenesis and control of infection and inflammation. This study confirms that treatment with VEGF / insulin skin substitutes can reduce AGEs even in skin with advanced glycation (AGE levels are roughly twice as high as those present in healthy skin). These results indicate that the wound healing effect of VEGF / insulin skin substitutes may be mediated by AGE reduction.

[0293] Example 7: VEGF / insulin skin substitutes are non-tumorigenic 20mm 2 of non-transduced skin substitutes (control) or 20 mm 2 Tumor formation was assessed after subcutaneous implantation of the skin substitutes in the interscapular region of 6-week-old nude mice (BALB / c nu / nu). Tumor growth was measured at weekly intervals. Skin substitutes transduced with Ad-CAG-VEFG-INS adenovirus showed no tumor formation 4 months after implantation in nude mice (data not shown). Positive control nude mice injected with MCF-7 human breast cancer cells showed tumor growth 1 month after subcutaneous injection (data not shown).

[0294] Example 8: Evaluation of the cytogenetic properties of human keratinocyte lines for the production of skin substitutes As described in the above examples, the production of skin substitutes involves first culturing human keratinocytes of the HaCaT cell line in a low calcium medium to generate a basal layer suitable for transduction, followed by their differentiation. HaCaT cells closely resemble normal human keratinocytes in terms of their growth and differentiation potential; however, they are immortalized cell lines with some chromosomal abnormalities. Although HaCaT cells retain a stable chromosomal content and remain non-tumorigenic, experiments were performed to characterize the cytogenetic characteristics of the cells after culturing in a low calcium and serum-free medium to cultivate the basal layer.

[0295] HaCat line was grown for 4 weeks under basal layer culture conditions in fetal bovine serum-free low calcium (0.03 mM) medium and named HaLow cells (serum-free low calcium HaCat). Starting from passage 2, when cells were in culture for a total of 3 months, the chromosomal composition of HaLow cells was followed during the growth period. Semi-confluent cell cultures were treated with 0.08 μg / ml KaryoMAX™ Colcemid™ (ThermoFisher 15212012) for 2 hours at 37°C. Cells were detached by subsequent treatment with Recombinant Trypsin EDTA Solution (Sartorius 03-079-1A 10 min), centrifuged, and cell pellets were resuspended in a hypotonic solution of 75 mM KC1. After incubation at room temperature for 15 min, cells were fixed with three changes of methanol / acetic acid (3:1), spread onto glass slides, and G-banded after 16 h. Usually, 15 metaphases were analyzed by microscopy, and at least five karyograms were generated.

[0296] As shown in Figure 12, cytogenetic analysis of cells at passage 7 showed the following karyogram: 65, XXX, +1, -2, add(3)(p25), -3, add(4)(p15), -4, -4, -5, 6, -7, +8, i(9)(q10), -9, -10, +der(11)del11(q23), +11, +12, +13, -14, 15, +16, +17, +18, +19, +20, +21, +22, +6mar, +min. Most cells were hypotriploid with an average of 65 chromosomes resulting from complete or partial monosomy of the chromosomes involved in the formation of the marker chromosomes. All metaphases had an XO sex chromosome configuration (lacking the Y chromosome). This cytogenetic profile indicates that the cells adapt to growth under modified culture conditions, but the karyogram is consistent with cells originating from HaCaT cells. Together with the results in Example 7, this result indicates that after incubation in low calcium and serum-free, the cell line HaLow has certain cytogenetic characteristics and is non-tumorigenic when used in skin substitutes.

[0297] Example 9: Expression and release of epidermal growth factor from skin substitutes Epidermal growth factor (EGF) is a key promoter of wound repair and regeneration in diabetic foot ulcers (DFUs). In DFUs, the wound healing process is impeded by the accumulation of advanced glycation end products (AGEs) due to high blood glucose levels in diabetic patients. AGEs competitively bind to EGF receptors, thus inhibiting EGF binding and perpetuating the initial damage to vascular endothelial cells and fibroblasts. Current techniques for delivering growth factors, including EGF, to the wound environment are ineffective, mainly due to the very short in vivo half-life of EGF when administered directly into the extracellular matrix.

