Methods for treating epidermolysis bullosa with cell-free amniotic fluid compositions

Abstract

Provided herein is a method of treating a subject having epidermolysis bullosa, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising amniotic fluid substantially free of endogenous cells.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 254,918, filed October 12, 2021, and U.S. Provisional Patent Application No. 63 / 274,214, filed November 1, 2021, each of which is incorporated by reference herein in its entirety.

[0002] FIELD OF THEINVENTION The present disclosure relates to methods of treating epidermolysis bullosa (EB). [Background technology]

[0003] Epidermolysis bullosa (EB) is a group of rare genetic disorders that result in complications that can affect various parts of the body, including blistering of the skin and mucous membranes. EB is caused by mutations in genes involved in inter- or intra-layer adhesion of epithelia, mucous membranes, and skin. For example, dystrophic EB (DEB), including recessive dystrophic EB (RDEB), is caused by mutations in the COL7A1 gene, which encodes collagen VII protein. Junctional EB (JEB) is caused by mutations in the genes encoding laminin 332 protein, namely, the LAMA3, LAMB3, and / or LAMC2 genes. Several other genes are involved in different types of EB. The progressive nature of EB results in scarring and contractures, resulting in reduced mobility and fusion of fingers and toes, leading to mitt deformities, microstomia, significant disability, and skin cancer.

[0004] Currently, there is no cure for EB. Therefore, there is a need for an effective treatment for EB. Most research approaches have focused on gene therapy to replace the defective mutation, while other approaches include protein replacement therapy using recombinant proteins. Summary of the Invention

[0005] The present disclosure provides a method of treating a subject having epidermolysis bullosa (EB), the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising amniotic fluid substantially free of endogenous cells, thereby treating EB.

[0006] In some embodiments, the composition is substantially free of lanugo and vernix caseosa. In some embodiments, the composition is aseptic or sterilized. In some embodiments, the composition further comprises some (e.g., micronized, homogenized, divided into small pieces, freeze-dried) amniotic membrane and / or Wharton's gel. In some embodiments, the method comprises reconstituting the pharmaceutical composition in whole or in part from a freeze-dried composition, e.g., amniotic fluid, amniotic membrane, or Wharton's gel.

[0007] In some embodiments, the composition comprises a therapeutically effective amount of a protein. In some embodiments, the protein is one or more of collagen VII, keratin, laminin, and decorin. In some embodiments, the composition comprises a therapeutically effective amount of a cell-free mRNA. In some embodiments, the cell-free mRNA is a transcript or a fragment thereof of one or more genes selected from the group consisting of COL7A1, COL17A, COL17A1, KRT5, KRT14, KLHL24, PLEC, DST, EXPH5, ​​CD151, LAMA3, LAMB3, LAMC2, ITGA3, ITGA6, ITGB4, FERMT1, and DCN. In some embodiments, the composition comprises a therapeutically effective amount of one or more neurotrophins. In some embodiments, the one or more neurotrophins are selected from the group consisting of nerve growth factor, brain-derived neurotrophic factor, and neurotrophin-3.

[0008] In some embodiments, the composition further comprises a protein composition exogenous to the amniotic fluid. In some embodiments, the protein composition comprises one or more of collagen VII, keratin, laminin, and decorin. In some embodiments, the composition further comprises a cell-free mRNA composition exogenous to the amniotic fluid. In some embodiments, the cell-free mRNA composition comprises a transcript or fragment thereof of one or more genes selected from the group consisting of COL7A1, COL17A, COL17A1, KRT5, KRT14, KLHL24, PLEC, DST, EXPH5, ​​CD151, LAMA3, LAMB3, LAMC2, ITGA3, ITGA6, ITGB4, FERMT1, and DCN.

[0009] In some embodiments, the composition further comprises or is co-administered with a penetration enhancer. In some embodiments, the composition is allogeneic to the subject. In some embodiments, the subject is human. In some embodiments, the composition is administered to the subject topically, subcutaneously, intradermally, intravenously, intracorneally, or intraocularly. In some embodiments, the composition is formulated as an eye drop. In some embodiments, the composition is formulated as a skin gel.

[0010] In some embodiments, the compositions of the present disclosure, including but not limited to decorin, modulate the transforming growth factor (TGF) signaling pathway. In some embodiments, the compositions of the present disclosure promote wound healing. In some embodiments, the compositions of the present disclosure prevent, reduce or treat one or more signs, symptoms or conditions associated with EB. In some embodiments, the one or more signs, symptoms or conditions are selected from the group consisting of pain, pruritus, blisters, keratoderma, granulation, erosion, ulcers, pseudosyndactyly, open wounds, tissue scarring, tissue fibrosis, corneal opacity, corneal scarring, corneal ulcers, corneal abrasions, blepharitis, ectropion, synechiae, pterygium, caries, dilated cardiomyopathy, hypoalbuminemia, growth retardation, muscular dystrophy, osteopenia, osteoporosis, and post-streptococcal glomerulonephritis. [Brief description of the drawings]

[0011] [Figure 1] 1 shows migration of primary fibroblasts ("EB fibroblasts") taken from RDEB patients treated with acellular amniotic fluid composition ("acAF") and untreated controls at 0, 24, 48, and 120 hours after initiation of treatment. [Figure 2A] Shown is the cleft zone length (multiple relative to time 0) after treatment (or without treatment) with acellular amniotic fluid composition beginning at time 0. [Figure 2B] The percentage of cleft closure following treatment (or without treatment) with acellular amniotic fluid compositions beginning at time 0 is shown. [Diagram 3] Shown is phosphorylated SMAD3 (P-SMAD3) immunoblotting of total lysates of fibroblasts from recessive dystrophic epidermolysis bullosa ("RDEB") subjects ("EB fibroblasts") and normal human breast fibroblasts ("control fibroblasts") treated with or without acAF composition and / or recombinant decorin ("rdecorin"). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] The present disclosure is described more fully hereinafter. This disclosure may be embodied in many different forms and should not be construed as limited to the aspects set forth herein; rather, these aspects are provided so that this disclosure will satisfy applicable legal requirements.

[0013] I. Definition Unless otherwise defined, all technical and scientific terms have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0014] As used herein, a "subject" is an animal, such as a mammal, including primates (humans, non-human primates, such as monkeys), and non-primates (such as cows, dogs, horses, sheep, rabbits, cats, rats, or mice). In some embodiments of the invention, the subject is a human, such as a human with EB, or a human at risk of developing EB. In some aspects, the subject is a pediatric subject, such as a neonate, infant, or child. In other aspects, the subject is an adult subject.

