Activated amphibian skin

Abstract

The present disclosure describes compositions including activated amphibian skin having enhanced functional activity, methods of preparing the compositions, and methods of using the compositions for treating skin conditions and wound healing.

Description

ACTIVATED AMPHIBIAN SKINCROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of priority of U.S. Provisional Application 63 / 672,961 , filed on July 18, 2024, which is incorporated herein by reference in its entirety.TECHNICAL FIELD

[0002] The disclosure describes a novel method for activating the skin of amphibians and compositions comprising the activated skin for various uses.BACKGROUND

[0003] The skin, the largest organ of the mammalian body, is an outer covering of the body that serves three main functions: protection against microbes and excessive water loss, regulation of body temperature, and sensation of touch, heat, and cold. Mammalian skin has two primary layers, the epidermis, and the dermis. The epidermis, the outermost layer of the skin that prevents microbes from entering and keeps water in our body, is a stratified squamous epithelium composed of keratinocytes. The dermis is the layer of skin beneath the epidermis which serves as a location for the appendages of the skin and provides elasticity to the skin through an extracellular matrix composed of collagen fibers, elastic fibers, hyaluronan, and proteoglycans. The dermis and the epidermis are separated by a thin sheet of fibers called the basement membrane which regulates the flow of cells and molecules, such as cytokines and growth factors, between the dermis and epidermis, during the remodeling, repair, and regeneration process. Underneath the dermis is the hypodermis which is composed of loose connective tissue, such as fat, and elastin. The primary cells of the hypodermis include fibroblasts, macrophages, and adipocytes.

[0004] An injury or a disease creates an interruption of the morphology and function of an organ or tissue, such as the skin. The remodeling and repair of the organ or tissue following an injury is a complex wound healing process involving interactions between cells, growth factors, and extracellular matrix (ECM). The process in adult mammals involves well-known stages: homeostasis, inflammation, proliferation, maturation, and remodeling. During homeostasis, clotting takes place to stop the bleeding. Inflammation involves the recruitment of white blood cells, antibodies, nutrients, and enzymes to the affected area to accelerate wound healing. During proliferation, new healthy granulation tissues, such as new connective tissues and blood vessels, replace the wound. Maturation and remodeling take place after the wound is closed and involve the repair of the dermal tissues to improve their tensile strength.

[0005] In contrast to the repair process in which the goal is to re-establish function without regard to the exact placement of injured tissue, regeneration is the replacement of injured tissue with an exact copy such that both morphology and functionality are completely restored. As anexample, non-injured skin undergoes complete regeneration continually with the replacement of new cells. However, injured adult mammalian skin does not regenerate completely and heals with a scar.

[0006] In contrast to mammals, amphibians regenerate their skin structures including the dermis and secretion glands without forming a scar after a deep skin injury. Thus, the skin of amphibians can serve as a model for studying skin regeneration and as an agent for assisting in the treatment of skin, also it contains proteins and other factors essential for the growth of skin.SUMMARY

[0007] This Summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter.

[0008] The present disclosure describes activated amphibian skin having increased functional activity and compositions including activated amphibian skin conditions.

[0009] The present disclosure describes methods of activating amphibian skin.

[0010] The present disclosure also describes methods of using the activated amphibian skin, including activated amphibian extracellular matrix (ECM), to treat skin conditions, including treating wounds and inflammations, and for remodeling the dermal matrix ECM, regeneration and / or protection of of the skin barrier, and enhancing general induction of longevity genes.

[0011] The present disclosure also describes a method of using the activated amphibian skin to modulate the expression of genes involved in wound healing, inflammation, and regeneration and protection of the skin barrier by maintaining the integrity and functionality of the skin.BRIEF DESCRIPTION OF THE DRAWINGS

[0012] The Detailed Description is described with reference to the accompanying figures.

[0013] Fig. 1A shows the results of the treatment of third-degree burns using Matrix without laser pretreatment from day 1 to day 14 post-treatment. Matrix is decellularized extracellular matrix (ECM) of axolotl without light exposure (non-activated). Control is an untreated wound.

[0014] Fig. 1 B shows the results of the treatment of third-degree burns using Matrix with laser pretreatment from day 1 to day 14 post-treatment.

[0015] Fig. 1C shows the results of the treatment of third-degree burns using Matrix PLE without laser pretreatment from day 2 to day 14 post-treatment. Matrix PLE is decellularized ECM that was exposed to light (activated with broad spectrum light (BSL)) and obtained 60 hours of PLE.

[0016] Fig. 1 D shows the results of the treatment of third-degree burns using Matrix PLE with laser pretreatment from day 2 to day 14 post-treatment.

[0017] Fig. 2A shows the results of the treatment of deep dermal wounds using Matrix PLE without laser pretreatment from day 2 to day 10 post-treatment.

[0018] Fig. 2B shows the results of the treatment of deep dermal wounds using Matrix PLE with laser pretreatment from day 2 to day 10 post-treatment.

[0019] Fig. 3A shows the results of the treatment of full-thickness wounds using Matrix without laser pretreatment from day 1 to day 10 post-treatment.

[0020] Fig. 3B shows the results of the treatment of full-thickness wounds using Matrix with laser pretreatment from day 1 to day 10 post-treatment.

[0021] Fig. 4A shows the averaged results of the treatments of third-degree burns and the averaged results of the treatments of the wounds (wound*: deep dermal and full-thickness wounds) with and without laser pretreatment using Matrix (non-BSL) or Matrix PLE (BSL). The average percentage of epithelialization of third-degree burns is the average of the results of Figs 1A-1D. The average percentage of epithelialization of the wounds is the average of the deep dermal and full-thickness wounds of Figs. 2A-3B.

[0022] Fig. 4B shows the averaged results of the treatments of third-degree burns with and without laser pretreatment using Matrix (non-BSL) or Matrix PLE (BSL).

[0023] Fig. 4C shows the averaged results of the treatments of wounds (deep dermal and full-thickness wounds) with and without laser pretreatment using Matrix (non-BSL) or Matrix PLE (BSL).

[0024] Fig. 5A shows the results of the treatment of third-degree burns using Super Serum without laser pretreatment from day 1 to day 14 post-treatment. Super Serum contains a nonactivated skin sample in gel formulation. The non-activated skin sample is in the form of a decellularized extracellular matrix (ECM). Control is an untreated wound.

[0025] Fig. 5B shows the results of the treatment of third-degree burns using Super Serum with laser pretreatment from day 1 to day 14 post-treatment.

[0026] Fig. 5C shows the results of the treatment of third-degree burns using Super Serum PLE without laser pretreatment from day 2 to day 14 post-treatment. Super Serum PLE is a solution containing a skin sample in gel formulation activated with BSL and obtained between 60 hrs PLE.

[0027] Fig. 5D shows the results of the treatment of third-degree burns using Super Serum PLE with laser pretreatment from day 2 to day 14 post-treatment.

[0028] Fig. 6A shows the results of the treatment of deep dermal wounds using Super Serum PLE without laser pretreatment from day 2 to day 10 post-treatment.

[0029] Fig. 6B shows the results of the treatment of deep dermal wounds using Super Serum PLE with laser pretreatment from day 2 to day 10 post-treatment.

[0030] Fig. 7A shows the results of the treatment of full-thickness wounds using Super Serum without laser pretreatment from day 1 to day 10 post-treatment.

[0031] Fig. 7B shows the results of the treatment of full-thickness wounds using Super Serum with laser pretreatment from day 1 to day 10 post-treatment.

[0032] Fig. 8A shows the averaged results of the treatments of third-degree burns and the averaged results of the treatments of the wounds (wound: deep dermal and full-thickness wounds) with and without laser pretreatment using Super Serum (non-BSL) or Super Serum PLE (BSL). The average percentage of epithelialization of third-degree burns is the average of the results of Figs 5A-5D. The average percentage of epithelialization of the wounds is the average of the deep dermal and full-thickness wounds of Figs. 6A-6B.

[0033] Fig. 8B shows the averaged results of the treatments of third-degree burns with and without laser pretreatment using Super Serum (non-BSL) or Super Serum PLE (BSL).

[0034] Fig. 8C shows the averaged results of the treatments of wounds (deep dermal and full-thickness wounds) with and without laser pretreatment using Super Serum (non-BSL) or Super Serum PLE (BSL).

[0035] Fig. 9A, 9B, 9C, and 9D show exemplary study designs for wound healing in animals.DETAILED DESCRIPTION

[0036] The terms “a,” “an,” “the” and similar referents used in the context of describing the invention (especially in the context of the claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0037] The term “activated” biological sample, such as “an activated skin sample”, “activated skin”, or “a sample of activated skin (thereof)”, refers to an activated amphibian skin with increased functional activity for performing a function, such as inducing gene or protein expression required for wound healing, inhibiting inflammation, or for treating other skin conditions. The increase in functional activity is compared to a control sample that has not been activated.

[0038] The term “activation light” refers to light that has been used to activate a biological sample. The activation light can be a broad spectrum light (BSL) having a wavelength ranging from 100 nm to 1 mm and includes infrared (IR), visible, and ultraviolet (UV) light. UV light includes UVC (100 nm to 280 nm), UVB (280 nm to 320 nm), and UVA (320 nm to 400 nm). Visible light has a wavelength of 400 nm to 750 nm and includes violet (400 nm to 450 nm), indigo (450 nm to 485 nm), blue (485 nm to 500 nm), green (500 nm to 565 nm), yellow (565 nm to 590 nm), orange (590 nm to 625 nm), and red (625 nm to 750 nm). IR light includes IRA (750 to 1 .4 pm), IRB (1.4 IR pm to 3 pm), and IRC, also known as far-IR (3 pm to 1 mm).

[0039] The term “antigen” refers to a molecule that is a toxin or is foreign to the subject and induces an immune response in the form of the production of antibodies against the molecule. The compositions or activated skin described herein can have reduced antigenicity as compared to native skin, such that it can be used in a subject.

[0040] The term “bioactivity” refers to biological effects. A substance is bioactive or has bioactivity includes a substance having a biological function.

[0041] The term "biocompatible" refers to a product and its normal degradation products in vitro, ex vivo, or in vivo that are substantially non-toxic and non-carcinogenic to a cell, tissue, organ, organism, or subject within useful, practical, and / or acceptable tolerances. The term "cytocompatible" refers to a product that can sustain the viability and growth of a population of cells.

[0042] The term “biomaterial” refers to a material suitable for in vitro, ex vivo, or in vivo use. As an example of in vivo use, the biomaterial is suitable for administering to a subject in need thereof. The material can be synthetic or natural. The compositions and activated skin described herein are examples of biomaterial.

[0043] The term "carrier" or “excipient” refers to a substance added to a composition that does not affect the active compound in the composition. The carrier can be a diluent. The excipient can be a substance added to the composition to facilitate the administration of the composition.

[0044] The term “cosmetics” refers to products (excluding pure soap) intended to be applied to the human body for cleansing, beautifying, promoting attractiveness, or altering the appearance. Examples of cosmetic benefits can include improving the appearance of skin such as improving the appearance of wrinkling and fine lines, removing oil and excess sebum, reducing the appearance of skin blemishes, cleansing, and conditioning the skin, toning, and tightening the skin, soothing irritation, and refreshing and cooling the skin.

[0045] The term “drugs,” “pharmaceuticals,” or “therapeutics” refers to articles intended for use in the diagnosis, cure, mitigation, treatment, or prevention of disease and articles (other than food) intended to affect the structure or any function of the body of man or other animals.

[0046] The term "derive", "derived," or "derives" refers to a product obtained from any stated source by any useful method. For example, an activated skin derived from an amphibian refers to an activated skin obtained from a member of the amphibian class.

[0047] The term “exogenous” refers to a product that originated outside of the organism, tissue, cell, organ, or subject. In contrast, the term “endogenous” refers to a product that originated from the organism, cell, tissue, organ, or subject.

[0048] The term “extracellular matrix” or “ECM” refers to a natural scaffolding having a three- dimensional structure including biomolecules and minerals that provide biochemical support to surrounding cells. The ECM includes structural and non-structural biomolecules, such as collagens, elastins, laminins, glycosaminoglycans, proteoglycans, antimicrobials, chemoattractants, cytokines, and / or growth factors. The ECM can be obtained from various sources of tissues including the skin and non-cutaneous tissues.

