Method for increasing therapeutic protein levels and / or for improving cell function using amniotic fluid composition

Abstract

Methods for increasing the levels of one or more therapeutic proteins in subjects having epidermolysis bullosa (EB), and methods for increasing cell adhesion and / or attachment in EB subjects, by administering a therapeutically effective amount of a pharmaceutical composition containing amniotic fluid that is absent or substantially absent in the amniotic fluid containing endogenous cells, to subjects. The methods can increase the production of collagen, type VII collagen, collagen alpha-1 (VII) chain, COL7A1 mRNA, laminin, laminin 332, laminin subunit alpha-3, laminin subunit beta-3, laminin subunit gamma-2, LAMA3 mRNA, LAMB3 mRNA, LAMC2 mRNA, decorin, TUBB3 mRNA, tubulin beta-3 class III, and / or functional fragments of any of these. Methods for increasing corneal nerve regeneration in subjects by administering a therapeutically effective amount of a pharmaceutical composition containing amniotic fluid that is absent or substantially absent in the amniotic fluid containing endogenous cells, are also provided.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the benefit of priority to U.S. Provisional Application No. 63 / 458,617, filed on April 11, 2023. The content of each of the foregoing applications is hereby incorporated by reference in its entirety.

[0002] Sequence Listing This application includes a sequence listing submitted electronically with this specification. The sequence listing file was created on April 10, 2024, named "E114015_1040_SL.xml", and its size is 4.56 kb. The entire content of the sequence listing within the XML file is hereby incorporated by reference.

[0003] The present disclosure relates to methods of increasing the levels of therapeutic proteins such as collagen, laminin, and decorin and improving the cellular functions of epidermolysis bullosa (EB).

Background Art

[0004] Epidermolysis bullosa (EB) is a group of rare genetic conditions that result in complications that can affect various parts of the body, including blister formation in the skin and mucous membranes. EB is caused by mutations in genes involved in adhesion between or within the epithelial, mucosal, and skin layers. For example, dystrophic epidermolysis bullosa (DEB), including recessive dystrophic EB (RDEB), is caused by mutations in the COL7A1 gene that encodes type VII collagen protein. Junctional epidermolysis bullosa (JEB) is caused by mutations in genes that encode 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 causes scarring and contractures, leading to decreased mobility, fusion of fingers and toes, mittens deformity, microstomia, significant disabilities, and skin cancer.

[0005] Currently, there is no treatment for EB. Therefore, an effective treatment for EB is needed. Most research approaches focus on gene therapy that replaces the defective mutation, while other approaches include protein replacement therapy using recombinant proteins. [Overview of the project] [Problems that the invention aims to solve]

[0006] This disclosure provides a method for increasing the level of one or more therapeutic proteins in a subject having epidermolysis bullosa (EB), comprising administering a therapeutically effective amount of a pharmaceutical composition containing amniotic fluid substantially free of endogenous cells to the subject. [Means for solving the problem]

[0007] In some embodiments of the methods provided herein, the production of one or more therapeutic proteins is increased in a subject. In embodiments, one or more therapeutic proteins are selected from the group consisting of collagen, laminin, decorin, and tubulin. In some embodiments, the method increases type VII collagen or its functional fragment and / or laminin 332 or its functional fragment and / or tubulin, which includes tubulin beta-3 class III or its functional fragment. In some embodiments, the production of COL7A1 mRNA, collagen alpha-1(VII) chain protein, LAMA3 mRNA, LAMB3 mRNA, LAMC2 mRNA, laminin subunit alpha-3, laminin subunit beta-3, laminin subunit gamma-2, decorin, TUBB3 mRNA, tubulin beta-3 class III, and / or its functional fragment is increased in a subject. In some embodiments, the production of COL7A1 mRNA, collagen alpha-1(VII) chain, LAMA3 mRNA, LAMB3 mRNA, LAMC2 mRNA, laminin subunit alpha-3, laminin subunit beta-3, laminin subunit gamma-2, decorin, TUBB3 mRNA, tubulin beta-3 class III, and / or functional fragments thereof is increased at the site of chronic and / or acute wounds in the subject.

[0008] In another embodiment, a method is provided for increasing cell adhesion and / or attachment in subjects having epidermolysis bullosa (EB). The method comprises administering a therapeutically effective amount of a pharmaceutical composition to a subject, comprising amniotic fluid substantially free of endogenous cells.

[0009] In some embodiments, a therapeutically effective amount of the pharmaceutical composition enhances cell adhesion and bonding without altering cell proliferation.

[0010] In some embodiments, the method increases cell adhesion and / or attachment independently of cell proliferation.

[0011] In another embodiment, a method is provided for increasing corneal nerve regeneration in a subject. The method involves administering a therapeutically effective amount of a pharmaceutical composition to a subject, comprising amniotic fluid substantially free of endogenous cells. In some embodiments, the subject has epidermolysis bullosa (EB).