[0298] Expression and release of EGF into the medium was assessed in cultures of skin substitutes transduced with adenovirus expressing human EGF (Ad-CMV-hEGF) using methods essentially as described in the Examples above, except that the skin substitutes were transduced with adenovirus expressing human EGF. The skin substitutes were cultured with 5×106 The skin substitutes were transduced with 100% IFU (infectious units) of adenovirus (Ad-CMV-hEGF, GenBank: BC113461). After 72 h of incubation, the culture medium was collected from the cultures of transduced and non-transduced skin substitutes, and the hEGF protein secreted from the skin substitutes into the culture medium was quantified by a commercially available enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems).

[0299] As shown in FIG. 13, the concentration of human epidermal growth factor (hEGF) was significantly higher in the culture medium of skin substitutes transduced with adenovirus expressing hEGF (Ad-CMV-hEGF) than in the culture medium of the non-transduced control.

[0300] These results demonstrate that the skin substitutes provided herein, when transduced with an adenovirus carrying a polynucleotide encoding hEGF, can effectively express and release hEGF into the culture medium.

[0301] These results support that other recombinant growth factors, such as EGF, can be used to produce skin substitutes that release growth factors during wound healing by genetically modifying cells derived from the skin substitute with adenoviruses that express and release the growth factors. Without wishing to be bound by theory, the results support that other recombinant growth factors can also be used in combination with insulin in the skin substitute, whereby insulin release from the skin substitute into the wound inhibits the presence of AGEs and allows activation of the EGF receptor.

[0302] The present invention is not intended to be limited in scope to the specific disclosed embodiments, which are provided, for example, to illustrate various aspects of the invention. Various modifications to the compositions and methods described will become apparent from the descriptions and teachings herein. Such variations can be practiced without departing from the scope and spirit of the disclosure and are intended to fall within the scope of the disclosure.

[0303] Sequence Listing TIFF2024531827000007.tif212165TIFF2024531827000008.tif212165TIFF202 4531827000009.tif232165TIFF2024531827000010.tif220165TIFF20245318270 00011.tif228165TIFF2024531827000012.tif228165TIFF2024531827000013.t if233165TIFF2024531827000014.tif222165TIFF2024531827000015.tif210165

Claims

1. 1. A skin substitute comprising a stratified epidermis including a basal layer, a spinous layer, a granular layer, and a stratum corneum, the cells of the stratified epidermis express recombinant growth factors and recombinant insulin; The skin substitute.

2. (a) The recombinant growth factor and recombinant insulin are secretable from cells of the stratified epidermis. (b) the recombinant insulin is or comprises recombinant human insulin; (c) recombinant insulin (i) the amino acid sequence shown in SEQ ID NO: 5; (ii) a functional variant having an amino acid sequence that has at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 5; or (iii) A two-chain form of (i) or (ii) comprising an A chain and a B chain, optionally wherein the A chain and the B chain are linked by a disulfide bond. Including, (d) the recombinant insulin is an AspB10 insulin analogue containing a histidine to aspartic acid mutation at position 10 in the B chain of modified human proinsulin compared to the wild-type insulin shown in SEQ ID NO: 5; (e) the skin substitute comprises a polynucleotide encoding proinsulin that includes at least one furin recognition sequence in place of the endopeptidase Arg31-Arg32 cleavage site or the endopeptidase Lys64-Arg65 cleavage site; and / or (f) recombinant insulin (i) the amino acid sequence shown in SEQ ID NO:6; (ii) a functional variant having an amino acid sequence that has at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 6; or (iii) A two-chain form of (i) or (ii) comprising an A chain and a B chain, optionally wherein the A chain and the B chain are linked by a disulfide bond. comprising or having 10. The skin substitute of claim 1.