[0049] The term "decellularized extracellular matrix" or "decellularized ECM" refers to ECM prepared by removing and / or devitalizing cells from ECM found in multicellular organisms, for example, amphibians or mammals. Decellularized ECM is substantially free of intact cells, lysed cells, and cellular components including cellular and nuclear debris such that the decellularized ECM exhibits reduced immunogenicity so that it can be administered to a subject, for example, a mammalian subject, as a non-toxic xenograft or biomaterial. A decellularized ECM that is “substantially free of immunogenic components” refers to an ECM in which immunogenic components are at a level that is not sufficient to induce an adverse immune response in a subject.

[0050] The term "isolated" refers to being separated or removed from its native surroundings, such that it is substantially free from components that accompany it in its naturally-occurring state. For example, a cell or a protein can be isolated from its naturally- occurring state.

[0051] The term “immunogenic” refers to relating to or producing an immune response. The term “immunogenicity” refers to the ability of a foreign substance, such as an antigen, to provoke an immune response in a subject. The compositions and activated skin described herein can have reduced immunogenicity as compared to the native skin, such that it can be used as a biomaterial in a subject.

[0052] The term "non-toxic" refers to a product that causes little or no adverse reaction or substantial harm to cells and tissues in vitro or ex vivo, and / or does not cause a substantial adverse or undesirable reaction or substantial harm to cells and tissues in the body (in vivo).

[0053] The term “prevent” or “prevention” refers to the prevention of the onset, recurrence, or spread of a condition or one or more symptoms of the condition. As an example, the condition could be a skin condition. The term includes the administration of a product described herein before the onset of symptoms in particular to subjects at risk of developing a condition, such as a skin condition. The term includes the inhibition or reduction of one or more symptoms associated with the skin condition. The term “prevention” can be used interchangeably with the term “prophylactic treatment”.

[0054] The term “retain structural and functional integrity" used with reference to the ECM refers to retaining sufficient structure and function to permit and support the use of the matrix as a substrate for the growth of cells in vivo, ex vivo, or in vitro. For example, the decellularized ECM retains the structure and functional properties of a naturally occurring ECM enabling its use as a biomaterial.

[0055] The terms “scaffold” and “bioscaffold” are used interchangeably to refer to a substrate on which cells can grow in vitro, ex vivo, and / or in vivo. A scaffold or bioscaffold is an example of a biomaterial.

[0056] The terms “skin” and “skin sample” are used interchangeably to refer to the skin or a sample of skin from a subject.

[0057] The term “skin conditions” includes skin conditions that require therapeutic “drug” treatment including diseases, defects, and injuries including wounds, such as full-thickness wounds, deep dermal wounds, and burns.

[0058] The term “cosmetic skin conditions” includes skin conditions that are related to tone, clarity, radiance, brightness, elasticity, thinning, firmness, pore size, inflammation, and / or hydration of the skin.

[0059] The term "subject" refers to an animal, for example, a mammal. Examples of mammals include humans, dogs, cats, horses, cows, goats, sheep, pigs, and non-human primates. A subject in need of treatment or a subject in need thereof includes a subject having a disease or condition that needs to be treated. A subject in need thereof also includes a subject that needs treatment and / or prevention of a skin condition.

[0060] The term "therapeutically effective amount" refers to an amount of a product or composition that provides a therapeutic benefit in the treatment, prevention, or management of a condition or disease, such as a skin disease, an injury to the skin, or a wound, for example, a drug product. The term “therapeutically effective amount” also includes that amount of a compound that, when administered, is sufficient to prevent the development of, or alleviate to some extent, one or more of the signs or symptoms of the disorder or disease being treated.

[0061] The term "treatment" or "treating" in the context of administering a product, such as a biomaterial, to a subject refers to administering the product to achieve a desirable clinical / medical end-point, including alleviating symptoms of a disease or condition. Examples of such desirable end-points associated with skin disease or condition include wound healing, tissue closure, bulking tissue, preventing tissue adhesion, providing structural support to tissue, providing a protective barrier, and / or correcting a defect. Administering the product also includes applying the product to a subject.

[0062] The term “xenogenic” refers to a product derived or originated from a member of another species.

[0063] The term “amphibians” refers to cold-blooded vertebrate animals that include frogs, toads, newts, salamanders, and caecilians. They have an aquatic gill-breathing larval stage followed by a terrestrial lung-breathing adult stage. Amphibians include the class of amphibians and the orders of Anura (frogs or toads), Urodela (newts or salamanders), and Apoda (caecilians). In embodiments, the amphibians described herein are young or neotenic amphibians. A young amphibian includes a young frog, such as a froglet, tadpole, or larval stage young Apoda. The amphibian can be a young amphibian and the young amphibian can include a larval stage of any order of amphibian. The amphibian can be neotenic.

[0064] The term “salamanders” refers to a group of amphibians characterized by a lizard-like appearance and having a tail throughout life. The families of salamanders include the Ambystomatidae (mole salamanders), Amphiumidae (Congo eels), Cryptobranchidae (giant salamanders), Dicamptodontidae (Pacific giant salamanders), Hynobiidae (Asiatic salamanders), Plethodontidae (lungless salamanders), Proteidae (mudpuppies and olms), Rhyacotritonidae (torrent salamanders), Salamandridae (newts and true salamanders), and Sirenidae (sirens). The Ambystomatidae family includes Ambystoma altamirani, Ambystoma amblycephalum, Ambystoma andersoni, Ambystoma annulatum, Ambystoma barbourin, Ambystoma bishop, Ambystoma bombypellum, Ambystoma californiense, Ambystoma cingulated, Ambystoma dumerilii, Ambystoma flavipiperatum, Ambystoma gracile, Ambystoma granulosum, Ambystoma jeffersonianum, Ambystoma laterale, Ambystoma leorae, Ambystoma lermaense, Ambystoma mabeei, Ambystoma macrodactylum, Ambystoma maculatum, Ambystoma mavortium, Ambystoma mexicanum, Ambystoma opacum, Ambystoma ordinarium, Ambystoma rivulare, Ambystoma rosaceum, Ambystoma silvense, Ambystoma subsalsum, Ambystoma talpoideum, Ambystoma taylori, Ambystoma texanum, Ambystoma tigrinum, and Ambystoma velasci.

[0065] The families of salamanders are grouped under the order Urodela (or Caudata). The term “Urodele” refers to a salamander of the order Urodela, in the class Amphibia. The amphibians can be from the orders Urodela, Anura, and Apoda. Urodeles begin life as aquatic animals in a larval state, and some undergo metamorphosis from a juvenile form with gills to an adult, terrestrial, air-breathing form with lungs. During metamorphosis, a Urodele's physical features are altered in preparation for life on land. These alterations include caudal fin resorption, thickening of the skin, the development of dermal glands, and resorption of gills. Sexual maturity also occurs during this time in most Urodeles. However, some families of Urodeles are "neotenic," which means that individuals of such families, even after reaching sexual maturity, retain their juvenile aquatic form throughout their lives. The axolotl (Mexican walking fish), Ambystoma mexicanum, and / or hybrids of A. mexicana and A. tigrinum are examples of neotenic salamanders. Instead of becoming a terrestrial amphibian, an adult axolotl remains aquatic and gilled. However, under certain circumstances, an axolotl will undergo metamorphosis and transform into a terrestrial form.

[0066] Axolotls possess pigment cells, called chromatophores, that are responsible fortheir colors. The chromatophores of axolotls include melanophores containing eumelanin, xanthophores containing pteridines, and iridophores containing crystallized purines. Eumelanin is a black-brown pigment; pteridine is a yellow and reddish pigment; and crystallized purine is an iridescent white pigment. These pigments which are encoded by their respective genesprovide the different phenotypes of axolotls, such as wild-type, golden albino, leucistic, and melanistic.

[0067] Axolotls have the ability to fully regenerate lost or damaged body parts including organs, limbs, and parts of the central nervous system, throughout their entire life. Axolotls undergo rapid re-epithelialization during wound healing and limb regeneration, both of which are scar-less processes. The axolotl wound healing process resembles the scar-free healing process of mammalian fetal and embryonic wounds. Such wounds exhibit re-epithelialization and basement membrane reformation that occur at a faster rate than do the corresponding events in postnatal mammals.

[0068] Although the skin structure of amphibians is similar to mammals, Urodeles and anuran amphibians (frogs and toads) can regenerate their skin structures including the dermis and secretion glands without forming any scar after a deep skin injury. Moreover, the skin of amphibians contains ECM, which is rich in growth factors, which are favorable for wound healing. The ECM is a three-dimensional network of extracellular macromolecules and minerals including collagen, enzymes, glycoproteins, and hydroxyapatite which provide structural and biochemical support to surrounding cells. The ECM can include a combination of fibrous and network-type collagens. Examples of various types of collagens, such as one or more of type I, II, III, IV, V, and VI collagens. The ECM can also include elastin and / or elastic fibers. The ECM can also include laminin, fibronectin, hyaluronan, chondroitin sulfate, or both, and / or one or more proteoglycan, glycoprotein, glycosaminoglycan, or any combination thereof. The components and structure of the ECM play an important role in the healing process because the ECM components create scaffolding which provides the structural architecture of the matrix required for the healing process. Moreover, the ECM components are involved in stimulating the adhesion and migration of cells during the healing process as well as mediating the interactions among the cells and between the cells and the matrix, or between ECM proteins during the healing process. Further, the ECM components also serve as a reservoir and modulator of the action of the cytokines and growth factors to regulate wound repair activities.

[0069] The present disclosure describes activated skin from an amphibian that can be used to treat various skin conditions. The activated amphibian skin has enhanced functional activity as evidenced by gene expression studies in the human skin model. The activated amphibian skin modulates the expression of one or more wound healing genes including NRG1 (neuregulin-1), TIAM1 (TIAM Rac1 associated GEF 1), TM7SF2 (transmembrane 7 superfamily member 2), GLRX (glutaredoxin), F3 (coagulation factor III, tissue factor), FETUB (fetuin B), and LEP (leptin). The activated amphibian skin modulates the expression of one or more genes associated with inflammation including CASP1 (caspase 1), CCL2 (C-C motif chemokine ligand 2), CSF2 (colony stimulating factor 2), CXCL5 (C-X-C motif chemokine ligand 5), CXCL8 (C-X-Cmotif chemokine ligand 8), DEFB4A (defensin beta 4A), IL18 (interleukin 18), IL1 A (interleukin 1 alpha), IL1 B (interleukin 1 beta), IL1 RL1 (interleukin 1 receptor like 1), IL1 RN (interleukin 1 receptor antagonist), IL24 (interleukin 24), IL33 (interleukin 33), JUN (Jun proto-oncogene, AP-1 transcription factor subunit), LTB4R (leukotriene B4 receptor), MYD88 (MYD88 innate immune signal transduction adaptor), NFKB1 (nuclear factor kappa B subunit 1), NFKB2 0, NLRP1 (NLR family pyrin domain containing 1), PTGS1 (prostaglandin-endoperoxide synthase 1), PTGS2 (prostaglandin-endoperoxide synthase 2), PYCARD (ASC; a bipartite protein containing a pyrin domain (PYD) and a caspase recruitment domain (CARD)), TLR3 (toll like receptor 3), TLR5 (toll like receptor 5), PYDC1 (pyrin domain containing 1), TNFSF10 (TNF superfamily member 10), and IL6 (interleukin 6). Moreover, the activated amphibian skin modulates the expression of one or more skin barrier genes including SCARB1 (scavenger receptor class B member 1), SPRR3 (small proline-rich protein 3), SPRR4 (small proline-rich protein 4), KLK5 (kallikrein-related peptidase 5), AQP3 (aquaporin 3), CAPN1 (calpain 1), ICAM1 (intercellular adhesion molecule 1), ALOX12B (arachidonate 12-lipoxygenase, 12R type), ALOXE3 (arachidonate lipoxygenase 3), AQP9 (aquaporin 9), BLMH (bleomycin hydrolase), TLR2 (tolllike receptor 2), CERS3 (ceramide synthase 3), FLG (filaggrin), and EPHX3 (epoxide hydrolase 3).

[0070] The activated amphibian skin modulates the hyaluronan genes including HAS2 (hyaluronan synthase 2), HAS3 (hyaluronan synthase 3), AQP3 (aquaporin 3), and CD44 (P- glycoprotein 1). Hyaluronan is a naturally occurring glycosaminoglycan component of the ECM. It plays an important role in CD44-mediated cell signaling, wound healing, tissue regeneration, and ECM composition.