[0012] In some embodiments, the composition is substantially free of vellus hair and vernix caseosa. In some embodiments, the composition is sterile or sterilized. In some embodiments, the composition further comprises amniotic membrane and / or Wharton's jelly (e.g., micronized, homogenized, finely divided, or freeze-dried). In some embodiments, the method involves reconstituting all or part of a pharmaceutical composition from a freeze-dried composition, e.g., amniotic fluid, amniotic membrane, or Wharton's jelly.

[0013] In some embodiments, the composition contains a therapeutically effective amount of protein. The protein may be endogenous and / or exogenous to the amniotic fluid. In some embodiments, the protein is one or more of type VII collagen, keratin, laminin, and decorin. In some embodiments, the composition contains a therapeutically effective amount of cell-free mRNA. The cell-free mRNA may be endogenous and / or exogenous to the amniotic fluid. In some embodiments, 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. In some embodiments, the composition contains a therapeutically effective amount of one or more neurotrophins. In some embodiments, one or more neurotrophins are selected from the group consisting of nerve growth factor, brain-derived neurotrophic factor, and neurotrophin-3.

[0014] In some embodiments, the composition further comprises or is administered concurrently with a penetration enhancer. In some embodiments, the composition is homogeneous with respect 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 subject has a corneal wound, and the composition is administered topically to the site of the corneal wound. In some embodiments, the composition is formulated as eye drops. In some embodiments, the composition is formulated as a skin gel.

[0015] In some embodiments, but not limited to, compositions of the Disclosure comprising decorin modulate the transforming growth factor (TGF) signaling pathway. In some embodiments, the methods provided herein promote wound healing. In some embodiments, the methods provided herein prevent, alleviate, or treat one or more signs, symptoms, or conditions associated with EB in a subject. In some embodiments, one or more signs, symptoms, or conditions are selected from the group consisting of pain, pruritus, vesicles, keratosis, granulation, erosion, ulceration, pseudosyndactyly, open wounds, tissue scarring, histofibrosis, corneal opacity, corneal scarring, corneal ulceration, corneal abrasion, blepharitis, ectropion, blepharoplasty, pterygium, caries, dilated cardiomyopathy, hypoalbuminemia, dysplasia, muscular dystrophy, osteopenia, osteoporosis, and post-streptococcal glomerulonephritis. In certain embodiments, the compositions alleviate or treat corneal opacity in a subject.

[0016] In one embodiment, a method is provided herein for increasing the production of one or more therapeutic proteins (e.g., collagen, laminin, decorin, tubulin) in a subject having epidermolysis bullosa (EB), the method comprising administering a therapeutically effective amount of a sterile pharmaceutical composition containing cell-free amniotic fluid to the subject. A patent or application file shall include at least one drawing made in color. A copy of this patent or patent application publication accompanied by the color drawing shall be provided by the Patent Office upon request and payment of the necessary fees. [Brief explanation of the drawing]

[0017] [Figure 1] Figure showing the migration of primary fibroblasts obtained from cell-free amniotic fluid composition ("acAF") and recessive dystrophic epidermolysis bullosa (RDEB) patients treated with untreated controls ("EB-fibroblasts") at 0, 24, 48, and 120 hours after the start of treatment. [Figure 2A] Figure showing the gap region distance (fold change relative to time 0) after treatment with cell-free amniotic fluid composition (or no treatment) starting at time 0. [Figure 2B] Figure showing the percentage of gap closure after treatment with cell-free amniotic fluid composition (or no treatment) starting at time 0. [Figure 3] Figure showing a phosphorylated SMAD3 (P-SMAD3) immunoblot of whole cell lysates of fibroblasts derived from recessive dystrophic epidermolysis bullosa (RDEB) subjects ("EB-fibroblasts") and normal human breast fibroblasts ("control fibroblasts") treated in the presence or absence of acAF composition and / or recombinant decorin ("rDecorin"). [Figure 4] Figure showing a dose-dependent increase in laminin expression in primary JEB cells (designated as cells 19 and 90) by acAF as evaluated by Western blotting.

[0018] [Figure 5] Figure showing ultracentrifugation of acAF with distinct brown pellets at the bottom of the tube.

[0019] [Figure 6] Figure showing the detection of exosomes in the acAF fraction by CD24 as an exosome marker as evaluated by Western blot analysis.

[0020] [Figure 7] Figure showing enhanced collagen 7 expression in primary DEB cells (designated as cells 45 and 57) treated with the acAF fraction.

[0021] [Figure 8A] This is a figure showing the improvement of adhesion of DEB cells (indicated by RLU) by acAF treatment. The effect of acAF on cell adhesion was confirmed by statistical analysis showing a significant difference in adhesion rates between cell types (p = 0.0380*). [Figure 8B] This is a figure showing the number of cells (indicated by RLU) across all test conditions. The significance levels are shown as follows: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

[0022] [Figure 9] This is a figure showing the time course of recovery from corneal abrasions in 9-week-old COL7A1 expression-reduced ("C7Hypo") mice. After injury, acAF solution (upper panel) or placebo (lower panel) was topically administered 6 times a day, and slit-lamp photographs were taken daily using fluorescein solution under cobalt blue light to monitor corneal recovery.