[0015] As used herein, "prevention" or "preventing", when used in relation to a disease (e.g., EB), refers to a reduction in the likelihood of developing a disease or associated signs, symptoms, conditions, or complications, or a reduction in the severity of a disease or associated signs, symptoms, conditions, or complications for a population with the same risk factors. Not developing a disease or delaying the time to develop associated signs, symptoms, conditions, or complications by days, weeks, months, or years is considered effective prevention. Prevention may require administration of two or more doses of a pharmaceutical composition comprising a cell-free AF ​​as described elsewhere herein.

[0016] As used herein, the term "treating" or "treatment" in the context of treating a disease refers to a beneficial or desired result in a subject, such as reducing at least one associated sign, symptom, condition, or complication, e.g., pain or itching associated with a skin condition, e.g., a blister. "Treatment" also refers to prophylactic treatment, such as prevention of a disease or at least one sign, symptom, condition, or complication associated with a disease. "Treatment" may also mean extending survival compared to expected survival in the absence of treatment.

[0017] II. Compositions Comprising Cell-Free Amniotic Fluid Provided herein are compositions comprising acellular amniotic fluid ("acAF") for use in treating a subject with EB according to the methods of the present disclosure.

[0018] A. Preparation of Cell-Free Amniotic Fluid Compositions Amniotic fluid ("AF") surrounds the fetus during pregnancy, providing it with a nutrient environment and composition for optimal growth and development. The amniotic membrane, also called the amnion, is the inner layer of the placenta and contains a basement membrane and an avascular interstitial matrix. Wharton's gel is the mucous connective tissue of the umbilical cord. Human amniotic fluid, amnion, Wharton's gel, and related tissues and compositions can be obtained from tissues and / or fluids provided by / from human donors with informed consent after delivery of the fetus, placenta, and said tissues and / or fluids. In certain embodiments, amniotic fluid, amnion, Wharton's gel, and / or related tissues and compositions are provided by a healthy human mother during a routine Caesarean delivery. Thus, obtaining human amniotic fluid, amniotic membrane, Wharton's gel, and related tissues and compositions for use in the methods of the present disclosure can be performed without harm (including death) to the donor (mother), infant, or newborn, and does not require induced termination of pregnancy. Each donor is tested using FDA-approved methods and found to be non-reactive to Hepatitis B, Hepatitis C, Human Immunodeficiency Viruses types 1 and 2, Human T-Lymphotropic Viruses 1 and 2, Syphilis, West Nile Virus, and Zika. Non-human amniotic fluid, amniotic membrane, Wharton's gel, and related materials can be obtained from non-human animal subjects according to methods known in the art.

[0019] As used herein, "acellular amniotic fluid" (acAF) or "cell-free amniotic fluid" refers to amniotic fluid that is substantially free of endogenous cells. As used herein, "substantially free" refers to a state in which cells are substantially absent, contain less than 1-10 cells / ml, or are cell-free. In certain embodiments, the acAF or acAF compositions provided herein are cell-free. Without being bound by theory, crude human amniotic fluid contains approximately 5x10 4The number of endogenous cells in the acAF or acAF compositions provided herein may be less than 0.1% or 0% of the cells in the crude amniotic fluid obtained from the donor. As used herein, "endogenous cells" refers to cells that are endogenously present in the crude amniotic fluid obtained from the donor. Cell-free amniotic fluid can be obtained by removing endogenous cells from the amniotic fluid and can be produced by any means known to those skilled in the art, such as applying centrifugation alone, filtration alone, in-line filtration alone, a combination of centrifugation and any type of filtration, or a combination of centrifugation and in-line filtration to an amniotic fluid sample obtained from a subject. Any step in the preparation of cell-free amniotic fluid can include irradiation, such as UV light or gamma radiation. Cell-free amniotic fluid can be sterilized by standard methods, such as filtration, e.g., sterile filtration, irradiation, or a combination thereof or a combination thereof. In a particular embodiment, acAF is prepared by (i) gamma-irradiating crude amniotic fluid obtained from healthy donors during Caesarean section with informed consent; (ii) centrifuging the irradiated amniotic fluid at 1400xg for 15 minutes at 4°C; (iii) collecting the supernatant and adjusting its pH to 7.4; and (iv) successively filtering the supernatant through 40 micron and then 0.2 micron filters. The acAF prepared by this method according to steps (i)-(iv) above is cell-free.

[0020] In some embodiments, the acellular amniotic fluid, in addition to being substantially acellular, is substantially free of lanugo, vernix (also called vernix caseosa), and / or debris. For example, the acAF provided herein can contain less than 2%, less than 1%, less than 0.5%, less than 0.1%, or 0% lanugo, vernix caseosa, and / or debris compared to crude amniotic fluid obtained from a donor. In certain embodiments, the acAF provided herein is free of lanugo, vernix caseosa, and / or debris.

[0021] In some embodiments, the pH of the acellular amniotic fluid composition is adjusted to a therapeutically desirable level or in the range of 6.5 to 8.5.

[0022] The acellular amniotic fluid or a composition comprising acellular amniotic fluid can be lyophilized. The lyophilized composition can be reconstituted into a solution by adding a solvent used in the art, such as saline. The composition can be diluted or concentrated. In some embodiments, the composition is aseptic or sterilized. The composition can be sterilized by subjecting all or part of the composition to any sterilization means known in the art, such as filtration, e.g., sterile filtration, irradiation, or a combination thereof.

[0023] B. Protein and / or mRNA Composition In some embodiments, the cell-free amniotic fluid compositions of the present disclosure comprise a therapeutically effective amount of protein and / or mRNA. In some embodiments, the composition comprises a therapeutically effective amount of protein. As used herein, a "therapeutically effective amount" or "effective amount" refers to an amount of a composition (e.g., protein, mRNA) effective to produce a desired pharmacological, therapeutic or prophylactic result, e.g., to treat EB.

[0024] In some embodiments, the protein in the composition is one or more of collagen VII, keratin, and laminin. In some embodiments, the composition comprises a therapeutically effective amount of mRNA, e.g., cell-free mRNA. As used herein, "cell-free mRNA" refers to extracellular mRNA that exists outside of cells. In some embodiments, the mRNA is a transcript or a fragment thereof of one or more genes selected from the group consisting of COL7A1, COL17A, COL17A1, KRT5, KRT14, KLHL24, PLEC, DST, EXPH5, ​​CD151, LAMA3, LAMB3, LAMC2, ITGA3, ITGA6, ITGB4, and FERMT1. In some embodiments, the cell-free amniotic fluid composition comprises one or more of collagen VII, collagen VII mRNA, keratin protein, keratin mRNA, laminin protein, and laminin mRNA. EB can be caused by mutations in one or more genes, including, but not limited to, COL7A1, COL17A, COL17A1, KRT5, KRT14, KLHL24, PLEC, DST, EXPH5, ​​CD151, LAMA3, LAMB3, LAMC2, ITGA3, ITGA6, ITGB4, and FERMT1. Thus, the cell-free amniotic fluid compositions of the present disclosure, including one or more of the proteins or mRNAs described above, can provide a therapeutic composition to replenish one or more of the missing, dysfunctional, or non-functional proteins in an EB subject.