[0071] The activated amphibian skin modulates the elastin gene, ELN. The primary function of elastin is to allow tissues to stretch and return to their original size. It is one of the most abundant proteins in the human body.

[0072] The activated amphibian skin modulates the collagen genes including COL1A1 (collagen type 1 alpha 1 chain) and COL3A1 (collagen type 3 alpha 1 chain). Collagen provides structure, support, elasticity, and strength to the skin. Collagen is the main structural protein of the ECM. It helps fibroblasts form in the dermis which helps new cells to grow. It plays a role in replacing dead skin cells and provides a protective covering for an organ.

[0073] The activated amphibian skin modulates the sirtuin (SIRT) genes including SIRT1, SIRT3, SIRT5, SIRT6, and SIRT7. Sirtuins are important regulators of the DNA damage response.

[0074] The activated amphibian skin modulates the inflammatory genes including TNF-a (tumor necrosis factor-alpha), NFKB1 (nuclear factor kappa B subunit 1), NFKB2 (nuclear factor kappa B subunit 2), matrix metalloproteinase (MMP), such as MMP1 , MMP2, MMP3, MMP7,MMP9, MMP10, MMP11 , and IL-1 B (interleukin-1 beta). These genes regulate functions related to inflammation.

[0075] The activated amphibian skin modulates the longevity genes including FOXO1 (forkhead box protein 01) and FOXO3 (forkhead box protein 03) and the regeneration genes including MARCKS (myristoylated alanine-rich C-kinase substrate) and MARCKSL1 (myristoylated alanine-rich C-kinase substrate like 1).

[0076] The activated amphibian skin also modulates the expression of TIMM 44 (translocase of inner mitochondrial membrane 44), INO80 (chromatin remodeling complexes, ATPases), ATP5F1 B (ATP synthase F1 subunit beta), POLE (DNA polymerase epsilon), and GOLGB1 (giantin or golgin B member 1). These genes are involved in wound healing. Their more specific functions are described below.

[0077] The gene ATP5F1B, also known as ATP5PB, encodes a subunit of the mitochondrial ATP synthase, specifically the ATP synthase F1 subunit beta. This enzyme, located in the mitochondrial membrane, is crucial for cellular energy production.

[0078] The INO80 complex is involved in the repair of DNA damage. It helps in the regulation and stabilization of broken DNA ends during the repair process, ensuring that the genetic material is accurately repaired and maintained.

[0079] The role of GOLGB1 is important in cellular function and health, contributing to a range of cellular activities from protein trafficking to maintaining cellular architecture and responding to cellular stress.

[0080] During the wound healing process, particularly in the proliferation phase, rapid cell division is required to replace lost or damaged cells. Upregulating POLE could enhance the fidelity and speed of DNA replication in these cells, potentially speeding up the tissue repair process.

[0081] Energy-intensive processes like cell proliferation and migration are essential for effective wound healing. By boosting mitochondrial function and energy production, the upregulation of TIMM44 could support these dynamic cellular activities, leading to faster and more efficient wound closure and tissue regeneration.

[0082] The expression of these genes is upregulated or downregulated by 0.5-fold to 2000- fold change as compared to the expression of these genes modulated by a control. The control is either amphibian skin that has not been activated by light (without light exposure) or a sample without amphibian skin. The expression of these genes is upregulated or downregulated as compared to the control by a change of 0.75 to 2000-fold, 1.0 to 1800-fold, 1.5 to 1900-fold, 2.0 to 900-fold, 2.0 to 1700-fold, 2.0 to 1600-fold, 2.0 to 1500-fold, 2.0 to 1400-fold, 2.0 to 1300- fold, 2.0 to 1200-fold, 2.0 to 1100-fold, 2.0 to 1000 fold, 2.0 to 900-fold, 2.0 to 800-fold, 2.0 to 700-fold, 2.0 to 600-fold, 2.0 to 500-fold, 2.0 to 400-fold, 2.0 to 300-fold, 2.0 to 200-fold, 2.0 to100-fold, 2.0 to 75-fold, 2.0 to 60.0-fold, 2.0 to 50-fold, 2.0 to 40-fold, 2.0 to 30-fold, 2.0 to 20- fold, 2.0 to 10-fold, or 2.0 to 5-fold.

[0083] The skin of any amphibian can be activated by exposing it to light. The activated skin can be obtained from an amphibian. In embodiments, the activated skin can be obtained from a young amphibian, such as a young Anura, neotenic Urodele (neotenic newt or salamander), or young Apoda. In embodiments, the activated skin is obtained from a neotenic salamander, such as an axolotl. Moreover, axolotls possess different pigments in their skin which are responsible for an axolotl’s skin color (phenotype). The present disclosure also describes skin compositions obtained from the different phenotypes of axolotls such as wild-type, golden albino, leucistic, or a combination thereof.

[0084] The skin of a living amphibian can be activated using an activation light. The activation light can be shined from one or more directions with respect to the amphibian. For example, the activation light can be shined from above, from the right or left side, from the bottom, or from a combination of directions with respect to an amphibian living in a container of water. The activation light can be broad spectrum light having a wavelength of 100 nm to 1 mm which includes UV, visible, and IR light. The activation light can have various ranges of wavelengths including 100 nm to 750 nm (UV and visible light), 280 nm to 800 nm (UVB, UVA, visible, and IRA), 100 nm to 400 nm (UVC, UVB, and UVA), 400 nm to 750 nm (visible light), or 400 nm to 800 nm (visible and IRA). The activation light can also have a wavelength of 400 nm to 450 (violet), 450 nm to 485 nm (indigo), 485 nm to 500 nm (blue), 500 nm to 565 nm (green), 565 nm to 590 nm (yellow), 590 nm to 625 nm (orange), 625 nm to 750 nm (red), or a combination of these wavelengths. The activation light can include UVA, UVB, UVC, red, orange, yellow, green, blue, indigo, violet, IRA, or a combination thereof.

[0085] The power of the activation light can be 15 watts to 35 watts, 20 watts to 30 watts, 20 watts, 21 watts, 22 watts, 23 watts, 24 watts, 25 watts, 26 watts, 27 watts, 28 watts, 29 watts, or 30 watts.

[0086] The skin of the amphibian can be exposed to (shined with) the activation light for 10 minutes (mins) to 10 hours (hrs). The amphibian can be exposed to the light for 15 mins to 8 hrs, 30 mins to 7 hrs, 45 mins to 6 hrs, 60 mins to 5 hrs, 75 mins to 4 hrs, 80 mins to 3 hrs, 85 mins, 90 mins, 95 mins, 100 mins, 105 mins, 110 mins, 115 mins, 120 mins, 125 mins, 130 mins, 135 mins, 140 mins, 145 mins, 150 mins, 155 mins, 160 mins, 165 mins, 170 mins, or 180 mins. In embodiments, the skin of the amphibian is exposed to activation light for 60 mins to 180 mins. In embodiments, the skin of the amphibian is exposed to activation light for 120 mins.

[0087] The activated skin of the amphibian can be harvested 60 mins to 90 hrs post-light exposure (PLE), for example, the activated skin of the amphibian can be harvested after waiting 60 mins to 90 hrs after light exposure. The activated skin of the amphibian can be harvested 60mins to 84 hrs, 60 mins to 70 hrs, 60 mins to 60 hrs, 60 mins to 50 hrs, 60 mins to 40 hrs, 60 mins to 30 hrs, 60 mins to 20 hrs, 2 hrs to 60 hrs, 2 hrs to 48 hrs, 2 hrs to 36 hrs, 2 hrs to 24 hrs, 60 mins, 2 hrs, 4hrs, 8 hrs, 12 hrs, 24 hrs, 36 hrs, 48 hrs, or 60 hrs PLE. In embodiments, the skin of the amphibian is harvested 12 hrs to 84 hrs PLE. PLE, the activated skin can be removed from the amphibian by degloving. Skin can be removed from the limbs, torso, head, and tail.

[0088] The present disclosure describes compositions comprising activated skin obtained from PLE amphibians. The compositions can include the skin from one or more limbs, belly, back, head, tail, or a combination thereof from one or more amphibians. The activated skin compositions described herein can be from a single amphibian body part or a mixture containing varying amounts, such as 0.01% to 99% of skin from the limbs, belly, back, head, and tail of the amphibian in the amount. The activated skin compositions described herein can also contain skin from only a limb, only one or more limbs, only the belly, only the back, only the head, or only the tail.

[0089] The compositions described herein can also include activated skin from one axolotl of a specific phenotype or a combination of varying amounts (0.01% to 99%) of different phenotypes of axolotls, such as wild-type, golden albino, or leucistic. Moreover, the different body parts of these axolotl phenotypes can be mixed to form a composition.

[0090] In embodiments, the activated skin compositions described herein can be decelluarized to obtain decellularized activated ECM. Decellularization of the ECM is the removal of cells and cellular components from the ECM of a biological sample, such as a tissue, while retaining the ECM proteins and the native ECM architecture or structure for effective use as a biomaterial. Methods for decellularizing amphibian ECM are described in WO 2023 / 154873 and WO 2023 / 154876, which are incorporated by reference in their entirety.

[0091] In embodiments, the activated skin compositions described herein can be prepared into various forms including a powder containing the micronized particulates, which can be reconstituted with water, a buffered solution, or any suitable liquid for use as a solution, a paste, a liquid, an extract, a cream, a lotion, a serum, an emollient, an ointment, gel, gelatin, a hydrogel, a dispersion, or an emulsion. The activated skin compositions described herein also can be gelatinized to form gelatin as described in WO 2023 / 154876, which is incorporated by reference in its entirety. The activated skin can be gelatinized after decellularization or without decellularization.

[0092] The present disclosure describes activated skin compositions including one or more carriers or excipients. Examples of carriers and excipients include saline, emulsion, a mixture of organic solvents with water, calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils, petrolatum, lanolin, mineral oil, dimethicone,humectant, and polyethylene glycols. Examples of humectants include glycerin, lecithin, and propylene glycol. In embodiments, the compositions described herein include cosmetic, or pharmaceutical compositions containing one or more cosmetically, or pharmaceutically acceptable carriers or excipients, respectively.

[0093] The compositions described herein can include one or more agents such as one or more therapeutic agents. Examples of therapeutic agents include known drugs such as retinoic acid, corticosteroids, antifungals, antivirals, antibiotics, antiseptics, local anesthetics, and antineoplastics.

[0094] The compositions described herein can include one or more agents such as one or more cosmetic agents. Examples of cosmetic agents include antioxidants, peptides, alpha or beta hydroxy acids, retinol, vitamins, plant extracts, skin clarifying agents such as arbutin, moisturizing agents such as hyaluronic acid, emollients, carbohydrates, glycoproteins, and / or polymers. The one or more agents can include a combination of agents. The agent can be exogenous or xenogenic to the compositions or activated skin described herein.

[0095] Examples of one or more peptides and proteins include growth factors, cytokines, and chemokines. Examples of growth factors include fibroblast growth factors (FGFs) including acidic FGF, basic FGF, FGF8, and FGF10; ciliary neurotrophic factor (CNTF); epidermal growth factor (EGF); granulocyte-macrophage colony-stimulating factor (GM-CSF); hepatocyte growth factor (HGF); insulin-like growth factors 1 and 2 (IGF-1 and IGF-2); keratinocyte growth factor (KGF); nerve growth factor (NGF); neurotrophins such as neurotrophin-3, neurotrophin-4, neurotrophin-5; platelet-derived growth factor (PDGF); vascular endothelial growth factor (VEGF); stromal-derived factor 1 alpha (SDF-1 alpha); and transforming growth factor-alpha and -beta (TGF-a and TGF-|3). Examples of cytokines and chemokines include tumor necrosis factor-alpha (TNF-a), interleukin-1 alpha and beta (IL-1 a and IL-1 P), interleukin-6 (IL-6), interleukin-7 (IL-7), interleukin-18 (IL-18), CCL2, CCL3, CCL5, CXCL1 , CXCL4, CXCL5, CXCL7, CXCL8, and CXCL12. Examples of cosmetic peptides include acetyl hexapeptide, acetyl tetrapeptide, palmitoyl pentapeptide, and palmitoyl oligopeptide.

[0096] Examples of one or more therapeutic agents include antimicrobials and antiinflammatory agents. Examples of antimicrobials include antibiotics such as penicillin, streptomycin, amoxicillin, cephalexin, clindamycin, dicloxacillin, and doxycycline. Other antimicrobials include anti-microbial peptides, silver salts, clotrimazole, miconazole, and ketoconazole. Examples of anti-inflammatory agents include nonsteroidal anti-inflammatory drugs (NSAIDs) such as salicylic acid, ibuprofen, naproxen, colchicine, fenoprofen, sulindac, diflunisal, diclofenac, indoprofen, and sodium salicylamide.