3. 10. The skin substitute of claim 1, wherein the cells of the stratified epidermis that express recombinant growth factors and recombinant insulin comprise cells of the stratum basale. (i) at least one furin recognition sequence is in place of the endopeptidase Arg31-Arg32 cleavage site and the endopeptidase Lys64-Arg65 cleavage site; (ii) at least one furin recognition sequence has the consensus sequence: RXRR (SEQ ID NO:8), where X is any amino acid; or RXKR (SEQ ID NO: 9), where X is any amino acid. Including, (iii) at least one furin recognition sequence is RTKR (SEQ ID NO: 10) or RQKR (SEQ ID NO: 42); 3. The skin substitute of claim 2.

5. 2. The skin substitute of claim 1, wherein the recombinant insulin comprises an A chain set forth in SEQ ID NO: 36 and a B chain set forth in SEQ ID NO:

41.

6. 2. The skin substitute of claim 1, wherein the recombinant growth factor is selected from the group consisting of epidermal growth factor (EGF), erythropoietin (EPO), fibroblast growth factor (FGF), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), hepatocyte growth factor (HGF), insulin-like growth factor (IGF), platelet-derived growth factor (PDGF), transforming growth factors alpha and beta, vascular endothelial growth factor (VEGF), epidermal growth factor receptor (EGFR), fibroblast growth factor receptor (FGFR), and any isoforms or alternatively spliced variants thereof.

7. 2. The skin substitute of claim 1, wherein the recombinant growth factor is VEGF or an isoform or alternatively spliced variant thereof.

8. (i) VEGF is a polypeptide sequence having at least 85%, or at least about 85%, at least 90%, or at least about 90%, or at least 95%, or at least about 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 7; or Its sequence lacks a signal peptide Contains, or the amino acid sequence set forth in SEQ ID NO: 7, or Its sequence lacks a signal peptide Including, (ii) VEGF comprises a polypeptide sequence having at least 85%, or at least about 85%, at least 90%, or at least about 90%, or at least 95%, or at least about 95% sequence identity to the sequence set forth in SEQ ID NO: 44, or comprises the polypeptide sequence set forth in SEQ ID NO: 44; 8. The skin substitute of claim 7.

9. (a) The recombinant growth factor and recombinant insulin are encoded by a bicistronic expression cassette comprising a polynucleotide encoding the recombinant growth factor and a polynucleotide encoding the recombinant insulin, separated by a bicistronic element. (b) the polynucleotide encoding the recombinant growth factor and the polynucleotide encoding the recombinant insulin are operably linked to a promoter; (c) the cells of the stratified epidermis continuously secrete the recombinant growth factor and recombinant insulin for up to or about 2 days, up to or about 3 days, up to or about 4 days, up to or about 5 days, up to or about 6 days, up to or about 7 days, up to or about 8 days, up to or about 9 days, up to or about 10 days, up to or about 11 days, up to or about 12 days, up to or about 13 days, or up to or about 14 days; and / or (d) The cells of the stratified epidermis are cells differentiated from keratinocytes.

10. The skin substitute of claim 1.

10. 10. The skin substitute of claim 9, wherein the bicistronic element is an IRES.

11. 10. The skin substitute of claim 9, wherein the promoter is a CAG promoter.

12. 10. The skin substitute of claim 9, wherein the polynucleotide encoding the recombinant growth factor is upstream of the polynucleotide encoding the recombinant insulin in the bicistronic expression cassette.

13. 10. The skin substitute of claim 9, wherein the keratinocytes are human keratinocytes and / or HaCaT keratinocyte cells.

14. A bicistronic expression cassette comprising polynucleotides encoding a recombinant human growth factor and recombinant insulin.

15. (a) The recombinant insulin is or comprises recombinant human insulin. (b) the recombinant insulin encoded therein is (i) is or comprises the amino acid sequence set forth in SEQ ID NO:5; or (ii) a functional variant having an amino acid sequence that has at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 5; (c) the encoded recombinant insulin comprises the amino acid sequence shown in SEQ ID NO: 5; (d) the encoded recombinant insulin is an AspB10 insulin analogue containing a histidine to aspartic acid mutation at position 10 in the B chain of modified human proinsulin compared to the wild-type insulin shown in SEQ ID NO: 5; (e) the polynucleotide encoding the recombinant insulin encodes a proinsulin containing at least one furin recognition sequence in place of the endopeptidase Arg31-Arg32 cleavage site or the endopeptidase Lys64-Arg65 cleavage site; (f) the recombinant insulin encoded by (i) is or comprises the amino acid sequence set forth in SEQ ID NO:6; or (ii) is a functional variant having an amino acid sequence that has at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 6; and / or (g) the polynucleotide encoding the recombinant insulin comprises the sequence set forth in SEQ ID NO: 2 or a sequence having at least 85%, or at least about 85%, at least 90%, or at least about 90%, or at least 95%, or at least about 95% sequence identity to the sequence set forth in SEQ ID NO: 2; 15. The bicistronic expression cassette of claim 14.