[0023] [Figure 10] This is a figure showing the collagen 7 (COL7) mRNA levels (fold change relative to placebo-treated eyes) in acAF-treated eyes of non-injured C7Hypo mice, 9-week-old and 13-week-old C7Hypo mice with corneal abrasions, and 9-week-old wild-type (WT) mice with corneal abrasions.

[0024] [Figure 11] This is a figure showing the tubulin beta 3 class III (TUBB3) mRNA levels (fold change relative to placebo-treated eyes) in acAF-treated eyes of non-injured C7Hypo mice, 9-week-old and 13-week-old C7Hypo mice with corneal abrasions, and 9-week-old wild-type (WT) mice with corneal abrasions.

Modes for Carrying Out the Invention

[0025] The Disclosure is described in more detail below. The Disclosure may be embodied in many different forms and should not be construed as being limited to the forms described herein, but rather these forms are provided to satisfy the legal requirements to which the Disclosure applies.

[0026] I. Definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which the invention pertains. The terms used herein in describing the invention are for the purpose of describing specific embodiments only and are not intended to limit the invention.

[0027] As used herein, “Subject” refers to animals such as mammals, including primates (e.g., humans, non-human primates, e.g., monkeys) and non-primates (e.g., cattle, dogs, horses, sheep, rabbits, cats, rats, or mice). In some embodiments of the present invention, the subject is a human being, such as a person who has EB or is at risk of developing EB. In some embodiments, the subject is a pediatric subject, such as a newborn, infant, or child. In other embodiments, the subject is an adult subject.

[0028] As used herein, the terms “treat” or “cure” in the context of treating a disease (e.g., EB) mean a beneficial or desired outcome, such as reducing at least one associated sign, symptom, condition, or complication in the subject, e.g., a skin condition, e.g., pain or itching associated with blisters. “Cure” also means a preventive treatment, such as the prevention of the disease or the prevention of at least one sign, symptom, condition, or complication associated with the disease. Thus, “Cure” may mean a reduced likelihood of developing the disease or an associated sign, symptom, condition, or complication, or a reduced severity of the disease or an associated sign, symptom, condition, or complication, compared to a population with the same risk factors but not receiving the treatment described herein. Not developing the disease, or delaying the time to develop an associated sign, symptom, condition, or complication by several days, weeks, months, or years, is considered an effective treatment. Treatment may require the administration of two or more doses of a pharmacochemical composition containing cell-free AF ​​as described elsewhere herein. “Treatment” may also mean an extension of survival compared to expected survival in the absence of treatment.

[0029] II. Compositions containing cell-free amniotic fluid Compositions comprising cell-free amniotic fluid ("acAF") for use in treating subjects having EB according to the methods of the present disclosure are provided herein.

[0030] A. Preparation of cell-free amniotic fluid composition Amniotic fluid ("AF") surrounds the fetus during pregnancy and provides an environment of nutrients and compositions for optimal growth and development of the fetus. The amniotic membrane, also called the amnion, is the inner layer of the placenta and includes the basement membrane and avascular stromal matrix. Howardon's jelly is the mucoid connective tissue of the umbilical cord. Human amniotic fluid, amniotic membrane, Howardon's jelly, and related tissues and compositions can be obtained from such tissues and / or fluids delivered by / from a human donor based on informed consent, after delivery of the fetus, placenta, and tissues and / or fluids. In certain embodiments, the amniotic fluid, amniotic membrane, Howardon's jelly, and / or related tissues and compositions are provided by a healthy human mother during routine cesarean section delivery. Therefore, obtaining human amniotic fluid, amniotic membrane, Howardon's jelly, and related tissues and compositions for use in the methods of this disclosure can be carried out without causing harm (including death) to the donor (mother), infant, or neonatal, and does not require the termination of pregnancy induction. Each donor has been tested using FDA-approved methods and found to be non-reactive to hepatitis B, hepatitis C, human immunodeficiency virus types 1 and 2, human T lymphotropic virus types 1 and 2, syphilis, West Nile virus, and Zika. Non-human amniotic fluid, amniotic membrane, Howardon's jelly, and related materials can be obtained from non-human animal subjects according to methods known in the art.