[0097] Examples of cosmetic agents include one or more glycoproteins including proteoglycans which are proteins covalently attached to glycosaminoglycans (GAGs),antioxidants, ascorbic acid, vitamin C, alpha hydroxy acids (AHAs), beta hydroxy acids (BHAs), exfoliants, skin whitening agents, light diffusers, UV absorbing agents, sunscreens, moisturizers, anti-wrinkle ingredients, and oil absorbing agents. Examples of AHAs include glycolic acid, lactic acid, malic acid, tartaric acid, and citric acid. Examples of BHAs include salicylic acid.

[0098] The compositions described herein can also include one or more natural and / or synthetic polymers. Natural polymers can be from an animal source or a non-animal source such as a plant source. Examples of natural polymers include natural polymers such as collagen, chitosan, alginate, glycosaminoglycans, fibrin, and hyaluronic acid. Examples of synthetic polymers include polyethylene, polyethylene glycol (PEG), polyethylene terephthalate (PET, or PETE), polytetrafluoroethylene (PTFE), polylactic acid (PLA), polyglycolic acid (PGA), polyethylene glycol (PEG), polyethylene glycol) diacrylate (PEG diacrylate), poly(hydroxy acids), polydioxanone, polycaprolactone, poly(ortho esters), poly(anhydrides), polyphosphazene, poly(amino acids), pseudo-poly(amino acids), conductive polymers (such as polyacetylene, polypyrrole, polyaniline), polyurethane, polystyrene, and nitinol.

[0099] The polymer of the compositions described herein can be biocompatible, biodegradable, and / or bioabsorbable, and can be a random copolymer, block copolymer, or blend of monomers, homopolymers, copolymers, and / or heteropolymers that contain these monomers. Exemplary biodegradable or bioabsorbable polymers include polylactides, polyglycolides, polycaprolactones, polydioxanes, and their random and block copolymers. A biodegradable and / or bioabsorbable polymer can contain a monomer selected from the group consisting of glycolide, lactide, dioxanone, caprolactone, trimethylene carbonate, ethylene glycol, and lysine. The biodegradable and / or bioabsorbable polymers can contain bioabsorbable and biodegradable linear aliphatic polyesters such as polyglycolide (PGA) and its random copolymer poly(glycolide-co-lactide-) (PGA-co-PLA). Other examples of suitable biocompatible polymers include polyhydroxyalkyl methacrylate, ethylmethacrylate, polyvinylpyrrolidone, and polyacrylamides. Other suitable bioabsorbable materials are biopolymers which include collagen, gelatin, alginic acid, chitin, chitosan, fibrin, hyaluronic acid, dextran, polyamino acid, polylysine, and copolymers of these materials. Any combination of polymers and copolymers or blend thereof of the above examples can also be included in the composition.

[0100] The compositions described herein can also include protectives, adsorbents, demulcents, emollients, preservatives, antioxidants, moisturizers, buffering agents, solubilizing agents, skin-penetration enhancers, and surfactants.

[0101] Any skin penetration enhancer can be added to the compositions or activated skin described herein, provided the skin penetration enhancer is safe and can effectively facilitate the passage of the desired substances across the skin membrane. Examples of skinpenetration enhancers include dimethyl sulphoxide (DMSO), monoglycerides, C10-C20 fatty acid esters including ethyl palmitate and isopropyl myristate; acyl lactylates such as caproyl lactylic acid and lauroyl lactylic acid; dimethyl lauramide; dodecyl (lauryl) acetate; lactate esters such as lauryl lactate, and myristyl lactate; monoalkyl ethers of polyethyleneglycol and their alkyl or aryl carboxylic acid esters and carboxymethyl ethers such as polyethylene glycol-4 lauryl ether (Laureth-4) and polyethylene glycol-2 lauryl ether (Laureth-2); Myreth-3, myristyl sarcosine, and methyl laurate; polypropylene glycol, polyethylene glycol, lecithin, urea, amino acids, 1-dodecylhexahydro-2H-azepine 2-one (Azone), oleic acid, linoleic acid, isopropyl linoleate, oleyl alcohol, 1-dodecyl-azacycloheptan-2-one, butanediol, and 2-(2- Ethoxyethoxy)ethanol (Transcutol).

[0102] The compositions and activated skin described herein can be prepared as a dry powder, a solution, a paste, a liquid, an extract, a cream, a lotion, a serum, an emollient, an ointment, a dispersion, gel, hydrogel, gelatinized composition, or an emulsion. The compositions and activated skin can be prepared into a variety of suitable shapes and sizes as they can be formed, laminated, homogenized, and reconstituted. They can be formed into two- dimensional or three-dimensional shapes. They can be formed into a sheet, mesh, graft, plug, or any shape or form for use. The sheets can include backing with or without an adhesive. The backing can be biodegradable or non-biodegradable. Two or more sheets can be laminated together or somehow attached. The sheets can be oriented in the same direction, different direction, or at an angle. There can be two to fifteen layers of sheets. The sheets can be from different sources of amphibians.

[0103] In embodiments, the powder containing the micronized particulates can be reconstituted with water, a buffered solution, or any suitable liquid and / or carriers to form a solution, a paste, a liquid, an extract, a cream, a lotion, a serum, an emollient, an ointment, a dispersion, or an emulsion. The solution can be a buffered solution, similar to that used for the decellularization process.

[0104] The activated skin described herein has been shown to have enhanced functional activity as compared to control. Accordingly, the compositions and activated skin described herein can be used in vitro, ex vivo, and / or in vivo. Because the activated skin has various bioactivities that are involved in regenerative cellular processes, the activated skin and the compositions described herein can be used as a biomaterial for wound healing, regeneration of tissue, scaffold for growing cells, and treating and preventing various skin conditions. Since the activated skin contains ECM, it can also serve as a scaffold, cells can grow on the ECM to enhance the regeneration and wound healing of tissues and organs. The activated skin can serve as a biomaterial that can also be applied to, coated on, and / or infused in an implant or a medical device for introduction into a subject. The medical device can be a patch, a bandage, orany suitable device for delivering the biomaterial. Thus, the biomaterial can be used as a material for any xenogenic transplantation or xenograft.

[0105] The biomaterial can also serve as a system or device for delivering agents to tissues and organs for wound healing and treating and / or preventing various skin conditions. The endogenous agents, such as growth factors, in the biomaterial, can be delivered via immediate release or controlled release. Moreover, exogenous or xenogenic agents described added to the biomaterial can be delivered via immediate release or controlled release.

[0106] Carriers for immediate or sustained release preparations include polymers. The polymers can be biodegradable, and / or bioabsorbable. As an example, for controlled release, the biomaterial can be coated with polymers such as acrylic polymer, acrylic / methacrylic copolymer, cellulose acetate phthalate (CAP), Opadry®, and Ethocel™. For immediate release, the biomaterial can be coated with cellulosic polymers, such as hydroxypropyl methylcellulose (HPMC), hydroxypropyl cellulose (HPC), hydroxyethylcellulose (HEC), methyl cellulose (MC), and sodium carboxymethyl cellulose (NaCMC); vinyl derivatives, such as polyvinyl pyrrolidone (PVP), polyvinyl pyrrolidone-polyvinyl acetate copolymer, polyvinyl alcohol (PVA), and polyvinyl alcohol-polyethylene glycol copolymer; acrylic polymers, such as Eudragit®; or glycols such as polyethylene glycols.

[0107] The activated skin and compositions described herein can be used to treat skin conditions including wounds and inflammations and for remodeling the dermal matrix ECM, regenerating and / or protecting the skin barrier, and enhancing the general induction of longevity. The activated skin and compositions described herein can be prepared as a formulation for treating and / or preventing various skin conditions including inflamed skin and / or injured skin having a wound or disrupted skin barrier. The formulation can be in the form of a dry powder, a solution, a paste, a liquid, an extract, a cream, a lotion, a serum, an emollient, an ointment, a dispersion, a gel, a hydrogel, a gelatin, or an emulsion.

[0108] Examples of inflammatory skin conditions include psoriasis; dermatitis, such as contact dermatitis, atopic dermatitis (eczema), seborrheic dermatitis, nummular dermatitis, generalized exfoliative dermatitis, stasis dermatitis, lichen simplex chronicus; disorders of hair follicles and sebaceous glands, such as acne, rosacea and rhinophyma, perioral dermatitis, and pseudofolliculitis barbae; and inflammatory reactions, such as drug eruptions, erythema multiforme, erythema nodosum, and granuloma annulare. Other skin conditions needing treatment include fine lines and / or wrinkles, aging, redness, abrasion, burns, cuts, infection, razor bumps, scars, uneven skin tone, pain, stretch marks, skin elasticity and / or firmness, skin hydration, and hyperpigmentation. The burns include acute thermal burns including first, second, or third-degree burns.

[0109] The activated skin and the compositions described herein can also be used in a skincare regimen for protecting the skin from damage including UV rays and environmental pollution and as an aesthetic agent for improving the appearance of the skin.

[0110] The activated skin and compositions described herein can be administered to the target site topically, or by injection, implantation, microneedling, or radiofrequency microneedling, or using an ablative fractional laser. The activated skin and compositions described herein can be administered to the subject before, during, or after a dermatological or cosmetic procedure, such as dermabrasion, microdermabrasion, and ablative laser resurfacing. The dermatological or cosmetic procedure includes procedures wherein at least one cell of the stratum corneum is removed. The activated skin and compositions described herein can also be delivered as an injectable or with a dermal or subdermal implant such as a volume filler, hyaluronic acid, or other dermal matrix protein including collagen or elastin, either naturally occurring, bioengineered, or recombinantly produced. The activated skin and compositions described herein can be administered alone or in combination with one or more agents described herein, such as growth factors, peptides, and proteins. The activated skin and compositions can also be administered with toxins, such as botulinum toxin.

[0111] The activated skin and compositions described herein can reduce inflammation, reduce scarring, reduce keloid formation, reduce or diminish the severity of scarring and keloid formation, and / or reduce healing time for wound healing and various dermatological and cosmetic procedures. The activated skin and compositions described herein can also be used to restore lost dermal matrix or subdermal volume.

[0112] The activated skin and compositions described herein can be used to treat wounds and enhance wound healing without any scarring. The compositions described herein can be applied topically, for example directly to the wound or indirectly, by applying it to a substrate that is used to cover the wound.

[0113] Before treatment with the activated skin or compositions described herein, the wound site can be treated using laser, micro-coring, microneedling, or any dermatological or cosmetic procedures wherein at least one cell of the stratum corneum is removed. These procedures can assist in the delivery of the activated blastemas and enhance wound healing without scarring. Treatment with laser includes treating the wound with an ablative fractional laser. Microneedling includes treating the wound with radiofrequency microneedling.

[0114] The present disclosure also describes methods of using the activated skin and compositions described herein to modulate one or more mammalian genes involved in wound healing, inflammation, and / or protection of the skin. The expression of the one or more mammalian genes can be upregulated or downregulated. The mammalian genes can be involved in wound healing of the skin, inflammation of the skin, and / or protection of the skinbarrier. The method includes administering the activated skin or compositions described herein to cells, in vitro, in vivo, or ex vivo to upregulate or downregulate genes. The cells can be in a 3- D skin model, in a cell culture, or in a subject. The subject can be a mammal and the mammal can be a human.

[0115] The present disclosure describes kits including the activated skin or compositions described herein for the various uses. The kits can include sterilized activated skin or a composition thereof in any shape and form. The kits can include a solution for reconstituting the activated skin for use. The kits can include a device for administering the activated skin or composition to a subject. The kits can include an implant to be coated with the activated skin or composition before being implanted in a subject. The kits can include components for the various uses described herein.

[0116] The present disclosure also describes a medical device comprising the activated skin or compositions described herein for the various uses. A medical device can be a material or an object used directly or indirectly to apply the activated skin or compositions described herein. As an example, a medical device can be used to apply the activated skin or compositions described herein on the skin of a subject.

[0117] The present disclosure also describeds methods of preparing activated amphibian skin described herein. The method comprises exposing an amphibian to broad spectrum activation light for a period to activate the skin, and harvesting the activated skin from the amphibian to obtain activated amphibian skin or a sample thereof.