[0025] In some embodiments, the cell-free amniotic fluid composition comprises a modulator of the TGF signaling pathway, including but not limited to decorin. The composition may comprise decorin protein and / or transcripts (e.g., mRNA, cell-free mRNA) or fragments thereof or fragments thereof of DCN. Decorin belongs to the low molecular weight leucine-rich proteoglycan family and can, in particular, regulate the TGF signaling pathway and / or interact with fibronectin, epidermal growth factor (EGF) receptor, and TGFβ. As used herein, "regulating the TGF signaling pathway" refers to regulating (e.g., increasing or decreasing) the expression or function of molecules involved in TGF signaling, such as TGFα, TGFβ, EGF receptor, TGFβ receptor, downstream effectors, and other TGF-related proteins.

[0026] In some embodiments, the composition comprises a therapeutically effective amount of one or more neurotrophins. In some embodiments, the one or more neurotrophins are selected from the group consisting of nerve growth factor, brain-derived neurotrophic factor, and neurotrophin-3.

[0027] C. Compositions exogenous to amniotic fluid In some embodiments, the compositions of the present disclosure include exogenous moieties or agents that do not naturally occur in amniotic fluid. For example, in some embodiments, the compositions further include amniotic membrane and / or Wharton's gel. The amniotic membrane and / or Wharton's gel may be micronized, homogenized, divided into small pieces, or lyophilized before being added to the acellular amniotic fluid composition. In some embodiments, the acellular amniotic fluid composition may be lyophilized before the incorporation of the amniotic membrane and / or Wharton's gel composition.

[0028] The inclusion of amniotic membrane or Wharton's gel in the acellular amniotic fluid compositions of the present disclosure increases the concentration of therapeutic protein or mRNA, for example, collagen VI, keratin, or laminin, in the composition.

[0029] A composition exogenous to amniotic fluid can be a molecule that is not present in the amniotic fluid, or, alternatively, a composition exogenous to amniotic fluid can be a molecule that can be identified in the amniotic fluid (e.g., type VII collagen protein) but that is added to the acellular amniotic fluid composition.

[0030] Thus, in some embodiments, the composition further comprises a protein composition that is exogenously added to the cell-free amniotic fluid composition, hi some embodiments, a recombinant protein of interest or a purified protein of interest can be added to the composition.

[0031] In some embodiments, the protein composition comprises one or more of collagen VII, keratin, laminin, and decorin. In some embodiments, the composition comprises a cell-free mRNA composition that is exogenously added to the cell-free amniotic fluid composition. In some embodiments, the cell-free mRNA composition comprises a transcript or fragment thereof of one or more genes selected from the group consisting of COL7A1, COL17A, COL17A1, KRT5, KRT14, KLHL24, PLEC, DST, EXPH5, ​​CD151, LAMA3, LAMB3, LAMC2, ITGA3, ITGA6, ITGB4, FERMT1, and DCN.

[0032] In some embodiments, the composition comprises a neurotrophin composition exogenously added to the cell-free amniotic fluid composition. In some embodiments, the neurotrophin composition comprises at least one neurotrophin protein or mRNA. In some embodiments, the at least one neurotrophin is selected from the group consisting of nerve growth factor, brain-derived neurotrophic factor, and neurotrophin-3.

[0033] In some embodiments, the concentration of therapeutic molecules, for example therapeutic proteins or mRNAs, in the composition can be increased or decreased as required, compared to the concentration in the amniotic fluid obtained from a donor or subject. Protein or mRNA concentration can be increased, for example, by adding amniotic membrane, Wharton's gel, recombinant protein, purified protein, or exogenously prepared form of the protein or mRNA of interest. Additionally or alternatively, the concentration of the protein or mRNA of interest can be decreased compared to the original concentration. Protein or mRNA concentration can be decreased, for example, by adding an acceptable diluent, thereby diluting the protein or mRNA of interest in the composition.

[0034] For example, the concentration of collagen VII can be measured in acellular amniotic fluid composition, and then additional amounts of amniotic membrane, Wharton's gel, recombinant collagen VII, and / or purified collagen VII protein can be added to increase the concentration of collagen VII to a therapeutically desirable level.For example, the collagen VII concentration in acellular amniotic fluid composition can be about 0.3mg / mL, which can be increased to about 0.6mg / mL, about 0.9mg / mL, about 1.2mg / mL, about 1.5mg / mL, about 1.8mg / mL, about 2.1mg / mL, about 2.4mg / mL, about 2.7mg / mL, about 3mg / mL, or more.Alternatively, collagen VII concentration can be decreased by adding a vehicle, such as saline, to the composition. For example, the type VII collagen concentration in the acellular amniotic fluid composition may be about 300 ng / mL, which can be reduced to about 270 ng / mL, 240 ng / mL, about 210 ng / mL, about 180 ng / mL, about 150 ng / mL, about 120 ng / mL, about 90 ng / mL, about 60 ng / mL, about 30 ng / mL, or less. Similar procedures can be applied to increase or decrease the concentration of other therapeutic molecules, such as laminin 332 and keratin, in the acellular amniotic fluid composition.

[0035] In some embodiments, the pH of the composition comprising the acellular amniotic fluid and the exogenous composition can be adjusted to a therapeutically desirable level, or, for example, in the range of 6.5 to 8.5. In some embodiments, the composition comprising the acellular amniotic fluid and the exogenous composition can be lyophilized. The lyophilized composition can be reconstituted into a solution by adding a solvent used in the art, for example, saline.