[0031] As used herein, “cell-free amniotic fluid” (acAF) or “cell-free amniotic fluid” refers to amniotic fluid that is substantially free of endogenous cells. As used herein, “substantially cell-free” means a state in which cells are essentially absent, for example, containing less than 1 to 10 cells / ml or no cells at all. In certain embodiments, the acAF or acAF composition provided herein does not contain endogenous cells. As used herein in the context of acAF, “endogenous” cells refer to cells contained in crude amniotic fluid obtained from a donor. While we do not wish to be bound by theory, crude human amniotic fluid is approximately 5 × 10 4It may contain cells / ml. The number of endogenous cells in 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. In certain embodiments, acAF or acAF compositions do not contain cells. As used herein, “endogenous cells” refers to cells that were endogenously present in the crude amniotic fluid obtained from the donor. Cell-free amniotic fluid can be obtained by removing endogenous cells from (crude) amniotic fluid and can be produced by any means known to those skilled in the art, such as applying centrifugation alone, filtration alone, continuous filtration alone, a combination of centrifugation and any type of filtration, or a combination of centrifugation and continuous filtration to an amniotic fluid sample obtained from a subject. Any step in preparing cell-free amniotic fluid may include irradiation, such as UV light or gamma rays. Cell-free amniotic fluid can be sterilized by standard methods such as filtration, e.g., sterile filtration, irradiation, or a combination thereof. In certain embodiments, acAF is prepared by (i) irradiating crude amniotic fluid obtained from a healthy donor during a cesarean section with gamma rays based on informed consent, (ii) centrifugating the irradiated amniotic fluid at 1400 × g for 15 minutes at 4°C, (iii) collecting the supernatant and adjusting its pH to 7.4, and (iv) continuously filtering the supernatant through a 40 μm filter, and then a 0.2 μm filter. In certain embodiments, acAF is prepared by (i) centrifugating crude amniotic fluid at 1400 × g for 15 minutes at 4°C, (ii) collecting the supernatant and irradiating it with UV-C, (iii) adjusting its pH to 7.4, and (iv) continuously filtering the supernatant through a 40 μm filter, and then a 0.2 μm filter. acAF from different donors can be combined to create a pooled form of acAF. The acAF prepared by this procedure following steps (i) to (iv) above is cell-free.

[0032] In some embodiments, cell-free amniotic fluid is substantially cell-free and substantially free of vellus hair, vernix caseosa (also known as vernix caseosa), and / or necrotic tissue fragments. For example, the acAF provided herein may contain less than 2%, less than 1%, less than 0.5%, less than 0.1%, or 0% of vellus hair, vernix caseosa, and / or necrotic tissue fragments relative to the crude amniotic fluid obtained from the donor. In certain embodiments, the acAF provided herein does not contain vellus hair, vernix caseosa, and / or necrotic tissue fragments.

[0033] In some embodiments, the pH of the cell-free amniotic fluid composition is adjusted to a therapeutically desired level or range of 6.5 to 8.5.

[0034] Cell-free amniotic fluid or compositions containing cell-free amniotic fluid can be freeze-dried. The freeze-dried composition can be reconstituted into a solution by adding a solvent used in the art, such as physiological saline. The composition can be diluted or concentrated. In some embodiments, the composition is sterile or sterilized. The composition can be sterilized by subjecting the whole or a portion of the composition to any sterilization method known in the art, such as filtration, such as sterile filtration, irradiation, or a combination thereof.

[0035] The amniotic fluid compositions provided herein may have therapeutic effects, such as increasing the expression of therapeutic proteins (e.g., collagen, COL7, laminin, laminin 332, decorin, tubulin, TUBB3), promoting cell adhesion and / or attachment, promoting wound healing, promoting nerve regeneration, and / or alleviating or preventing one or more symptoms or signs associated with EB. Such therapeutic effects of the amniotic fluid compositions provided herein may be independent of exosomes or extracellular vesicles contained in the composition or crude amniotic fluid.

[0036] B. Protein and / or mRNA composition The cell-free amniotic fluid compositions of this disclosure may contain therapeutically effective amounts of protein and / or mRNA. As used herein, “therapeutically effective amount” or “effective amount” means the amount of a composition (e.g., protein, mRNA) that is effective in producing the intended pharmacological, therapeutic, or prophylactic outcome, for example, to treat EB. The therapeutically effective amounts of protein and / or mRNA contained in the acAF composition may be endogenous or exogenous. As used herein, “endogenous” means that it is present in the crude amniotic fluid obtained from the donor. As used herein, “exogenous” means that it is not naturally present in the crude amniotic fluid and / or is added to the amniotic fluid or amniotic fluid composition.

[0037] In some embodiments, the composition contains a therapeutically effective amount of protein, which may be exogenous and / or endogenous to the amniotic fluid. The protein in the composition may be one or more of type VII collagen, keratin, and laminin.

[0038] In some embodiments, the composition comprises a therapeutically effective amount of mRNA, such as cell-free mRNA, which may be exogenous and / or endogenous to the amniotic fluid. As used herein, “cell-free mRNA” refers to extracellular mRNA present outside the cell. The mRNA may be 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, and FERMT1. In some embodiments, the cell-free amniotic fluid composition comprises one or more of type VII collagen protein, type VII collagen (COL7A1) mRNA, keratin protein, keratin mRNA, laminin protein, and laminin mRNA.