[0118] In embodiments, the method comprises exposing the amphibian to activation light for 10 minutes (mins) to 10 hours (hrs), 15 mins to 8 hrs, 30 mins to 7 hrs, 45 mins to 6 hrs, 60 mins to 5 hrs, 75 mins to 4 hrs, 80 mins to 3 hrs, 85 mins, 90 mins, 95 mins, 100 mins, 105 mins, 110 mins, 115 mins, 120 mins, 125 mins, 130 mins, 135 mins, 140 mins, 145 mins, 150 mins, 155 mins, 160 mins, 165 mins, 170 mins, or 180 mins.

[0119] In embodiments, the method comprises harvesting the activated skin from the amphibian 60 mins to 90 hrs post-light exposure (PLE), 60 mins to 84 hrs PLE, 60 mins to 70 hrs PLE, 60 mins to 60 hrs PLE, 60 mins to 50 hrs PLE, 60 mins to 40 hrs PLE, 60 mins to 30 hrs PLE, 60 mins to 20 hrs PLE, 2 hrs to 60 hrs PLE, 2 hrs to 48 hrs PLE, 2 hrs to 36 hrs PLE, 2 hrs to 24 hrs PLE, 60 mins PLE, 2 hrs PLE, 4hrs PLE, 8 hrs PLE, 12 hrs PLE, 24 hrs PLE, 36 hrs PLE, 48 hrs PLE, or 60 hrs PLE.

[0120] The activated skin described herein can be decellularized to obtain an activated decellarized ECM.

[0121] The activated skin described herein can be gelatinized to obtain gelatinized activated skin. Moreover, the activated skin described herein can be gelatinized after decellularizing toobtain gelatinized decellularized ECM. In embodiments, the activated skin can be gelatinized with or without decellularizing.

[0122] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0123] Numbers expressing ranges or quantities of ingredients, constituents, reaction conditions, and so forth used in the specification and claims are to be understood as being modified by the term "about." When further clarity is required, the term “about” has the meaning reasonably ascribed to it by a person skilled in the art when used in conjunction with a stated numerical value or range, i.e. denoting somewhat more or somewhat less than the stated value or range, to within a range of ± 20% of the stated value; ± 15% of the stated value; ± 10% of the stated value; ± 5% of the stated value; ± 4% of the stated value; ± 3% of the stated value; ± 2% of the stated value; ± 1% of the stated value; or ± any percentage between 1% and 20% of the stated value.

[0124] As will be understood by one of ordinary skill in the art, each embodiment disclosed herein can comprise, consist essentially of, or consist of its particular stated element, step, ingredient, or component. Thus, the terms “include” or “including” should be interpreted to recite: “comprise, consist of, or consist essentially of.” The transition term “comprise” or “comprises” means includes, but is not limited to, and allows for the inclusion of unspecified elements, steps, ingredients, or components, even in major amounts. The transitional phrase “consisting of’ excludes any element, step, ingredient, or component not specified. The transition phrase “consisting essentially of’ limits the scope of the embodiment to the specified elements, steps, ingredients, or components and to those that do not materially affect the embodiment. In embodiments, the lack of a material effect of a step is evidenced by the lack of a statistically-significant reduction in the process step in removing cellular debris such as DNA from the sample. Lack of a material effect of an embodiment can include a lack of a statistically- significant improvement in using the activated amphibian skin for wound healing.

[0125] Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. The description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to havespecifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as individual numbers within that range, for example, 1 , 2, 2.5, 2.7, 3, 4, 5, 5.1 , 5.3, 5.8 and 6. Moreover, any ranges cited herein are inclusive.

[0126] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member may be referred to and claimed individually or in any combination with other members of the group or other elements found herein.

[0127] The following exemplary embodiments and examples illustrate exemplary methods provided herein. These exemplary embodiments and examples are not intended, nor are they to be construed, as limiting the scope of the disclosure. It will be clear that the methods can be practiced otherwise than as particularly described herein. Numerous modifications and variations are possible in view of the teachings herein and, therefore, are within the scope of the disclosure.EXEMPLARY EMBODIMENTS

[0128] The following are exemplary embodiments:1 . Activated amphibian skin or a sample thereof, wherein the activated skin has increased functional activity as compared to a control skin or a control skin sample.2. The activated amphibian skin or a sample thereof, wherein the activated skin or sample thereof is obtained from an amphibian exposed to activation light, and wherein the control skin or skin sample is obtained from an amphibian that was not exposed to activation light.3. The activated skin or a sample thereof of embodiment 1 or 2, wherein the activation light is a broad spectrum light (BSL) source including wavelength in the range of 100 nm to 1 mm, 100 nm to 800 nm, 100 nm to 750 nm, 280 nm to 800 nm, 100 nm to 400 nm, 400 nm to 750 nm, or 400 nm to 800 nm.4. The activated skin or a sample thereof of any one of embodiments 1-3, wherein the increase in functional activity of the activated skin or sample thereof is based on changes in gene expression in human skin induced by the activated skin.5. The activated skin or a sample thereof of any one of embodiments 1-4, wherein the increased functional activity includes modulation in expression of one or more genes in human skin as compared to a control by a change of 0.75 to 2000-fold, 1.0 to 1800-fold, 1.5 to 1900-fold, 2.0 to 900-fold, 2.0 to 1700-fold, 2.0 to 1600-fold, 2.0 to 1500-fold, 2.0 to 1400-fold, 2.0 to 1300-fold, 2.0 to 1200-fold, 2.0 to 1100-fold, 2.0 to 1000 fold, 2.0 to 900-fold, 2.0 to 800-fold, 2.0 to 700-fold, 2.0 to 600-fold, 2.0 to 500-fold, 2.0 to 400-fold, 2.0 to 300-fold, 2.0 to 200-fold, 2.0 to 100-fold, 2.0 to 75-fold, 2.0 to 60.0-fold, 2.0 to 50- fold, 2.0 to 40-fold, 2.0 to 30-fold, 2.0 to 20-fold, 2.0 to 10-fold, or 2.0 to 5-fold.The activated skin or a sample thereof of any one of embodiments 1-5, wherein the increased functional activity includes upregulation or downregulation of the expression of one or more genes in human skin. The activated skin or a sample thereof of any one of embodiments 1-6, wherein the increased functional activity includes upregulation or downregulation of the expression of one or more human genes associated with wound healing, inflammation, regeneration of the skin barrier, and / or protection of the skin barrier. The activated skin or a sample thereof of any one of embodiments 1-7, wherein the increased functional activity includes modulation in expression of one or more of the following human genes: NRG1 , TIAM1 , TM7SF2, GLRX, F3, FETUB, LEP, CASP1 , CCL2, CSF2, CXCL5, CXCL8, DEFB4A, IL18, IL1A, IL1 B, IL1 RL1 , IL1 RN, IL24, IL33, JUN, LTB4R, MYD88, NFKB1 , NFKB2, NLRP1 , PTGS1, PTGS2, PYCARD, TLR3, TLR5, PYDC1 , TNFSF10, IL6, SCARB1 , SPRR3, SPRR4, KLK5, AQP3, CAPN1 , ICAM1 , ALOX12B, ALOXE3, AQP9, BLMH, EPHX3, TIMM44, INO80, ATP5F1B, POLE, GOLGB1 , TLR2, CERS3, FLG, HAS2, HAS3, AQP3, CD44, ELN, COL1A1 , COL3A1 , SIRT1 , SIRT3, SIRT5, SIRT6, SIRT7, TNF-a, NFKB1 , NFKB2 MMP1 , MMP2, MMP3, MMP7, MMP9, MMP10 and MMP11 and IL1B, FOXO1 and FOXO3, MARCKS, and MARCKSL1 . The activated skin or a sample thereof of any one of embodiments 1-8, wherein the increased functional activity includes modulation in expression of: one or more genes associated with wound healing including NRG1 , TIAM1 , TM7SF2, GLRX, F3, FETUB, LEP, TIMM44, INO80, ATP5F1 B, POLE, and GOLGB1 ; one or more genes associated with inflammation including CASP1 , CCL2, CSF2, CXCL5, CXCL8, DEFB4A, IL18, IL1 A, IL1 B, IL1RL1 , IL1 RN, IL24, IL33, JUN, LTB4R, MYD88, NFKB1 , NFKB2, NLRP1 , PTGS1 , PTGS2, PYCARD, TLR3, TLR5, PYDC1 , TNFSF10, and IL6; and / or one or more genes associated with regeneration and / or protection of the skin barrier including SCARB1 , SPRR3, SPRR4, KLK5, AQP3, CAPN1 , ICAM1, ALOX12B, ALOXE3, AQP9, BLMH, and EPHX3. The activated skin or a sample thereof of any one of embodiments 1 -9, wherein the amphibian is a frog, toad, newt, or salamander. The activated skin or a sample thereof of any one of embodiments 1-10, wherein the amphibian is a young amphibian, and optionally, wherein the young amphibian includes a froglet, tadpole, Urodele, or larval stage young Apoda. The activated skin or a sample thereof of any one of embodiments 1-11 , wherein the amphibian is a neotenic Urodele.The activated skin or a sample thereof of any one of embodiments 1-12, wherein the amphibian is an axolotl. The activated skin or a sample thereof of any one of embodiments 1-13, wherein the amphibian is an axolotl having one of the following phenotypes: wild-type, golden albino, or leucistic. The activated skin or sample thereof of any one of embodiments 1-14, wherein the activated skin or sample thereof includes activated skin from one or more limbs, belly, back, head, tail, or a combination thereof from one or more amphibians. The activated skin or a sample thereof of any one of embodiments 1-15, wherein the activated skin or sample thereof includes decellularized ECM. A composition including the activated skin or a sample thereof of any one of embodiments 1-16 and a carrier. The composition of embodiment 17, wherein the composition is a pharmaceutical composition or cosmetic composition and the carrier is a pharmaceutically acceptable carrier or cosmetically acceptable carrier. The composition of embodiment 17 or 18, wherein the composition further includes one or more agents xenogenic to the activated skin. The composition of any one of embodiments 17-19, wherein the composition further includes one or more agents including peptides, proteins, drugs, nutrients, retinoids, emollients, steroids, carbohydrates, glycoproteins, polymers, or a combination thereof; and optionally, wherein the proteins or peptides include growth factors, cytokines, or chemokines; optionally, wherein the one or more polymers includes synthetic or natural polymers or copolymers; optionally, wherein the drugs include retinoic acid, corticosteroids, antifungals, antivirals, antibiotics, antiseptics, local anesthetics, and antineoplastics; and optionally, wherein the drugs include neomycin, polymyxin B, bacitracin, or a combination thereof. The composition of any one of embodiments 17-20, wherein the composition is in the form of a gel, dry powder, paste, solution, serum, extract, cream, lotion, dispersion, or emulsion. A method of preparing the activated skin or a sample thereof of any one of embodiments 1-16, wherein the method includes exposing the amphibian to broad spectrum activation light for a period to obtain activated skin and harvesting the activated skin from the amphibian. The method of embodiment 22, wherein the method includes exposing the amphibian to activation light for 10 minutes (mins) to 10 hours (hrs), 15 mins to 8 hrs, 30 mins to 7 hrs, 45 mins to 6 hrs, 60 mins to 5 hrs, 75 mins to 4 hrs, 80 mins to 3 hrs, 85 mins, 90 mins,95 mins, 100 mins, 105 mins, 110 mins, 115 mins, 120 mins, 125 mins, 130 mins, 135 mins, 140 mins, 145 mins, 150 mins, 155 mins, 160 mins, 165 mins, 170 mins, or 180 mins. The method of embodiment 22 or 23, wherein the method includes harvesting the activated skin from the amphibian 60 mins to 90 hrs post-light exposure (PLE), 60 mins to 84 hrs PLE, 60 mins to 70 hrs PLE, 60 mins to 60 hrs PLE, 60 mins to 50 hrs PLE, 60 mins to 40 hrs PLE, 60 mins to 30 hrs PLE, 60 mins to 20 hrs PLE, 2 hrs to 60 hrs PLE, 2 hrs to 48 hrs PLE, 2 hrs to 36 hrs PLE, 2 hrs to 24 hrs PLE, 60 mins PLE, 2 hrs PLE, 4hrs PLE, 8 hrs PLE, 12 hrs PLE, 24 hrs PLE, 36 hrs PLE, 48 hrs PLE, or 60 hrs PLE. The method of any one of embodiments 22-24, wherein the method further includes decellularizing the activated skin or a sample thereof to obtain an activated skin or a sample thereof including decellularized ECM. The method of any one of embodiments 22-24, wherein the method further includes gelatinizing the activated skin or a sample thereof to obtain gelatinized activated skin or a sample thereof. A method of treating or preventing a skin condition of a subject including administering the activated skin or a sample thereof of any one of embodiments 1-16 or the composition of any one of embodiments 17-21 to the subject. The method of embodiment 27, wherein the skin condition includes inflamed skin or injured skin including a wound or disrupted skin barrier. The method of embodiment 27 or 28, wherein the skin condition includes psoriasis, dermatitis, such as contact dermatitis, atopic dermatitis (eczema), seborrheic dermatitis, nummular dermatitis, generalized exfoliative dermatitis, stasis dermatitis, lichen simplex chronicus; disorders of hair follicles and sebaceous glands, including acne, rosacea, rhinophyma, perioral dermatitis, and pseudofolliculitis barbae; and inflammatory reactions, such as drug eruptions, erythema multiforme, erythema nodosum, and granuloma annulare. The method of embodiment 27 or 28, wherein skin condition includes fine lines and / or wrinkles, aging, redness, abrasion, burn, cut, infection, razor bumps, scars, uneven skin tone, pain, stretch marks, skin elasticity and / or firmness, skin hydration, and hyperpigmentation. The method of embodiment 27 or 28, wherein the skin condition includes acute thermal burns including first, second, or third-degree burns. The method of embodiment 27 or 28, wherein preventing a skin condition includes protecting the skin from damages caused by UV rays and / or environmental pollution.33. The method of embodiment 27 or 28, wherein the method further includes treating the skin with a laser before administering the activated skin or a sample thereof.34. The method of embodiment 33, wherein the skin is treated with ablative fractional laser.35. The method of any one of embodiments 27-31 , wherein the activated skin or a sample thereof is delivered to the subject by injection.36. A method of modulating expression of one or more mammalian genes of the skin, wherein the method includes administering the activated skin or a sample thereof of any one of embodiments 1-16 or the composition of any one of embodiments 17-21 to mammalian cells to upregulate or downregulate one or more genes of the mammalian skin.37. The method of embodiment 36, wherein the method upregulates or downregulates one or more genes associated with wound healing, inflammation of the skin, regeneration of the skin barrier, and / or protection of the skin barrier.38. The method of any one of embodiments 36 or 37, wherein the cells include in vitro, in vivo, or ex vivo cells.39. The method of any one of embodiments 36-38, wherein the mammalian cells are human cells.40. The method of any one of embodiments 36-39, wherein the cells are in a subject.EXAMPLES