[0036] D. Penetration enhancers In some embodiments, the composition further comprises or is co-administered with a penetration enhancer. As used herein, a "penetration enhancer" is an agent that enhances the penetration of drugs through epithelial barriers, e.g., the corneal barrier, and alters the integrity of the epithelial cell layer. In some embodiments, the penetration enhancer is formulated for ocular delivery. In some embodiments, the penetration enhancer that can be included in the composition or co-administered with the composition includes cyclodextrin, dimethyl sulfoxide (DMSO), ethylenediaminetetraacetic acid (EDTA), sodium glycocholate and related cholates, Tween 20 (non-ionic polysorbate surfactant), Bridge 35 (polyoxyethylene lauryl ether), saponin, or bile salts. Without intending to be bound by theory, penetration enhancers such as EDTA and cholates can transiently loosen tight junctions between epithelial adjacent cells, e.g., the corneal epithelium. Thus, when applied topically, e.g., to the eye, the penetration enhancer can enhance the delivery of therapeutic molecules, e.g., proteins, peptides, or mRNA, through the epithelium.

[0037] In some embodiments, the penetration enhancer included in the composition or co-administered with the composition can be a chemical penetration enhancer. As used herein, a "chemical penetration enhancer" is an agent that enhances transdermal drug delivery by disrupting the stratum corneum and / or other skin components. Chemical penetration enhancers included in the composition or co-administered with the composition via transdermal drug delivery include pyrrolidones, alcohols, esters, water, ester sulfoxides (e.g., dimethyl sulfoxide) and their derivatives, hydrocarbons, terpenes and derivatives, azones and their analogs, amides (including ureas and their derivatives), fatty acids, surfactants (nonionic, cationic, and anionic), oleodendrimers, ionic liquids, and deep eutectic solvents.

[0038] III. Amount, Route, and Timing of Administration A subject (eg, a human subject having EB or at risk of developing EB) can be administered a therapeutic amount of a pharmaceutical composition comprising cell-free amniotic fluid.

[0039] In some embodiments, the composition is administered to the subject topically, subcutaneously, intradermally, intravenously, intracorneally, or intraocularly.

[0040] In some embodiments, administration is repeated, for example, hourly, twice daily, daily, twice weekly, weekly, once every two weeks, or once monthly.In some embodiments, administration is for a prescribed period, for example, for one month, two months, three months, four months, five months, six months, one year, longer, or indefinitely.After initial treatment, subsequent treatments can be administered less frequently than initial treatment.

[0041] In some embodiments, the composition is administered in one dose, or in two or more doses. In some embodiments, the number, frequency, or amount of subsequent administrations depends on the achievement of a desired therapeutic effect. In some embodiments, the composition is administered to the subject with the frequency and amount required to achieve a therapeutic effect. In some embodiments, the subject can be monitored for the desired therapeutic effect and for undesired side effects associated with the administration of the composition.

[0042] In some embodiments, the composition is formulated for delivery to a target organ, for example, to the eye or to the skin. In some embodiments, the composition is formulated as an eye drop for topical administration. In some embodiments, the composition is formulated as a skin gel, ointment, or cream.

[0043] IV. Treatment of Subjects with Epidermolysis Bullosa Provided herein are methods of treating a subject with EB or at risk of developing EB, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising amniotic fluid substantially free of endogenous cells, thereby treating EB. As used herein, a "therapeutically effective amount" or "effective amount" refers to an amount of a composition (e.g., an acAF composition) effective to produce a desired pharmacological, therapeutic or prophylactic result. For example, in the methods of treating a subject with EB provided herein, an effective amount refers to an amount effective to reduce one or more signs, symptoms, or conditions associated with EB, such as downregulating the TGF signaling pathway; enhancing cell proliferation, migration, or adhesion; enhancing wound healing; reducing or preventing one or more ocular symptoms of EB (e.g., pain, pruritus, corneal opacity, corneal scarring, corneal ulcers, corneal abrasions, blepharitis, ectropion, synechiae, pterygium, and vision loss); reducing or preventing one or more skin symptoms (e.g., erythroderma, erythroblastosis ... For example, an amount effective to reduce or prevent one or more neurological symptoms of EB (e.g., neuropathic pain, pruritus, blister, keratoderma, granulation, erosion, ulcer, pseudosyndactyly, open wound, tissue scar, and tissue fibrosis); reduce or prevent one or more neurological symptoms of EB (e.g., neuropathic pain, pruritus, and muscle weakness); and / or reduce or prevent one or more incidents, signs, or symptoms of dental caries, dilated cardiomyopathy, hypoalbuminemia, growth retardation, muscular dystrophy, osteopenia, osteoporosis, and post-streptococcal glomerulonephritis. For example, if a given clinical treatment is considered effective when there is at least a certain percentage reduction in a measurable parameter associated with EB, then a therapeutically effective amount of a composition (e.g., an acAF composition) for the treatment of EB is the amount necessary to obtain at least a certain percentage reduction in that parameter. Those skilled in the art can select a particular percentage, or a range of percentages, of reduction in a measurable parameter that will make the treatment effective according to the clinical and scientific circumstances.

[0044] In some embodiments, the composition is allogeneic to the subject. In some embodiments, the subject is a human. For example, the disclosed methods include treating a human subject with EB or at risk of developing EB, the methods include administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising human amniotic fluid substantially free of endogenous cells.

[0045] In some embodiments, the compositions of the present disclosure supplement or replace one or more proteins that are defective or absent in cells, tissues, or organs of subjects with EB. In some embodiments, the proteins that are supplemented or replaced are one or more of collagen VII, keratin, laminin, decorin, or neurotrophins. In some embodiments, the compositions of the present disclosure supplement or replace one or more mRNAs that are defective or absent in cells, tissues, or organs of subjects with EB. In some embodiments, the mRNAs that are supplemented or replaced are one or more of the following mRNAs: COL7A1, COL17A, COL17A1, KRT5, KRT14, KLHL24, PLEC, DST, EXPH5, ​​CD151, LAMA3, LAMB3, LAMC2, ITGA3, ITGA6, ITGB4, FERMT1, DCN, or variants thereof. In some embodiments, the composition comprises a therapeutic cell-free mRNA that is taken up by cells of a subject that has EBs for de novo production of a protein that is defective or missing in the EBs, i.e., to cause the cells to produce a replacement protein in place of the defective or missing protein.