[0039] 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. Therefore, the cell-free amniotic fluid compositions of this disclosure, comprising one or more of the above-mentioned proteins and / or mRNAs, can provide therapeutic compositions to compensate for one or more deficiencies, reductions, dysfunctions, or non-functional proteins in subjects with EB. Furthermore, the cell-free amniotic fluid compositions of this disclosure can increase (e.g., stimulate) the production of one or more mRNAs and / or proteins disclosed herein, such as COL7A1, COL17A, COL17A1, KRT5, KRT14, KLHL24, PLEC, DST, EXPH5, ​​CD151, LAMA3, LAMB3, LAMC2, ITGA3, ITGA6, ITGB4, FERMT1, or any functional fragment thereof, or a gene-encoded protein or its functional fragment, and can increase EB targets, such as type VII collagen, keratin, or laminin-deficient or reduced proteins. As used herein, “functional fragment” means a fragment of a polynucleotide (e.g., mRNA) or polypeptide that retains at least partially the function of a full-length polynucleotide or polypeptide. In certain embodiments, administering a cell-free amniotic fluid composition to an EB subject can increase (e.g., stimulate) the production of COL7A1 mRNA, collagen alpha-1(VII) chain protein, and / or functional fragments thereof in the subject, thereby increasing collagen levels in the subject (in certain embodiments, levels of type VII collagen or its functional fragments). For example, administration of a cell-free amniotic fluid composition can increase levels of collagen, type VII collagen, collagen alpha-1(VII) chain protein, COL7A1 mRNA, and / or functional fragments thereof systemically or at the site of chronic and / or acute wounds in the subject.In certain embodiments, administration of a cell-free amniotic fluid composition to an EB subject can increase (e.g., stimulate) the production of LAMA3 mRNA, LAMB3 mRNA, LAMC2 mRNA, laminin subunit alpha-3, laminin subunit beta-3, laminin subunit gamma-2, and / or functional fragments thereof in the subject, thereby increasing laminin levels in the subject (in certain embodiments, levels of laminin 332 or its functional fragments). For example, administration of a cell-free amniotic fluid composition can increase the levels of laminin, laminin 332, laminin subunit alpha-3, laminin subunit beta-3, laminin subunit gamma-2, LAMA3 mRNA, LAMB3 mRNA, LAMC2 mRNA, and / or functional fragments thereof systemically or at the site of chronic and / or acute wounds in the subject.

[0040] In some embodiments, the cell-free amniotic fluid (acAF) composition includes, but is not limited to, decorin, a modulator of the TGF signaling pathway. The composition may include the decorin protein and / or transcript (e.g., mRNA, cell-free mRNA) or fragments thereof of DCN. Decorin is a protein belonging to the small leucine-rich proteoglycan family and, among other things, can modulate the TGF signaling pathway and / or interact with fibronectin, epidermal growth factor (EGF) receptor, and TGF-β. As used herein, “modulate the TGF signaling pathway” means modulating (e.g., increasing or decreasing) the expression or function of molecules involved in TGF signaling, such as TGF-α, TGF-β, EGF receptor, TGF-β receptor, downstream effectors, and other TGF-related proteins.

[0041] In some embodiments, the acAF compositions provided herein, for use according to the methods provided herein, for example, include a decoratedoline core protein. As used herein, “decolin core protein” refers to the protein core of a decoratedoline proteoglycan, which includes a protein core and one or more carbohydrate glycosaminoglycan (GAG) side chains. The decoratedoline core protein may have several different structural domains: 1) a short signal sequence of about 16 amino acids; 2) a propeptide of about 14 amino acids; 3) a glycosaminoglycan (GAG) acceptor region having a chondroitin / dermatan sulfate chain substituted with the Ser-4 residue of the mature core protein; 4) a variable cysteine ​​globular domain; 5) a leucine-rich domain having three N-linked oligosaccharide binding sites; and 6) a carboxyl-terminal globular domain. Mature decoratedoline molecules typically lack a propeptide domain. The decoratedoline core protein may be about 40 kDa, containing about 8 to 12 leucine-rich repeats (LRRs) of about 20 to 29 residues containing leucine.

[0042] In some embodiments, the acAF composition comprises decorin in different (e.g., one or more) glycosylated states. The glycosylated decorin may have one or more GAG ​​chains and a size of about 45–100 kDa, depending on the number and size of the GAG ​​chains. The composition may contain multiple decorin subtypes, each having a different number of GAG chains, GAG chains of different lengths, and / or proteoglycans of different sizes.

[0043] The acAF composition may contain a therapeutically effective amount of one or more neurotrophins. The one or more neurotrophins may be nerve growth factor, brain-derived neurotrophic factor, and / or neurotrophin-3.

[0044] C. Exogenous compositions for amniotic fluid In some embodiments, the compositions of the present disclosure include exogenous components or agents that were not naturally present in the amniotic fluid. For example, in some embodiments, the composition further includes amniotic membrane and / or Howartan's jelly. The amniotic membrane and / or Howartan's jelly may be micronized, homogenized, fragmented, or freeze-dried before being added to the cell-free amniotic fluid composition. In some embodiments, the cell-free amniotic fluid composition may be freeze-dried before incorporating the amniotic membrane and / or Howartan's jelly composition.