[0129] Example 1. Activating Axolotl Skin

[0130] Axolotls were submerged in 3 centimeters (cms) of water to leave minimal observable space between the top of their bodies and the water surface. The light was placed at 8 cms from the surface of the water. Light was shined from above on the axolotl living in water for 2 to 5 hrs to activate the axolotl’s skin. The source of the light was a ReptiSun® Compact Fluorescent lamp (10% UVB output, 30% UVA output, and full spectrum). At 36 hrs, 48 hrs, and 60 hrs, after light removal (post-light exposure, PLE), the axolotl was euthanized and the skin was harvested.

[0131] It was determined that 1 to 3 hrs, preferably 2 hrs of light exposure is optimal for activating axolotl skin.

[0132] Example 2. Harvesting Activated Skin from Axolotls

[0133] Degloving of the torso and limbs o Starting at the pithing incision below the gills, scissors were used to cut the skin all the way around the neck. Next, the skin around the tail was cut using a hole created from the removal of the hind legs. o Then, using the incision around the neck, the skin was cut along the edge of the dorsal fin until it met the incision around the tail.o The previous steps were repeated using the previous incision along the alternate side of the dorsal fin. o A final cut was made to remove the dorsal fin between the two vertical incisions previously created. o The tissue scissors were used to begin to carefully pry the skin away from the trunk muscle and cartilage, ensuring no tears were made. o This process was continued until the entire skin of the trunk had been loosened, removed, and maintained unity. o The torso was separated from the limbs using the tissue scissors. o The explanted skin was placed in a clean and treated container of water. o The tissue was rinsed and placed in the numbered, PBS-filled, transport bottle. o The tissue was subsequently decellularized and / or prepared into a gel formulation for use. The gel formulation can be applied directly or onto a disc for application on an injury. The process for decellularization and preparation into the gel formulations is described in WO 2023 / 154876, which is incorporated by references in its entirety.

[0134] Example 3. Genemarker Studies

[0135] Genemarker studies were conducted to determine how the different samples of activated axolotl skin modulate gene expression in human skin. A 3D in vitro skin model (MatTek EFT-400) containing epidermal keratinocytes and dermal fibroblasts was used. Differential gene expression was assessed after 24 hours of exposure to the materials (TMs) using mRNA-Seq. The TM groups are activated axolotl skin samples as prepared in Examples 1 and 2. The unique controls are non-activated (no light exposure) axolotl skin samples. The study design included nineteen TM groups, three replicates each (N=3). Sequencing was performed using Illumina's NextSeq 550Dx instrument. The following 37 test material groups were included in the study (N=3, Dose A through Dose C):1 . Vehicle Control #1 (no skin sample)2. TM #1 -Dose A through Dose C (Golden Albino, dorsal)3. TM #2-Dose A through Dose C (Golden Albino, belly (ventral))4. TM #3-Dose A through Dose C (Golden Albino, limb (leg))5. TM #4-Dose A through Dose C (Leucistic, dorsal)6. TM #5-Dose A through Dose C (Leucistic, belly)7. TM #6-Dose A through Dose C (Leucistic, limb)8. TM #7-Dose A through Dose C (wild-type, dorsal)9. TM #8-Dose A through Dose C (wild-type, belly)10. TM #9-Dose A through Dose C (wild-type, limb)11 . Unique control- 1a-Dose A through Dose C (Golden albino, dorsal)12. Unique control- 1 b-Dose A through Dose C (Golden Albino, belly)13. Unique control- 1c-Dose A through Dose C (Golden Albino, limb)14. Unique control-2a-Dose A through Dose C (Leucistic, dorsal)15. Unique control-2b-Dose A through Dose C (Leucistic, belly)16. Unique control-2c-Dose A through Dose C (Leucistic, limb)17. Unique control-3a-Dose A through Dose C (wild-type, dorsal)18. Unique control-3b-Dose A through Dose C (wild-type, belly)19. Unique control-3-Dose A through Dose C (wild-type, limb)

[0136] The results of genemarker studies are shown in Tables 1A-28. A 1.5-fold change or greater indicates that the change is clinically significant, group.

[0137] Table 1A: Golden Albino (GA) Back 36 hrs PLE vs. GA Back Without (W / O) Light Exposure

[0138] Table 1 B: GA Back 36 hrs PLE vs. Vehicle Control

[0139] Table 2A: GA Back 48 hrs PLE vs. GA Back W / O Light Exposure

[0140] Table 2B: GA Back 48 hrs PLE vs. Vehicle Control

[0141] Table 3A: GA Back 60 hrs PLE vs. GA Back vs. W / O Light Exposure

[0142] Table 3B: GA Back 60 hrs PLE vs.Vehicle Control

[0143] Table 4A: GA Belly 36 hrs PLE vs. GA Belly W / O Light Exposure

[0144] Table 4B: GA Belly 36 hrs PLE vs. Vehicle Control

[0145] Table 5: GA Belly 48 hrs PLE vs. Vehicle Control

[0146] Table 6A: GA Belly 60 hrs PLE vs. GA Belly W / O Light Exposure

[0147] Table 6B: GA Belly 60 hrs PLE vs. Vehicle Control

[0148] Table 7A: GA Limb 36 hrs PLE vs. GA Limb W / O Light Exposure

[0149] Table 7B: GA Limb 36 hrs PLE vs. Vehicle Control

[0150] Table 8A: GA Limb 48 hrs PLE vs. GA Limb W / O Light Exposure

[0151] Table 8B: GA Limb 48 hrs PLE vs. Vehicle Control

[0152] Table 9A: GA Limb 60 hrs PLE vs. GA Limb W / O Light Exposure

[0153] Table 9B: GA Limb 60 hrs PLE vs. Vehicle Control

[0154] Table 10A: Leucistic Back 36 hrs PLE vs. Leucistic Back Without (W / O) LightExposure

[0155] Table 10B: Leucistic Back 36 hrs PLE vs. Vehicle Control

[0156] Table 11A: Leucistic Back 48 hrs PLE vs. Leucistic Back Without (W / O) LightExposure

[0157] Table 11 B: Leucistic Back 48 hrs PLE vs. Vehicle Control

[0158] Table 12A: Leucistic Back 60 hrs PLE vs. Leucistic Back W / O Light Exposure

[0159] Table 12B: Leucistic Back 60 hrs PLE vs. Vehicle Control

[0160] Table 13A: Leucistic Belly 36 hrs PLE vs. Leucistic Belly W / O Light Exposure

[0161] Table 13B: Leucistic Belly 36 hrs PLE vs. Vehicle Control

[0162] Table 14A: Leucistic Belly 48 hrs PLE vs. Leucistic Belly W / O Light Exposure

[0163] Table 14B: Leucistic Belly 48 hrs PLE vs. Vehicle Control

[0164] Table 15: Leucistic Belly 60 hrs PLE vs. Vehicle Control

[0165] Table 16A: Leucistic Limb 36 hrs PLE vs. Leucistic Limb W / O Light Exposure

[0166] Table 16B: Leucistic Limb 36 hrs PLE vs. Vehicle Control

[0167] Table 17A: Leucistic Limb 48 hrs PLE vs. Leucistic Limb W / O Light Exposure

[0168] Table 17B: Leucistic Limb 48 hrs PLE vs. Vehicle Control

[0169] Table 18A: Leucistic Limb 60 hrs PLE vs. Leucistic Limb W / O Light Exposure

[0170] Table 18B: Leucistic Limb 60 hrs PLE vs. Vehicle Control

[0171] Table 19A: Wild-Type Back 36 hrs PLE vs. Wild-Type Back W / O Light Exposure

[0172] Table 19B: Wild-Type Back 36 hrs PLE vs. Vehicle Control

[0173] Table 20A: Wild-Type Back 48 hrs PLE vs. Wild-Type Back W / O Light Exposure

[0174] Table 20B: Wild-Type Back 48 hrs PLE vs. Vehicle Control

[0175] Table 21 A: Wild-Type Back 60 hrs PLE vs. Wild-Type Back W / O Light Exposure

[0176] Table 21 B: Wild-Type Back 60 hrs PLE vs. Vehicle Control

[0177] Table 22A: Wild-Type Belly 36 hrs PLE vs. Wild-Type Belly W / O Light Exposure | Gene Name | Linear Fold Change | Function |

[0178] Table 22B: Wild-Type Belly 36 hrs PLE vs. Vehicle Control

[0179] Table 23A: Wild-Type Belly 48 hrs PLE vs. Wild-Type Belly W / O Light Exposure

[0180] Table 23B: Wild-Type Belly 48 hrs PLE vs. Vehicle Control

[0181] Table 24A: Wild-Type Belly 60 hrs PLE vs. Wild-Type Belly W / O Light Exposure

[0182] Table 24B: Wild-Type Belly 60 hrs PLE vs. Vehicle Control

[0183] Table 25A: Wild-Type Limb 36 hrs PLE vs. Wild-Type Limb W / O Light Exposure

[0184] Table 25B: Wild-Type Limb 36 hrs PLE vs. Vehicle Control

[0185] Table 26A: Wild-Type Limb 48 hrs PLE vs. Wild-Type Limb W / O Light Exposure

[0186] Table 26B: Wild-Type Limb 48 hrs PLE vs. Vehicle Control

[0187] Table 27A: Wild-Type Limb 60 hrs PLE vs. Wild-Type Limb W / O Light Exposure| BLMH | -2.42773| Skin Barrier

[0188] Table 27B: Wild-Type Limb 60 hrs PLE vs. Vehicle Control

[0189] T able 28 Genes with 200-1000 + Linear Fold Change

[0190] Example 4. Animal Studies

[0191] The objective of these studies is to determine the ability of activated skin to enhance healing using a porcine wound model.