16. (a) at least one furin recognition sequence in place of the endopeptidase Arg31-Arg32 cleavage site and the endopeptidase Lys64-Arg65 cleavage site; (b) at least one furin recognition sequence has the consensus sequence: RXRR (SEQ ID NO:8), where X is any amino acid; or RXKR (SEQ ID NO: 9), where X is any amino acid. Including, (c) at least one furin recognition sequence is RTKR (SEQ ID NO: 10) or RQKR (SEQ ID NO: 42); 16. The bicistronic expression cassette of claim 15.

17. 15. The bicistronic expression cassette of claim 14, wherein the encoded recombinant growth factor is selected from the group consisting of epidermal growth factor (EGF), erythropoietin (EPO), fibroblast growth factor (FGF), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), hepatocyte growth factor (HGF), insulin-like growth factor (IGF), platelet-derived growth factor (PDGF), transforming growth factor alpha and beta, vascular endothelial growth factor (VEGF), epidermal growth factor receptor (EGFR), fibroblast growth factor receptor (FGFR), and any isoform or alternatively spliced variant thereof.

18. 15. The bicistronic expression cassette of claim 14, wherein the recombinant growth factor is VEGF or an isoform or alternatively spliced variant thereof.

19. (a) the polynucleotide encoding the growth factor comprises the sequence set forth in SEQ ID NO:4 or a sequence having at least 85%, or at least about 85%, at least 90%, or at least about 90%, or at least 95%, or at least about 95% sequence identity to the sequence set forth in SEQ ID NO:4; (b) the polynucleotide encoding the growth factor comprises the sequence set forth in SEQ ID NO: 7 or the sequence thereof lacking the signal peptide, or comprises a sequence having at least 85%, or at least about 85%, at least 90%, or at least about 90%, or at least 95%, or at least about 95% sequence identity to the sequence set forth in SEQ ID NO: 7, or the sequence thereof lacking the signal peptide; (c) the polynucleotide encoding the growth factor comprises the sequence set forth in SEQ ID NO: 44 or comprises a sequence having at least 85%, or at least about 85%, at least 90%, or at least about 90%, or at least 95%, or at least about 95% sequence identity to the sequence set forth in SEQ ID NO: 44; (d) the polynucleotide encoding the recombinant growth factor and the polynucleotide encoding the recombinant insulin are separated by a bicistronic element; and / or (e) the polynucleotide encoding the recombinant growth factor and the polynucleotide encoding the recombinant insulin are operably linked to a promoter; 19. The bicistronic expression cassette of claim 18.

20. 20. The bicistronic expression cassette of claim 19, wherein the bicistronic element is an IRES.

21. 15. The bicistronic expression cassette of claim 14, wherein the polynucleotide encoding the recombinant growth factor and the polynucleotide encoding the recombinant insulin are operably linked to the same promoter.

22. 20. The bicistronic expression cassette of claim 19, wherein the promoter is a CAG promoter.

23. 20. The bicistronic expression cassette of claim 19, wherein the polynucleotide encoding the recombinant growth factor is upstream of the polynucleotide encoding the recombinant insulin in the bicistronic expression cassette.

24. A vector comprising the bicistronic expression cassette of any one of claims 14 to 23. (a) a viral vector, (b) an adenoviral vector; (c) is a non-replicating type 5 adenovirus; and / or (d) a non-replicating adenovirus lacking or lacking functional E1 and E3 regions; 25. The vector of claim 24.