[0045] By including amniotic membrane or Wharton's jelly in the cell-free amniotic fluid composition of this disclosure, the concentration of therapeutic proteins or mRNAs in the composition, such as type VI collagen, keratin, or laminin, can be increased.

[0046] Exogenous compositions for amniotic fluid may be molecules not present in amniotic fluid. Alternatively, exogenous compositions for amniotic fluid may be molecules that can be identified in amniotic fluid (e.g., type VII collagen protein) but are added to cell-free amniotic fluid compositions.

[0047] Therefore, in some embodiments, the composition further comprises a protein composition exogenously added to the cell-free amniotic fluid composition. In some embodiments, the recombinant protein of the target or the purified protein of the target can be added to the composition.

[0048] In some embodiments, the protein composition comprises one or more type VII collagen, keratin, laminin, and decorin. In some embodiments, the composition comprises a cell-free mRNA composition exogenously added to a cell-free amniotic fluid composition. In some embodiments, the cell-free mRNA composition comprises transcripts or fragments 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.

[0049] In some embodiments, the composition comprises a neurotrophin composition exogenously added to a 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.

[0050] In some embodiments, the concentration of therapeutic molecules in the composition, such as therapeutic proteins or mRNA, can be increased or decreased as needed compared to the concentration in the amniotic fluid obtained from the donor or subject. The protein or mRNA concentration can be increased, for example, by adding the target protein or mRNA in the form of amniotic membrane, Wharton's jelly, recombinant protein, purified protein, or exogenously prepared nucleic acid. Additionally or alternatively, the concentration of the target protein or mRNA can be decreased compared to the original concentration. The protein or mRNA concentration can be decreased, for example, by adding an acceptable diluent, thereby diluting the target protein or mRNA in the composition.

[0051] For example, the concentration of type VII collagen can be measured in a cell-free amniotic fluid composition, and then the concentration of type VII collagen can be increased to a therapeutically desired level by adding additional amounts of amniotic membrane, Wharton's jelly, recombinant type VII collagen protein, and / or purified type VII collagen protein. For example, the concentration of type VII collagen in a cell-free amniotic fluid composition may be about 0.3 mg / mL, and this can be increased to about 0.6 mg / mL, about 0.9 mg / mL, about 1.2 mg / mL, about 1.5 mg / mL, about 1.8 mg / mL, about 2.1 mg / mL, about 2.4 mg / mL, about 2.7 mg / mL, about 3 mg / mL, or more. Alternatively, the concentration of type VII collagen can be decreased by adding a vehicle, such as physiological saline, to the composition. For example, the concentration of type VII collagen in a cell-free amniotic fluid composition may be approximately 300 ng / mL, which can be reduced to approximately 270 ng / mL, 240 ng / mL, 210 ng / mL, 180 ng / mL, 150 ng / mL, 120 ng / mL, 90 ng / mL, 60 ng / mL, 30 ng / mL, or less. A similar process can be applied to increase or decrease the concentrations of other therapeutic molecules in the cell-free amniotic fluid composition, such as laminin 332 and keratin.

[0052] In some embodiments, the pH of the composition comprising cell-free amniotic fluid and the exogenous composition can be adjusted to a therapeutically desired level or range, for example, 6.5 to 8.5. In some embodiments, the composition comprising cell-free 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, physiological saline.

[0053] D. Penetration enhancers In some embodiments, the composition further comprises or is administered with a penetration enhancer. As used herein, “penetration enhancer” is a reagent that facilitates the penetration of a drug through an epithelial barrier, such as the corneal barrier, and alters the integrity of the epithelial cell layer. In some embodiments, the penetration enhancer is formulated for ocular delivery. Examples of penetration enhancers that may be included in or administered with the composition in some embodiments include cyclodextrin, dimethyl sulfoxide (DMSO), ethylenediaminetetraacetic acid (EDTA), sodium glycocholate and related cholates, Tween 20 (nonionic polysorbate surfactant), Brij 35 (polyoxyethylene lauryl ether), saponins, or bile salts. While not wishing to be bound by theory, penetration enhancers such as EDTA and cholates can temporarily loosen tight junctions between adjacent cells in epithelium, such as the corneal epithelium. Therefore, when applied topically, for example to the eye, penetration enhancers can facilitate the delivery of therapeutic molecules, such as proteins, peptides, or mRNA, through the epithelium.

[0054] In some embodiments, the penetration enhancers included in or administered co-administered with the composition may be chemical penetration enhancers. As used herein, “chemical penetration enhancer” is a reagent that promotes transdermal drug delivery by disrupting the stratum corneum and / or other components of the skin. Chemical penetration enhancers that may be included in a composition or administered co-administered with the composition via transdermal drug delivery include pyrrolidone, alcohols, esters, water, ester sulfoxides (such as dimethyl sulfoxide) and their derivatives, hydrocarbons, terpenes and their derivatives, azone and its analogues, amides (including urea and its derivatives), fatty acids, surfactants (nonionic, cationic, and anionic), oleodendrimers, ionic liquids, and deep eutectic solvents.