[0192] Test Samples. Gel formulations and discs comprising non-activated axolotl skin and activated axolotl skin were applied directly to the different wounds (burns, full-thickness, anddeep dermal). The gel formulations contain decellularized ECM obtained from a non-activated axolotl skin sample (Super Serum) or activated axolotl skin sample (Super Serum PLE). The discs comprising decellularized ECM obtained from non-activated axolotl (Matrix) or activated axolotl skin (Matrix PLE) were applied to the different wounds. The axolotl skin samples were activated by exposing the axolotl skin to activation light for 2 hrs as described in Example 1 and harvested 60 hrs PLE as described in Example 2.

[0193] Experimental Animals. Porcine models were used for our experimental research due to the morphological similarities between swine skin and human skin. Two (2) female animals’ specific pathogen-free (B. G. Looper Farm 4673 Petra Mill Road Granite Falls, NC 28630) pigs weighing 40-45 kg were kept in-house for at least 5 days prior to initiating the experiment to allow the animals to acclimatize. One animal received third-degree burns and the other either full-thickness wounds or deep dermal wounds as described below. The animals were fed a basal diet ad libitum and housed individually in our animal facilities (meeting American Association for Accreditation of Laboratory Animal Care [AAALAC] accredited) with controlled temperature (19-21°C) and lighting (12h / 12h LD).

[0194] Wound Models

[0195] The wound models include the third-degree burn model, deep dermal wound model, and 20 mm full-thickness wound model.

[0196] Third Degree Burn Model. Figs. 9A and 9C show an exemplary study design for third-degree burn wounds. Third-degree burn wounds were made on the paravertebral and thoracic areas. Burn wounds were created by using a branding iron (L & H Manufacturing Company Mandan, North Dakota 58554) with a heat controller that was set to 300°C. The iron was held at a vertical position on the skin for 15 seconds, with pressure supplied by gravity, to make a burn wound of 27mm in diameter and with a depth of approximately 3mm (to subcutaneous tissue). The wounds were separated from one another by 5-7 cm of unwounded skin. The wounds were randomly assigned to the treatment groups with 3 wounds per treatment as seen in Figs. 37A and 37C. One of three wounds was first lasered prior to treatment.

[0197] 20 mm Full Thickness Wound Model. Fig. 9B shows an exemplary study design for full-thickness wounds. Twenty-four (24) full-thickness wounds were made on the paravertebral and thoracic area with a 20mm circular biopsy punch (Fig. 9B). The wounds were separated from one another by 5-7 cm of unwounded skin. The wounds were randomly assigned to treatment groups with 3 wounds per treatment as seen in Fig. 9B. The animal was treated within 20 minutes after the creation of the wound. One of three wounds was first lasered prior to treatment.

[0198] Deep Dermal Wound Model. Fig. 9D shows an exemplary study design for deep dermal wounds. Twenty-five (25) deep reticular dermal wounds measuring (22 mm x 22 mm x 3mm, L x W x D) were made in the paravertebral and thoracic area with a specialized electrokeratome fitted with a 22 mm blade. The wounds were separated from one another by 5- 7 cm of unwounded skin. The wounds were randomly assigned to treatment groups with 3 wounds per treatment as seen in Fig. 9D. One of three wounds was first lasered prior to treatment.

[0199] Treatment Regimen. Within the first hour after wounding, some of the wounds were treated with an erbium YAG ablative fractional laser. This YAG is used to create millions of tiny dots that represent columns of tissue that were removed. The columns act as channels to enhance the delivery of topical agents, allowing for better penetration into the wounded area. The laser was set at 15 mJ for micropulse energy at a rate of 300 Hz and a repeat delay of 30 seconds.

[0200] Immediately after lasering, all the third-degree burn wounds and deep dermal wounds received 200 L of the gel formulation containing the activated tissue that was spread with a sterile spatula to cover the wounded area and surrounding unwounded skin and covered with Tegaderm dressing. All the 20 mm full-thickness wounds received 200 pL of the gel formulation. Untreated Control wounds for the animals were covered with polyurethane film dressing (Tegaderm; 3M, St. Paul, MN). After application, all treatment groups were covered with Tegaderm.

[0201] All wounds were treated daily for 7 days. For the third-degree burn wounds, after 7 days, all wounds were covered with non-adherent gauze until the final assessment time. For the deep dermal wounds, after treatment application on Day 6 (Day 7 after wounding), all wounds were covered with non-adherent gauze and then Tegaderm dressing. For the 20 mm fullthickness wounds, on Day 7, wounds were treated with non-adherent gauze until the final assessment time. All dressings were secured in place with tape and covered with Coban wrap (3M, St. Paul MN).

[0202] Histological Assessment. On assessment times (Day 14 for third-degree burns and Day 10 for deep dermal wounds and full-thickness wounds), incisional biopsies were taken from each treatment group using a sterile scalpel. Biopsies were obtained through the center of the wounds including normal adjacent skin on both sides. These specimens were placed in formalin and then stained with hematoxylin and eosin (H&E). One section per block was analyzed. The specimens were then scanned on an Olympus VS120 slide scanner and blindly evaluated for the following parameters to determine a potential treatment response:

[0203] Percent of wound epithelialized (%). Measurement of the length of the wound surface that has been covered with epithelium.

[0204] Epithelial thickness (cell layers pm). The epithelial thickness may vary from area to area within the biopsy. The thickness of the epithelium in pm was measured on five equal distance points from each other in the biopsy and averaged.

[0205] White cell infiltrate. Measured by the presence and amount of subepithelial mixed leukocytic infiltrates. Mean Score: 1 = absent, 2 = mild, 3 = moderate, 4 = marked, 5 = exuberant.

[0206] Granulation Tissue Formation. The approximate amount of new granulation tissue formation (dermis) was graded as follows: 0 = 0, 0.5 = 1-10%, 1 = 11-30%, 2 = 31-50%, 3 = 51- 70%, 4 = 71-90%, 5= 91-100%.

[0207] Clinical Observations (Erythema).

[0208] Erythema - Indicative of the amount of inflammation present*

[0209] * Score: 1 = absent, 2 = mild, 3 = moderate, 4 = marked, 5 = exuberant

[0210] All animals exhibited no erythema (absent score) on all wounds throughout the duration of the study.

[0211] Digital Photography & Measurement of the Wound Contraction. Wounds from each group were photographed and the area of the wound was traced to measure wound contraction. The wound circumference was traced by digital imaging with Imaged and compared to Day 0 to determine the degree of wound contraction.

[0212] Results.

[0213] Histological Results.

[0214] The data were combined and analysis was performed to determine the treatment response. The percentage of epithelialization and the other histological parameters in wounds were plotted against days after treatment. Assessments for third-degree burn wounds, deep dermal wounds, and full-thickness wounds were on days 14, 10, and 10 respectively. Since there were only one, two, or three samples for each treatment group, only means were used for tabulation and graphs for presentation purposes.

[0215] Percentage of Epithelialization. The percentage of epithelialization represents the area of the wound area covered by a newly formed epidermis with one or more layers of keratinocytes, which is a good index for the speed of keratinocyte migration and the first step of the epithelialization. The percentages of epithelialization for the different trials using the different wound models treated with activated skin and compared to control are shown in Figs. 29A-31 B and 33A-35B. The results show that activated skin harvested PLE 60 hrs can be used with or without laser pretreatment to effectively treat different wounds. However, the wounds with laser pretreatment seem to heal better than without laser treatment.

[0216] Epithelial thickness. The epithelial thickness was a measure of an average thickness of five points of newly formed epithelium. Epithelial thickness reflects the process of keratinocyte proliferation, differentiation, and epidermal maturation.

[0217] White Cell Infiltration (WCI). WCI is used to assess the inflammation reaction.

[0218] Granulation Tissue Formation. The dermal reconstitution begins in about 3 to 4 days of injury with the hallmark of granulation tissue formation, which includes new blood vessel formation (angiogenesis), accumulation of fibroblasts, and collagen extracellular matrices. The granulation tissue formation measures the percentage of wound beds filled with newly formed granulation tissue.

[0219] Planimetry Measurements

[0220] The wound areas were combined for each treatment group at each time point (every 3- 4 days) and were analyzed for comparative quantitative analysis. A comparison of the wound area of different treatments using activated skin applied after laser application for third-degree burn wounds. On Day 14, the wound area is lower than the untreated control confirming that the activated skin is effective in treating wounds.

[0221] Tables 29A-29D show the results of the treatment of third-degree burns using Matrix or Matrix PLE with or without laser pretreatment. These results show the percent epithelialization of wounds on day 14 post-treatment. Matrix comprises decellularized ECM from axolotl skin sample that has not been activated. Control is an untreated wound. The “Spot #” indicates the spot on the animal.

[0222] Table 29A: Results of the treatment of third-degree burns using Matrix without laser pretreatment (Fig. 1A).

[0223] Table 29B: Results of the treatment of third-degree burns using Matrix with laser pretreatment (Fig. 1 B).

[0224] Table 29C: Results of the treatment of third-degree burns using Matrix PLE without laser pretreatment (Fig. 1C).

[0225] Table 29D: Results of the treatment of third-degree burns using Matrix PLE with laser pretreatment (Fig. 1 D).

[0226] Tables 30A and 30B show the results of the treatment of deep dermal wounds using Matrix PLE with and without laser pretreatment. These results show percent epithelialization on day 10 post-treatment of wounds treated with Matrix PLE compared with control. The “Spot #” indicates the spot on the animal.

[0227] Table 30A: Results of the treatment of deep dermal wounds using Matrix PLE without laser pretreatment (Fig. 2A).

[0228] Table 30B: Results ofthe treatment of deep dermal wounds using Matrix PLE with laser pretreatment (Fig. 2B).

[0229] Tables 31 A and 31 B show the results of the treatment of full-thickness wounds using Matrix with and without laser pretreatment. These results show percent epithelialization on day 10 post-treatment of wounds treated with Matrix compared with control.

[0230] Table 31A: Results of the treatment of full-thickness wounds using Matrix without laser pretreatment (Fig. 3A).

[0231] Table 31B: Results of the treatment of full-thickness wounds using Matrix with laser pretreatment (Fig. 3B).

[0232] Tables 32A-32C show the averaged results of the treatments of third-degree burns and wounds (deep dermal and full thickness wounds) with and without laser pretreatment using Matrix or Matrix PLE. The average percentage of epithelialization of third-degree burns is the average of the results of Tables 29A-29D. The average percentage of epithelialization of the wounds is the average of the deep dermal and full-thickness wounds of Tables 30A-31 B.

[0233] Table 32A: Averaged results of the treatments of third-degree burns with and without laser pretreatment using Matrix (non-BSL) or Matrix PLE (BSL) (Fig. 4A).*Wound: Deep Dermal and Full-Thickness wounds

[0234] Table 32B: Averaged results of the treatments of third-degree burns with and without laser pretreatment using Matrix (non-BSL) or Matrix PLE (BSL) (Fig. 4B).

[0235] Table 32C: Averaged results of the treatments of wounds (deep dermal and fullthickness wounds) with and without laser pretreatment using Matrix (non-BSL) or Matrix PLE (BSL) (Fig. 4C).

[0236] Tables 33A-33D show the results of the treatment of third-degree burns using Super Serum and Super Serum PLE with and without laser pretreatment from day 1 to day 14 posttreatment. Control is an untreated wound. The “Spot #” indicates the spot on the animal.

[0237] Table 33A: Results of the treatment of third-degree burns using Super Serum without laser pretreatment from day 1 to day 14 post-treatment (Fig. 5A).

[0238] Table 33B: Results of the treatment of third-degree burns using Super Serum with laser pretreatment from day 1 to day 14 post-treatment (Fig. 5B).

[0239] Table 33C: Results of the treatment of third-degree burns using Super Serum PLE without laser pretreatment from day 2 to day 14 post-treatment (Fig. 5C).

[0240] Table 33D: Results of the treatment of third-degree burns using Super Serum PLE with laser pretreatment from day 2 to day 14 post-treatment (Fig. 5D).

[0241] Tables 34A and 34B show the results of the treatment of deep dermal wounds using Super Serum PLE with and without laser pretreatment from day 2 to day 10 post-treatment. The “Spot #” indicates the spot on the animal.

[0242] Table 34A: Results of the treatment of deep dermal wounds using Super Serum PLE without laser pretreatment from day 2 to day 10 post-treatment (Fig. 6A).

[0243] Table 34B: Results of the treatment of deep dermal wounds using Super Serum PLE with laser pretreatment from day 2 to day 10 post-treatment (Fig. 6B).

[0244] Tables 35A and 35B show the results of the treatment of full-thickness wounds using Super Serum with and without laser pretreatment from day 1 to day 10 post-treatment. The “Spot #" indicates the spot on the animal.