[0046] In some embodiments, the acellular amniotic fluid composition modulates the TGF signaling pathway in a subject, which may result in an increase or decrease in the levels of TGFα, TGFβ, and / or other TGF-related proteins in the cells, tissues, body fluids, or organs of the subject. The TGFβ pathway is activated (e.g., upregulated) in some EB patients. Without intending to be bound by theory, activators of the TGFβ pathway are independent regulators (aggravating factors) of the clinical severity of EB, for example, independent of the amount of collagen 7 in the patient (Chacon-Solano et al. 2022 Matrix Biol. 111:189-206; Nystrom et al. 2015 EMBO Mol. Med. 7(9):1211-1228; Odorisio et al. 2014 Human Mol. Genet. 23:15; 3907-3922). In some embodiments, administration of a cell-free amniotic fluid composition provided herein improves or reduces the overall survival of a patient by 5-100%, 10-100%, 20-100%, 30-100%, 40-100%, 50-100%, 60-100%, 70-100%, 80-100%, 20-90%, 30-90%, 40-90%, 50-90%, 60-90%, or 70-90% (e.g., about 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, or 90-100%), e.g., about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, or at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% TGF signaling pathway downregulation (e.g., attenuate, reduce) The effect of acellular amniotic fluid on the TGF signaling pathway can be assessed by standard methods, such as those described in Odorisio et al. 2014 Human Mol. Genet. 23:15;3907-3922.For example, human corneal fibroblasts can be cultured in standard cell culture medium supplemented with 10% FBS (fetal bovine serum) until confluency.The medium is then replaced with test medium containing cell-free amniotic fluid composition at various dilutions in PBS (increasing from 1% composition and 99% PBS to 99% composition and 1% PBS), and the cells are incubated at 37°C for 0, 3, 6, 12, 24 and 48 hours.After the incubation period, TGFα, TGFβ, ACTA2 / α-SMA, SERPINE1 / PAI-1, BMP2K, decorin, TGFβ R2, ZEB1, IL7, MMP3, DKK2, tenascin-C, and / or other TGF-related proteins can be measured in total cell lysate or in supernatant by standard methods such as ELISA or Western blot. The amount of phosphorylated Smad3, phosphorylated Smad2, phosphorylated p38, phosphorylated ERK1 / 2, phosphorylated AKT in total cell lysates and their ratio to non-phosphorylated counterparts can be measured by immunoblotting, quantified by densitometry, and normalized to GAPDH as indicators of TGFβ pathway activity. Furthermore, the expression of TGFα, TGFβ, ACTA2 / α-SMA, SERPINE1 / PAI-1, BMP2K, decorin, TGFβ R2, ZEB1, IL7, MMP3, DKK2, tenascin-C, and / or other TGF-related mRNA molecules can be quantified by standard methods, such as mRNA isolation, cDNA synthesis, and subsequent real-time PCR quantification. The TGFβ receptor I inhibitor SB431542 can be included in the assay as a control.

[0047] In some embodiments, the compositions of the present disclosure are used to provide preventative, symptomatic, or therapeutic relief for signs or symptoms of EB according to the methods of the present disclosure. In some embodiments, the methods of the present disclosure prevent, reduce, or treat one or more signs, symptoms, or conditions associated with EB. In some embodiments, the compositions and methods of the present disclosure prevent, reduce, or treat one or more ocular symptoms of EB, such as corneal opacity, corneal scarring, corneal ulcers, corneal abrasions, blepharitis, ectropion, synechiae, pterygium, and vision loss. Additionally or alternatively, the compositions and methods of the present disclosure can prevent, reduce, or treat one or more skin symptoms of EB, such as blisters, keratodermas, granulation, erosions, ulcers, pseudosyndactyly, open wounds, tissue scarring, and tissue fibrosis. Additionally or alternatively, the compositions and methods of the disclosure can prevent, reduce, or treat one or more neurological symptoms of EB, such as neuropathic pain, itch, and muscle weakness.

[0048] In certain embodiments, the disclosed compositions and methods promote wound healing in EB subjects, such as wound healing of skin, cornea, or mucosal surfaces. Wound healing, the replacement of damaged or destroyed tissue with newly produced tissue or cells, is a biological process in organisms such as humans, accomplished through the steps of hemostasis, inflammation, proliferation, and remodeling (Sorg et al., 2017 Eur.Surg.Res.58,81-94). Chronic wounds, often characterized as wounds that remain open for more than three months, are estimated to cost the U.S. healthcare system alone $10-20 billion / year (Sen et al., 2009 Wound Repair Regen.17,763-771). Non-healing wounds cause significant morbidity and mortality, a burden rivaling that of cancer (Armstrong et al., 2007 Int.Wound J.4,286-287). Chronic wounds result from a disruption to one of the four orchestration steps that normal acute wound healing undergoes: hemostasis, inflammation, proliferation, and remodeling (Sorg et al., 2017 Eur. Surg. Res. 58, 81-94). Dysregulation of any of these steps leads to non-healing ulcers or excessive scar formation. Delayed, dysregulated, or dysfunctional wound healing, chronic wounds, and / or non-healing wounds are associated with EB and subjects with EB.

[0049] The compositions and methods provided herein may improve wound healing by about 10-100%, 20-100%, 30-100%, 40-100%, 50-100%, 60-100%, 70-100%, 80-100%, 20-90%, 30-90%, 40-90%, 50-90%, 60-90%, 70-90%, 100-1000%, 200-1000%, 300-1000%, 400-1000%, 500-1000%, 600-1000%, 700-1000%, 800-1000%, 900-1000%, 1000-1000%, 1100-1000%, 1200-1000%, 1300-1000%, 1400-1000%, 1500-1000%, 1600-1000%, 1700-1000%, 1800-1000%, 1900-1000%, 2100-1000%, 2200-1000%, 2300-1000%, 2400-1000%, 2500-1000%, 2600-1000%, 2700-1000%, 2800-1000%, 2900-1000%, 3000-1000%, 3100-1000%, 3200-1000%, 3300-1000%, 3400-1000%, 3500-1000%, 3600-1000%, 3700-1000%, 3800- 1000%, 500-1000%, 600-1000%, 700-1000%, 800-1000%, 200-900%, 300-900%, 400-900%, 500-900%, 600-900%, 700-900%, or more than 1000% (e.g., about 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, 90-100%, 100-200%, 200- 300%, 300-400%, 400-500%, 500-600%, 600-700%, 700-800%, 800-900%, 900-1000%, or more than 1000%), for example, about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, The wound healing may be promoted (e.g., enhanced) by 800%, 900%, 1000%, or more, or by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, or more. Wound healing may be measured by any standard method for assessing wound healing in vitro, ex vivo, or in vivo, including, but not limited to, scratch assays, cell proliferation assays, cell migration assays, cell detachment assays, cell adhesion assays, collagen lattice contraction assays, or functional assessment of repair tissue (e.g., tissue integrity). Exemplary assessment methods that can be used are provided in this disclosure.

[0050] In some embodiments, the one or more signs, symptoms, or conditions are selected from the group consisting of pain, pruritus, blisters, keratoderma, granulation, erosion, ulcers, pseudosyndactyly, open wounds, tissue scarring, tissue fibrosis, corneal scarring, blepharitis, ectropion, synechiae, pterygium, vision loss, caries, dilated cardiomyopathy, hypoalbuminemia, growth retardation, muscular dystrophy, osteopenia, osteoporosis, and post-streptococcal glomerulonephritis.