[0055] III. Dosage, route of administration, and timing of administration A therapeutic dose of the pharmaceutical composition containing cell-free amniotic fluid can be administered to subjects (for example, human subjects with EB or at risk of developing EB) by any dose, route, or timing that is suitable in clinical or experimental circumstances. A person skilled in the art can determine the dose, route, or timing for the subject.

[0056] For example, the composition can be administered topically, subcutaneously, intradermally, intravenously, intracorneally, or intraocularly to a subject. For example, to treat EB lesions of the eye, a cell-free amniotic fluid composition can be administered intracorneally or intraocularly to a subject.

[0057] The drug may be administered once, or it may be repeated, for example, every hour, 4 to 6 times a day, twice a day, daily, twice a week, weekly, every other week, or monthly. The drug may be administered for a predetermined period, for example, one month, two months, three months, four months, five months, six months, one year or longer, or indefinitely. After the initial treatment, subsequent treatments may be administered at a lower frequency compared to the initial treatment.

[0058] The composition can be administered in a single dose or in two or more doses. The number, frequency, or amount of subsequent doses may depend on achieving the desired therapeutic effect. In some embodiments, the composition is administered to the subject at the frequency and in the amount necessary to achieve the therapeutic effect. Furthermore, the subject can be monitored for desired therapeutic effects and undesirable side effects associated with the administration of the composition.

[0059] The composition can be formulated for delivery to a target organ, such as the eye or skin. In some embodiments, the composition is formulated as eye drops for topical administration. In some embodiments, the composition is formulated as a skin gel, ointment, or cream. The composition can be administered to a target in two or more different formulations.

[0060] IV. Increased therapeutic protein levels in subjects with epidermolysis bullosa This specification provides a method for increasing the levels of one or more therapeutic proteins in subjects who have or are at risk of developing EB. As used herein, “therapeutic protein” refers to proteins, including endogenous proteins, that may be beneficial to cells, tissues, organs, or bodily functions. Examples of therapeutic proteins include collagen (e.g., type VII collagen), laminin (e.g., laminin 332), decorin, and tubulin (e.g., tubulin beta-3 class III). While not wishing to be bound by theory, collagen (e.g., type VII collagen), laminin (e.g., laminin 332), decorin, and / or tubulin (e.g., tubulin beta-3 class III) may promote wound healing, such as corneal wound healing, or improve corneal opacity in EB subjects. Tubulin (e.g., tubulin beta-3 class III) may promote nerve regeneration in subjects. The method comprises administering a therapeutically effective amount of a pharmaceutical composition containing amniotic fluid substantially free of endogenous cells to a subject, thereby treating EB. As used herein, “effective dose” or “therapeutic effective dose” refers to the amount of a composition (e.g., an acAF composition) that is effective in increasing the amount of a target molecule (e.g., collagen, type VII collagen, laminin, laminin 332, decorin, tubulin, TUBB3) by a specific amount in the subject. Those skilled in the art can select a specific percentage or range of percentages of increase in a measurable parameter (e.g., the amount of collagen, type VII collagen, COL7A1 protein, collagen alpha-1(VII) chain protein, laminin, laminin 332, laminin subunit alpha-3, laminin subunit beta-3, laminin subunit gamma-2, LAMA3 mRNA, LAMB3 mRNA, LAMC2 mRNA, decorin, TUBB3 mRNA, tubulin beta-3 class III, or any functional fragment thereof) for which the administration is considered effective according to the clinical and scientific circumstances.

[0061] "Effective dose" or "therapeutic effective dose" may also refer to the amount of a composition (e.g., acAF composition) that is effective in producing the intended pharmacological, therapeutic, or prophylactic effect. For example, in a method for treating a subject having EB provided herein, the effective dose is an amount effective in reducing one or more signs, symptoms, or conditions associated with EB, e.g., downregulating the TGF signaling pathway; increasing cell proliferation, migration, or adhesion; increasing wound healing; reducing or preventing one or more ocular manifestations of EB (e.g., pain, pruritus, corneal opacity, corneal scarring, corneal ulcer, corneal abrasion, blepharitis, ectropion, blepharoplasty, pterygium, and vision loss); one or more EBs It contains an amount effective to alleviate or prevent the skin manifestations of EB (e.g., pain, itching, blistering, keratosis, granulation, erosion, ulceration, pseudosyndactyly, open wounds, tissue scarring, and histofibrosis); alleviate or prevent one or more neurological manifestations of EB (e.g., neuropathic pain, itching, and muscle weakness); and / or alleviate or prevent one or more events, signs, or symptoms of caries, dilated cardiomyopathy, hypoalbuminemia, dysplasia, 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, the therapeutically effective amount of the composition for the treatment of EB (e.g., the acAF composition) is the amount required to obtain at least a certain percentage reduction in that parameter. Those skilled in the art can select a certain percentage or range of percentages of reduction in a measurable parameter for which the treatment is considered effective according to the clinical and scientific circumstances.