[0245] Table 35A: Results of the treatment of full-thickness wounds using Super Serum without laser pretreatment from day 1 to day 10 post-treatment (Fig. 7A).

[0246] Table 35B: Results of the treatment of full-thickness wounds using Super Serum with laser pretreatment from day 1 to day 10 post-treatment (Fig. 7B).

[0247] Tables 36A-36C show the averaged results of the treatments of third-degree burns and the averaged results of the treatments of the wounds (deep dermal and full-thickness wounds) with and without laser pretreatment using Super Serum or Super Serum PLE. The average percentage of epithelialization of third-degree burns is the average of the results of Tables 33A-33D. The average percentage of epithelialization of the wounds is the average of the deep dermal and full-thickness wounds of Tables 34A-35B.

[0248] Table 36A: Averaged results of the treatments of third-degree burns and the averaged results of the treatments of the wounds with and without laser pretreatment using Super Serum (non-BSL) or Super Serum PLE (BSL) (Fig. 8A).Wound: Deep Dermal and Full-Thickness wounds

[0249] Table 36B: Averaged results of the treatments of third-degree burns with and without laser pretreatment using Super Serum (non-BSL) or Super Serum PLE (BSL) (Fig. 8B).

[0250] Table 36C: averaged results of the treatments of wounds (deep dermal and fullthickness wounds) with and without laser pretreatment using Super Serum (non-BSL) or Super Serum PLE (BSL) (Fig. 8C).ounWound: Deep Dermal and Full-Thickness wounds

[0251] The subject matter described above is provided by way of illustration only and should not be construed as limiting. Various modifications and changes may be made to the subject matter described herein without following the example embodiments and applications illustrated and described, and without departing from the true spirit and scope of the present disclosure, which is set forth in the following claims.

[0252] All publications, patents, and patent applications cited in this specification are incorporated herein by reference in their entirety as if each individual publication, patent, or patent application were specifically and individually indicated to be incorporated by reference. While the foregoing has been described in terms of various embodiments, the skilled artisan will appreciate that various modifications, substitutions, omissions, and changes may be made without departing from the spirit thereof.

Claims

CLAIMS1 . Activated amphibian skin, wherein the activated skin has increased functional activity as compared to a control skin, wherein the activated skin is obtained from an amphibian exposed to activation light, and wherein the control skin is obtained from an amphibian that was not exposed to activation light.

2. The activated skin of claim 1 , wherein the activation light is a broad spectrum light (BSL) source comprising wavelength in the range of 100 nm to 1 mm, 100 nm to 800 nm, 100 nm to 750 nm, 280 nm to 800 nm, 100 nm to 400 nm, 400 nm to 750 nm, or 400 nm to 800 nm.

3. The activated skin of claim 1 , wherein the increase in functional activity of the activated skin is based on changes in gene expression in human skin induced by the activated skin.

4. The activated skin of claim 1 , wherein the increased functional activity comprises modulation in expression of one or more genes in human skin as compared to a control by a change of 0.75 to 2000-fold, 1 .0 to 1800-fold, 1.5 to 1900-fold, 2.0 to 900-fold, 2.0 to 1700-fold, 2.0 to 1600-fold, 2.0 to 1500-fold, 2.0 to 1400-fold, 2.0 to 1300-fold, 2.0 to 1200-fold, 2.0 to 1100-fold, 2.0 to 1000 fold, 2.0 to 900-fold, 2.0 to 800-fold, 2.0 to 700- fold, 2.0 to 600-fold, 2.0 to 500-fold, 2.0 to 400-fold, 2.0 to 300-fold, 2.0 to 200-fold, 2.0 to 100-fold, 2.0 to 75-fold, 2.0 to 60.0-fold, 2.0 to 50-fold, 2.0 to 40-fold, 2.0 to 30-fold, 2.0 to 20-fold, 2.0 to 10-fold, or 2.0 to 5-fold.

5. The activated skin of claim 1 , wherein the increased functional activity comprises upregulation or downregulation of the expression of one or more genes in human skin.

6. The activated skin of claim 1 , wherein the increased functional activity comprises upregulation or downregulation of the expression of one or more human genes associated with wound healing, inflammation, regeneration of the skin barrier, and / or protection of the skin barrier.

7. The activated skin of claim 1 , wherein the increased functional activity comprises modulation in expression of one or more of the following human genes: NRG1 , TIAM1 , TM7SF2, GLRX, F3, FETUB, LEP, CASP1 , CCL2, CSF2, CXCL5, CXCL8, DEFB4A, IL18, IL1 A, IL1 B, IL1RL1 , IL1RN, IL24, IL33, JUN, LTB4R, MYD88, NFKB1 , NFKB2, NLRP1 , PTGS1 , PTGS2, PYCARD, TLR3, TLR5, PYDC1 , TNFSF10, IL6, SCARB1 , SPRR3, SPRR4, KLK5, AQP3, CAPN1 , ICAM1 , ALOX12B, ALOXE3, AQP9, BLMH, EPHX3, TIMM44, INO80, ATP5F1B, POLE, GOLGB1 , TLR2, CERS3, FLG, HAS2, HAS3, AQP3, CD44, ELN, COL1 A1 , COL3A1 , SIRT1 , SIRT3, SIRT5, SIRT6, SIRT7, TNF-a, NFKB1 , NFKB2 MMP1 , MMP2, MMP3, MMP7, MMP9, MMP10 and MMP11 and IL1 B, FOXO1 and FOXO3, MARCKS, and MARCKSL1.

8. The activated skin claim 1 , wherein the increased functional activity comprises modulation in expression of: one or more genes associated with wound healing comprising NRG1 , TIAM1 , TM7SF2, GLRX, F3, FETUB, LEP, TIMM44, INO80, ATP5F1 B, POLE, and GOLGB1 ; one or more genes associated with inflammation comprising CASP1 , CCL2, CSF2, CXCL5, CXCL8, DEFB4A, IL18, IL1A, IL1 B, IL1RL1 , IL1 RN, IL24, IL33, JUN, LTB4R, MYD88, NFKB1 , NFKB2, NLRP1 , PTGS1 , PTGS2, PYCARD, TLR3, TLR5, PYDC1 , TNFSF10, and IL6; and / or one or more genes associated with regeneration and / or protection of the skin barrier comprising SCARB1 , SPRR3, SPRR4, KLK5, AQP3, CAPN1 , ICAM1, ALOX12B, ALOXE3, AQP9, BLMH, and EPHX3.

9. The activated skin of claim 1 , wherein the amphibian is a frog, toad, newt, or salamander.

10. The activated skin of claim 1 , wherein the amphibian is a young amphibian, and optionally, wherein the young amphibian comprises a froglet, tadpole, Urodele, or larval stage young Apoda.11 . The activated skin of claim 1 , wherein the amphibian is a neotenic Urodele.

12. The activated skin of claim 1 , wherein the amphibian is an axolotl.

13. The activated skin of claim 1 , wherein the amphibian is an axolotl having one of the following phenotypes: wild-type, golden albino, or leucistic.

14. The activated skin of claim 1 , wherein the activated skin comprises activated skin from one or more limbs, belly, back, head, tail, or a combination thereof from one or more amphibians.

15. The activated skin of claim 1 , wherein the activated skin comprises decellularized ECM.

16. A composition comprising the activated skin of claim 1 and a carrier.

17. The composition of claim 16, wherein the composition is a pharmaceutical composition or cosmetic composition and the carrier is a pharmaceutically acceptable carrier or cosmetically acceptable carrier.

18. The composition of claim 16, wherein the composition further comprises one or more agents xenogenic to the activated skin.

19. The composition of claim 16, wherein the composition further comprises one or more agents comprising peptides, proteins, drugs, nutrients, retinoids, emollients, steroids, carbohydrates, glycoproteins, polymers, or a combination thereof; and optionally, wherein the proteins or peptides comprise growth factors, cytokines, or chemokines; optionally, wherein the one or more polymers comprises synthetic or natural polymers or copolymers; optionally, wherein the drugs comprise retinoic acid, corticosteroids,antifungals, antivirals, antibiotics, antiseptics, local anesthetics, and antineoplastics; and optionally, wherein the drugs comprise neomycin, polymyxin B, bacitracin, or a combination thereof.

20. The composition of claim 16, wherein the composition is in the form of a gel, dry powder, paste, solution, serum, extract, cream, lotion, dispersion, or emulsion.21 . A method of preparing the activated skin of any one of claims 1-16, wherein the method comprises exposing the amphibian to broad spectrum activation light for a period to obtain activated skin and harvesting the activated skin from the amphibian.

22. The method of claim 21 , wherein the method comprises exposing the amphibian to activation light for 10 minutes (mins) to 10 hours (hrs), 15 mins to 8 hrs, 30 mins to 7 hrs, 45 mins to 6 hrs, 60 mins to 5 hrs, 75 mins to 4 hrs, 80 mins to 3 hrs, 85 mins, 90 mins, 95 mins, 100 mins, 105 mins, 110 mins, 115 mins, 120 mins, 125 mins, 130 mins, 135 mins, 140 mins, 145 mins, 150 mins, 155 mins, 160 mins, 165 mins, 170 mins, or 180 mins.

23. The method of claim 21 , wherein the method comprises harvesting the activated skin from the amphibian 60 mins to 90 hrs post-light exposure (PLE), 60 mins to 84 hrs PLE, 60 mins to 70 hrs PLE, 60 mins to 60 hrs PLE, 60 mins to 50 hrs PLE, 60 mins to 40 hrs PLE, 60 mins to 30 hrs PLE, 60 mins to 20 hrs PLE, 2 hrs to 60 hrs PLE, 2 hrs to 48 hrs PLE, 2 hrs to 36 hrs PLE, 2 hrs to 24 hrs PLE, 60 mins PLE, 2 hrs PLE, 4hrs PLE, 8 hrs PLE, 12 hrs PLE, 24 hrs PLE, 36 hrs PLE, 48 hrs PLE, or 60 hrs PLE.

24. The method of claim 21 , wherein the method further comprises decellularizing the activated skin to obtain an activated skin comprising decellularized ECM.

25. The method of claim 21 , wherein the method further comprises gelatinizing the activated skin to obtain gelatinized activated skin.

26. A method of treating or preventing a skin condition of a subject comprising administering the activated skin of any one of claims 1-16 or the composition of any one of claims IT- 20 to the subject.

27. The method of claim 26, wherein the skin condition comprises inflamed skin or injured skin comprising a wound or disrupted skin barrier.

28. The method of claim 26, wherein the skin condition comprises psoriasis, dermatitis, such as contact dermatitis, atopic dermatitis (eczema), seborrheic dermatitis, nummular dermatitis, generalized exfoliative dermatitis, stasis dermatitis, lichen simplex chronicus; disorders of hair follicles and sebaceous glands, comprising acne, rosacea, rhinophyma, perioral dermatitis, and pseudofolliculitis barbae; and inflammatory reactions, such as drug eruptions, erythema multiforme, erythema nodosum, and granuloma annulare.

29. The method of claim 26, wherein skin condition comprises fine lines and / or wrinkles, aging, redness, abrasion, burn, cut, infection, razor bumps, scars, uneven skin tone, pain, stretch marks, skin elasticity and / or firmness, skin hydration, and hyperpigmentation.

30. The method of claim 26, wherein the skin condition comprises acute thermal burns comprising first, second, or third-degree burns.31 . The method of claim 26, wherein preventing a skin condition comprises protecting the skin from damages caused by UV rays and / or environmental pollution.

32. The method of claim 26, wherein the method further comprises treating the skin with a laser before administering the activated skin.

33. The method of claim 32, wherein the skin is treated with ablative fractional laser.

34. The method of claim 26, wherein the activated skin is delivered to the subject by injection.

35. A method of modulating expression of one or more mammalian genes of the skin, wherein the method comprises administering the activated skin of any one of claims 1- 16 or the composition of any one of claims 17-21 to mammalian cells to upregulate or downregulate one or more genes of the mammalian skin.

36. The method of claim 35, wherein the method upregulates or downregulates one or more genes associated with wound healing, inflammation of the skin, regeneration of the skin barrier, and / or protection of the skin barrier.

37. The method of claim 35, wherein the cells comprise in vitro, in vivo, or ex vivo cells.

38. The method of claim 35, wherein the mammalian cells are human cells.

39. The method of claim 35, wherein the cells are in a subject.

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