[0051] In some embodiments, the compositions of the present disclosure, i.e., acellular amniotic fluid compositions, are administered in combination with a second therapy known to be effective in treating EB or in preventing, alleviating, or treating one or more signs, symptoms, conditions, or complications associated with EB. The acellular amniotic fluid composition may be administered before, after, or simultaneously with the second therapy.

[0052] The second therapy may be an additional therapeutic agent. The acellular amniotic fluid composition and the additional therapeutic agent may be administered in combination in the same composition or may be co-administered as separate compositions. In some embodiments, the additional therapeutic agent is an anti-inflammatory agent, an analgesic agent, or an antibiotic. The acellular amniotic fluid composition may be administered in conjunction with the second therapy. Exemplary combination therapies include debridement, skin grafting, and gene therapy.

[0053] Working Example The following examples are offered by way of illustration and not by way of limitation. Example 1: Collagen VII and decorin concentrations in acellular amniotic fluid samples Cell-free amniotic fluid was obtained from five human donors without EB. The concentrations of collagen VII and decorin in the five cell-free amniotic fluid samples were measured by commercially available ELISA kits according to the manufacturer's instructions.

[0054] As shown in Table 1, collagen VII concentrations in acellular amniotic fluid samples range from about 77 ng / mL to about 987 ng / mL, with a mean concentration of 335 ng / mL. Dystrophic epidermolysis bullosa (DEB) is caused by mutations in the gene encoding collagen VII protein. The data indicate that acellular amniotic fluid compositions can be used to replenish therapeutic proteins, including collagen VII, in DEB patients. Concentrations of other defective or missing proteins, such as laminin and keratin, in the acellular amniotic fluid of EB patients can also be measured.

[0055] Furthermore, as shown in Table 1, the concentrations of decorin in the acellular amniotic fluid samples ranged from about 9 ng / mL to about 58 ng / mL, with an average concentration of 27 ng / mL. Decorin, in particular, is known to regulate the TGF signaling pathway and may play a therapeutic role in EB. The data indicate that acellular amniotic fluid compositions can be used to provide therapeutic proteins, including decorin, in EB patients. [Table 1]

[0056] Example 2: mRNA levels in cell-free amniotic fluid samples Levels of mutated or defective cell-free mRNAs involved in EB pathogenesis, such as COL7A1, LAMA3, LAMB3, and LAMC2, were analyzed based on data from 98 samples of cell-free amniotic fluid collected from subjects without EB.

[0057] Higher levels of COL7A1, LAMA3, LAMB3, and LAMC2 mRNA (detected with gene-specific probes) were present in cell-free amniotic fluid samples compared to control mRNA (detected with all gene binding probes), as shown in Table 2. Cell-free mRNA levels of genes defective or missing in other types of EB, such as COL17A, COL17A1, KRT5, KRT14, KLHL24, PLEC, DST, EXPH5, ​​CD151, ITGA3, ITGA6, ITGB4, and FERMT1, can also be analyzed.

[0058] The data in Table 2 show that cell-free mRNA that is missing or defective in EB subjects is present in high concentrations in cell-free amniotic fluid. The cell-free mRNA can be incorporated into cells of the EB subject, resulting in de novo production of the defective or missing protein in EB. [Table 2]

[0059] Example 3: Acellular amniotic fluid composition promotes wound healing Experimental Method Amniotic fluid (crude amniotic fluid) was obtained from healthy donors during Caesarean section with informed consent. Crude amniotic fluid was irradiated with gamma radiation and then centrifuged at 1400xg for 15 minutes at 4°C. The supernatant was collected, the pH was adjusted to 7.4, and then filtered sequentially through a 40 micron filter and a 0.2 micron filter to obtain sterile acAF.

[0060] To investigate the effect of acellular amniotic fluid composition ("acAF composition") on cell migration, an in vitro scratch assay was performed as previously described by Pitzurra 2020 J.Peridont.Res.55:287-295. Briefly, fibroblasts from recessive dystrophic epidermolysis bullosa (RDEB) patients ("EB-fibroblasts") were seeded at optimal density for culture saturation in specialized migration silicon inserts in serum-free medium with or without acAF composition (25% acAF in serum-free medium) and incubated overnight (in duplicate for each group). To segment the region of interest, a binary mask was created from the raw phase contrast image using ImageJ software, and the cleft / wound closure percentage was quantified as described in Youssefian et al., 2021 J.Invest.Dermatol.141(7):1754-1764. Migration rates and percentage of cleft closure were measured at 24, 48, and 120 hours after treatment.

[0061] result As shown in Figures 1, 2A, and 2B, the cleft area remained clearly detectable after 120 hours in untreated control cells. In contrast, closure occurred between 48 and 120 hours in fibroblasts treated with the acAF composition. These data demonstrate the ability of the acAF composition to promote fibroblast migration and accelerate wound healing.

[0062] Example 4: Cell-free amniotic fluid downregulates the transforming growth factor (TGF) signaling pathway Experimental Method Sterile acAF was prepared according to the procedure described in Example 3. The effect of acAF composition on the TGF signaling pathway in acellular amniotic fluid was evaluated by standard methods such as those described in Odorisio et al. 2014 Human Mol. Genet. 23:15; 3907-3922. Briefly, EB fibroblasts from RDEB patients described in Example 3 and control fibroblasts from healthy subjects ("control fibroblasts") were incubated in serum-free medium at optimal density for 24 hours with or without 25% acAF composition or recombinant decorin (positive control). Cells were harvested and lysed for 48 hours. To evaluate the activation of TGFβ signaling pathway, phosphorylation of SMAD3 was quantified by immunoblotting of total cell lysates. The intensity and the ratio of phosphorylated and non-phosphorylated SMAD3 were evaluated.

[0063] result Increased TGFβ signaling, as determined by increased phosphorylation of SMAD3, a downstream target of TGFβ, was evident in primary EB fibroblasts RDEB fibroblasts ( FIG. 3 , lane 1). Addition of recombinant decorin ("rdecorin"), an endogenous TGFβ inhibitor, resulted in a moderate reduction in SMAD3 phosphorylation ( FIG. 3 , lane 2). Importantly, EB fibroblasts RDEB primary fibroblasts treatment with 25% acAF composition for 24 hours showed a marked reduction in SMAD3 phosphorylation ( FIG. 3 , lane 3). Undetectable phosphorylated SMAD3 was observed in control fibroblasts both with and without treatment with decorin and / or acAF composition. These data indicate that the acAF composition of the present disclosure downregulates the TGF signaling pathway. Downregulation of the TGF pathway can treat EB in a subject and ameliorate signs and symptoms associated with EB, regardless of the amount of collagen in the subject's tissue.