26. (1) differentiating keratinocytes into stratified epidermis, the stratified epidermis comprising a basal layer, a spinous layer, a granular layer, and a stratum corneum; and (2) introducing the bicistronic expression cassette of any one of claims 14 to 23 or the vector of claim 24 or 25 into cells of said stratified epidermis to produce a skin substitute, wherein said skin substitute comprises recombinant growth factors and recombinant insulin, or (2) transducing cells of said stratified epidermis with the vector of claim 24 or 25 to produce a skin substitute, said skin substitute comprising growth factors and insulin. Including, A method for producing a skin substitute.

27. (a) the step of introducing or transducing is into cells of the basal layer. (b) prior to differentiation in step (1), culturing the keratinocytes in low calcium medium for 2 weeks, 3 weeks, 4 weeks, 5 weeks, or 6 weeks, optionally 4 weeks or about 4 weeks; and / or (c) the keratinocytes are human keratinocytes; 27. The method of claim 26.

28. 28. The method of claim 27, wherein the low calcium medium comprises a calcium concentration of 0.01 to 0.1 mM at the time of seeding said cells or during said culturing step.

29. 27. The method of claim 26, wherein step (1) comprises culturing keratinocytes on an extracellular matrix substrate.

30. (a) The method of claim 30, wherein the extracellular matrix substrate is collagen. (b) the culturing in step (1) is for about 23 to 28 days; and / or (c) the culturing in step (1) comprises a first incubation in a low calcium medium and a second incubation in a high calcium medium; 30. The method of claim 29.

31. 31. The method of claim 30, wherein the first incubation in the low calcium medium is for about 3 to 5 days and the second incubation in the high calcium medium is for about 20 to 23 days.

32. (a) The low-calcium medium contains 0.01 to 0.1 mM calcium. (b) the high-calcium medium contains 1.0 to 3.0 mM calcium; (c) the low calcium medium and the high calcium medium further contain EGF and BPE; (d) the low calcium medium and the high calcium medium contain 0.05 ng / mL to 1 ng / ml of EGF and 1 μg / ml to 100 μg / ml of BPE; (e) the low calcium medium and the high calcium medium contain 0.2 ng / ml or about 0.2 ng / ml EGF and 30 μg / ml or about 30 μg / ml BPE; (f) the high calcium medium further contains hydrocortisone; (g) the high calcium medium contains 0.1 to 1.0 μg / ml of hydrocortisone; and / or (h) the high calcium medium contains 0.4 μg / ml or about 0.4 μg / ml hydrocortisone; 31. The method of claim 30.

33. 27. A skin substitute produced by the method of claim 26.

34. 10. A cryopreserved skin substitute comprising the skin substitute of claim 1 and a cryoprotectant.

35. 35. The cryopreserved skin substitute of claim 34, wherein the cryoprotectant comprises human albumin (0.02 g / mL) and D-glucose (0.09 g / mL).

36. A skin substitute dressing comprising the cryopreserved skin substitute of claim 34 and absorbent gauze, wherein the cryopreserved skin substitute is placed on the absorbent gauze.

37. 37. A container comprising the skin substitute of any one of claims 1 to 13 or 33, the cryopreserved skin substitute of claim 34 or claim 35, or the skin substitute dressing of claim 36.

38. 36. A method for preparing a skin substitute dressing, comprising placing a skin substitute according to any one of claims 1 to 13 or 33 or a cryopreserved skin substitute according to claim 34 or 35 onto absorbent gauze.

39. A skin substitute as described in claims 1 to 13 or 33, a cryopreserved skin substitute as described in claim 34 or claim 35, or a skin substitute dressing as described in claim 36, for use in promoting wound healing comprising applying the skin substitute, cryopreserved skin substitute, or skin substitute dressing to a wound.

40. (a) The wound is selected from the group consisting of a sore, an open wound, an ulcer, and an abscess. (b) a skin substitute is applied to a wound in a diabetic patient; (c) the wound is a diabetic ulcer; (d) the wound is a diabetic foot ulcer; and / or (e) the wound is a venous leg ulcer; 40. The skin substitute, cryopreserved skin substitute, or skin substitute dressing of claim 39.

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