[0064] Example 5: Functional in vitro assays Proper cell proliferation, migration and adhesion are important cell functions for proper tissue homeostasis. These cell functions are usually altered in EB patients, leading to excessive blister formation and improper wound healing. Cells derived from EB subjects, such as fibroblasts, keratinocytes or ocular epithelial cells, show similar altered cell functions in vitro.

[0065] An exemplary in vitro model of EB (e.g., EB cells or EB cell lines) is established as follows. Cells such as fibroblasts, keratinocytes, and ocular epithelial cells are taken from human and non-human subjects with EB or with EB-causing mutations and cultured in vitro. EB cells are also generated by knock-in or knock-down of one or more genes or proteins associated with EB, such as COL7A1, LAMA3, LAMB3, LAMC2, COL17A, COL17A1, KRT5, KRT14, KLHL24, PLEC, DST, EXPH5, ​​CD151, ITGA3, ITGA6, ITGB4, FERMT1. Primary cell cultures are established from these EB cells and passaged as necessary. Immortalized EB cell lines are also established. The therapeutic effects of the compositions of the present disclosure are evaluated by cellular assays, including the following:

[0066] 1. Cell Proliferation Assay EB cells and control cells are cultured with the composition of the present disclosure, including acellular amniotic fluid with various concentrations or amounts of therapeutic molecules, such as the protein or mRNA of defective genes in EB.For example, cells are cultured in serum-free cell culture medium with various dilutions of acellular amniotic fluid, such as 100%, 75%, 50%, 25%, 10%, 1% or 0.3% acellular amniotic fluid.Cell proliferation is measured by methods known to those skilled in the art, such as DNA synthesis cell proliferation assay, metabolic cell proliferation assay (e.g., MTT assay), detection of proliferation markers, among others.

[0067] 2. Cell Migration Assay EBs are cultured with compositions of the present disclosure, including acellular amniotic fluid with various concentrations or amounts of therapeutic molecules, such as proteins or mRNAs of defective genes in EBs. For example, cells are cultured in serum-free cell culture medium with various dilutions of acellular amniotic fluid, such as 100%, 75%, 50%, 25%, 10%, or 1% acellular amniotic fluid. Cell migration properties are tested by methods known in the art, such as Boyden chambers, cell culture wounds, and scratch assays, among others.

[0068] 3. Cell Detachment Assay EBs are cultured with the composition of the present disclosure, which comprises acellular amniotic fluid with various concentrations or amounts of therapeutic molecules, such as the protein or mRNA of defective genes in EBs.For example, cells are cultured in serum-free cell culture medium, which comprises various dilutions of acellular amniotic fluid, such as 100%, 75%, 50%, 25%, 10%, or 1% acellular amniotic fluid.Cell detachment characteristics are measured by standard methods, such as those described in Loffek et al.2014 PLOS One 9(2):e87263 and Jackow et al.2016 J.Invest.Dermatol.136:1346-1354.

[0069] For example, fibroblasts or corneal epithelial cells are seeded in cell culture plates and cultured for 24 hours. Subsequently, cells are washed with phosphate-buffered saline (PBS) and treated with trypsin / EDTA (0.05 / 0.02%) for 10, 6, 4, 2, 1, and 0 minutes, and then washed again with PBS. Adherent cells are stained with 0.5% crystal violet in distilled water for 30 minutes, dissolved with 1% sodium dodecyl sulfate (SDS), and the percentage of adherent cells can be determined by measuring the absorbance at 540, 590, or 595 nm using a spectrophotometer. Results are expressed as percentages relative to 0 minutes (untreated with trypsin).

[0070] Additionally or alternatively, a centrifugal force assay is performed. Briefly, cell culture plates are coated with the composition of the present disclosure overnight, and EB cells are seeded for a period ranging from 10 minutes to several hours. Subsequently, the cell culture plates are centrifuged at different centrifugal forces, and non-adherent cells are washed with PBS. Adherent cells are fixed, stained with crystal violet, lysed, and the absorbance of the dye is measured using a spectrophotometer.

[0071] 4. Cell Adhesion Assay EBs are cultured with the composition of the present disclosure, which comprises acellular amniotic fluid with various concentrations or amounts of therapeutic molecules, such as proteins or mRNAs of defective genes in EBs. For example, cells are cultured in serum-free cell culture medium with various dilutions of acellular amniotic fluid, such as 100%, 75%, 50%, 25%, 10%, 1%, or 0% acellular amniotic fluid. Cell adhesion properties are measured by standard methods, including those described in Chen et al. 1999 Experim. Cell Res. 249(2):231-239. Briefly, cell culture plates are coated with the composition overnight. Fibroblasts or corneal epithelial cells are added and allowed to attach for a period of time, such as 1.5 hours at 37°C. Non-adherent cells are then removed by washing them with PBS. Adherent cells are stained with 0.5% crystal violet for 15 minutes, thoroughly washed with distilled water, solubilized in 1% SDS, and quantified by measuring absorbance.

[0072] 5. Collagen Lattice Contraction Assay EBs are cultured with compositions of the present disclosure, including acellular amniotic fluid with various concentrations or amounts of therapeutic molecules, such as proteins or mRNAs of defective genes in EBs. For example, cells are cultured in serum-free cell culture medium with various dilutions of acellular amniotic fluid, such as 100%, 75%, 50%, 25%, 10%, 1%, or 0% acellular amniotic fluid. Collagen lattice contractile properties are measured by standard methods, including those described in Odorisio et al. 2014 Human Mol. Genet. 23:15; 3907-3922. Briefly, collagen solutions are prepared in a 7:2:1 ratio with 3 mg / ml acid-soluble type I collagen (Symatese Biomateriaux, Chaponost, France), 5x DMEM and buffer (0.05 M NaOH, 2.2% NaHCO 3 ). 3 Collagen is produced by mixing collagen with serum-free medium (200 mM HEPES). The collagen solution is mixed with a cell suspension in serum-free medium, seeded in 6-well cell culture clusters (Costar; Corning, New York, USA) and allowed to gel for 30 min at 37° C. The final concentration of collagen can be 2.1 mg / ml. Serum-free DMEM is poured over the gel to prevent the surface from dehydrating. After 12 h of incubation, the gels are detached from each well and left floating. The surface area of ​​the gel samples is measured at detachment (time 0) and 24 and 48 h after detachment. Gel contraction can be expressed as a percentage of the initial lattice according to the following formula: A2 / A1×100, where A1 is the initial gel area and A2 is the area at the observed time interval. Three culture plates are used for each experimental group. Assays are performed for the following experimental groups: (1) gelation performed with 0.25 ng / ml recombinant human TGFβ1 (R&D Systems) (positive contraction control); (2) gelation performed with 200 nM recombinant human DCN (R&D Systems) and / or in the absence of TGFβ1 (0.25 ng / ml) (negative control); and (3) gelation with 0%, 50%, or 100% acAF composition (experimental groups).

[0073] Example 6: Treatment of epidermolysis bullosa in an in vivo animal model The compositions of the present disclosure comprising acellular amniotic fluid are administered to animal models of EB to test their therapeutic effects.Several EB animal models are available in various species, including cows, dogs, horses, sheep, CATs, rats, and mice.Exemplary EB animal models are described, for example, in Bruckner-Tuderman et al.2010 J.Invest.Dermatol.130:1485-1488, which is incorporated herein by reference in its entirety.EB animal models include knockout models, conditional knockout models, and reduced expression models.

[0074] The amniotic fluid and / or placental material is donated by a healthy mother during a routine Caesarean delivery. The collection of amniotic fluid or placental material does not inflict harm on the mother or newborn and does not require induced termination of the pregnancy. Each donor is tested using FDA approved methods and found to be non-reactive for Hepatitis B, Hepatitis C, Human Immunodeficiency Virus types 1 and 2, Human T-Lymphotropic Virus 1 and 2, Syphilis, West Nile Virus, and Zika. The acAF composition is prepared as provided in the present disclosure.

[0075] The composition is administered to the EB and control subjects as eye drops. Additionally or alternatively, the composition is delivered to the EB and control subjects by topical, subcutaneous, intradermal, intravenous, intracorneal or intraocular administration. Additionally or alternatively, the composition is formulated as gels and ointments, and a penetration enhancer is added and administered to the EB and control subjects. In some embodiments, the composition is combined with a pharmaceutical agent such as a drug to manage pain or inflammation, and is co-administered to the EB and control subjects.

[0076] In one example, a reduced expression mouse model producing approximately 10% wild type type VII collagen protein ("C7Hypo") described by Fritsch et al. 2008 J. Clin. Invest. 8;118(5):1669-1679 is used as the EB animal model. Briefly, central corneal epithelial defects of up to 2 mm diameter (defined with a trephine) are created in 8-10 week old C7Hypo mice (n=6 animals, but this number can be flexible depending on the availability of animals) without damaging the stroma in both eyes. Immediately after, the epithelial defects are measured, photographed, and graded using fluorescein staining to determine a baseline. 10 μL of the acAF composition is administered topically to the right eye and 10 μL of saline (control) is administered topically to the left eye. The topical product is allowed to sit on the cornea for a period of time (e.g., 5 minutes). Topical administration is repeated 6 times daily until the epithelial defect resolves. Corneal healing is compared between acAF composition-treated corneas and control, untreated corneas. At the end of the study, corneas are harvested for qPCR, Western Blot (WB), and immunofluorescence / immunohistochemistry staining to quantify mRNA expression and protein expression of collagen 7, TGFβ, α-SMA, decorin, Ki67, MMP9, tenascin-C, and / or β-III tubulin.

[0077] All citations to references, including, for example, citations to patents, patent application publications, and articles, are incorporated herein by reference in their entirety. The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described in any way.

Claims

1. A pharmaceutical composition comprising amniotic fluid substantially free of endogenous cells for use in a method for treating epidermolysis bullosa (EB) in a subject, wherein the treatment comprises administering a therapeutically effective amount of the pharmaceutical composition to the subject.

2. the pharmaceutical composition is substantially free of vellus hair and vernix caseosa, and / or 10. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition is sterile or sterilized.

3. 10. The pharmaceutical composition of claim 1, further comprising amniotic membrane and / or Wharton's gel.

4. The pharmaceutical composition of claim 1, wherein the use comprises reconstituting the pharmaceutical composition from freeze-dried amniotic fluid and, optionally, from amniotic membrane and / or Wharton's gelatin.

5. 10. The pharmaceutical composition of claim 1, wherein the composition comprises a therapeutically effective amount of a protein, optionally wherein the protein is one or more of type VII collagen, keratin, laminin, and decorin.

6. 2. The pharmaceutical composition of claim 1, wherein the composition comprises a therapeutically effective amount of cell-free mRNA, and optionally the cell-free mRNA is a transcript or fragment thereof of one or more genes selected from the group consisting of COL7A1, COL17A, COL17A1, KRT5, KRT14, KLHL24, PLEC, DST, EXPH5, ​​CD151, LAMA3, LAMB3, LAMC2, ITGA3, ITGA6, ITGB4, FERMT1, and DCN.

7. 2. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition comprises a therapeutically effective amount of one or more neurotrophin proteins or mRNAs, optionally wherein the one or more neurotrophins are selected from the group consisting of nerve growth factor, brain-derived neurotrophic factor, and neurotrophin-3.

8. 10. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition further comprises or is co-administered with a penetration enhancer.

9. 10. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition is allogeneic to the subject, and optionally the subject is a human.

10. 10. The pharmaceutical composition of claim 1, wherein the composition is administered to the subject topically, subcutaneously, intradermally, intravenously, intracorneally, or intraocularly.

11. 10. The pharmaceutical composition of claim 1, wherein the composition is formulated as an eye drop and / or as a skin gel.

12. 10. The pharmaceutical composition of claim 1, wherein the composition modulates a transforming growth factor (TGF) signaling pathway.

13. The pharmaceutical composition of claim 1 , wherein the composition promotes wound healing.

14. The pharmaceutical composition of claim 1, wherein the treatment prevents, alleviates, or treats one or more signs, symptoms, or conditions associated with epidermolysis bullosa, wherein the one or more signs, symptoms, or conditions are selected from the group consisting of pain, pruritus, blisters, keratoderma, granulation, erosion, ulcers, pseudosyndactyly, open wounds, tissue scarring, tissue fibrosis, corneal opacity, corneal ulcers, corneal abrasions, corneal scarring, blepharitis, ectropion, synechiae, pterygium, vision loss, dental caries, dilated cardiomyopathy, hypoalbuminemia, growth retardation, muscular dystrophy, osteopenia, osteoporosis, and post-streptococcal glomerulonephritis.

15. The pharmaceutical composition according to any one of claims 1 to 14, wherein the pharmaceutical composition is cell-free and sterile.

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