[0062] In some embodiments, the methods provided herein can increase collagen production in a subject. As used herein, “endogenous” collagen production refers to the production of collagen by the subject, including cells, tissues, or organs, as opposed to an increase in collagen levels due to external supply or administration of collagen. For example, the methods provided herein can increase the production of type VII collagen or its fragments in a subject. While not wishing to be bound by theory, type VII collagen consists of three main domains: a non-collagenous domain (NC-1); a collagenous domain (collagen alpha-1(VII) chain); and a second non-collagenous domain (NC-2). The collagen alpha-1(VII) chain is a protein encoded by the COL7A1 gene in humans. The collagen alpha-1(VII) chain consists of a triple-helical collagenous domain adjacent to two non-collagenous domains (NC-1 and NC-2) and functions as an anchoring line between the dermal-epidermal junction of the basement membrane. The interaction between the NC-1 domain of type VII collagen and several other proteins, including laminin 5 and type IV collagen, significantly contributes to the overall stability of the basement membrane. Mutations in COL7A1 cause all types of dystrophic epidermolysis bullosa (DEB), and the exact mutations vary based on the specific type or subtype. In recessive DEB, the presence of immature stop codon (PTC) causative mutations in both alleles results in the complete absence of type VII collagen, presenting with severe, numerous scarring and blistering. A combination of PTC-causing mutations and less frequent missense mutations can lead to a milder autosomal recessive form of DEB. Most dominant cases of DEB are due to glycine substitution mutations in the collagenous domain, substituting one of the glycines in the Gly-XY repeat triplet sequence. In summary, the exact severity of DEB reflects the combination of mutations in COL7A1 and their consequences at the mRNA and protein levels, in combination with the individual's genetic background and the impact of the modified gene on exposure to environmental trauma. In addition to hereditary EB, acquired epidermolysis bullosa (EBA) involves circulating autoantibodies in EBA patients recognizing epitopes in type VII collagen molecules.

[0063] In certain embodiments, the methods provided herein for administering acAF compositions increase the production of COL7A1 mRNA, collagen alpha-1(VII) chain protein, and / or its functional fragments in a subject. For example, the methods can increase the production of COL7A1 mRNA, collagen alpha-1(VII) chain, and / or its functional fragments at chronic and / or acute wound sites in a subject. The expression level of the COL7A1 gene (e.g., mRNA level) is measured by any standard method for measuring the mRNA level of a gene, including quantitative RT-PCR, Northern blotting, and sequential gene expression analysis (SAGE). The expression level of the collagen alpha-1(VII) chain is measured by any standard method for measuring the protein level, including Western blotting, ELISA, and dot blotting.

[0064] In some embodiments, the methods provided herein can increase the production of laminin, decorin, and / or tubulin in a subject. As used herein, “endogenous” production of laminin, decorin, and / or tubulin refers to the production of laminin by the subject, including cells, tissues, or organs, as opposed to an increase in laminin levels due to external supply or administration of laminin. For example, the methods provided herein can increase the production of laminin 332 or TUBB3 or fragments thereof in a subject. While not wishing to be bound by theory, laminins are a large family of glycoproteins present in various types of basement membranes and play a crucial role in regulating tissue construction and cellular function. Laminin molecules consist of three subunits (or chains)—alpha, beta, and gamma—linked by disulfide bonds to form a cruciate structure. Laminin 332, also known as laminin 5, is composed of three subunits: laminin subunit alpha-3, laminin subunit beta-3, and / or laminin subunit gamma-2, encoded by the LAMA3, LAMB3, and LAMC2 genes, respectively. Laminin 332 is an essential component of the dermal-epidermal junction, a specialized basement membrane region that adheres the epidermis to the dermis, thereby providing skin integrity and resistance to external mechanical forces. Mutations in the genes encoding the laminin subunits, such as the LAMA3, LAMB3, and LAMC2 genes, cause junctional epidermolysis bullosa (JEB), characterized by reduced dermal-epidermal adhesion, skin fragility, mechanically induced blistering, and chronic wounds. JEB can be divided into two subcategories: JEB-Herlitz (JEB-H), where extreme fragility of the skin and mucous membranes usually leads to death within the first few years of life, and milder forms are collectively referred to as JEB-other or non-Herlitz. JEB-H is caused by loss-of-function mutations in LAMA3, LAMB3, and LAMC2, resulting in the complete loss of laminin 332. JEB-other is associated with mutations in the three genes mentioned above or in COL17A1, the gene encoding type XVII collagen, the binding ligand for laminin 332.Rare cases of JEB are associated with integrin α6β4 deficiency and result in JEB accompanied by pyloric closure.

[0065] In certain embodiments, the methods provided herein increase the production of LAMA3 mRNA, LAMB3 mRNA, LAMC2 mRNA, laminin subunit alpha-3, laminin subunit beta-3, laminin subunit gamma-2, and / or any of these functional fragments in a subject for administration of an acAF composition. For example, the methods can increase the production of LAMA3 mRNA, LAMB3 mRNA, LAMC2 mRNA, laminin subunit alpha-3, laminin subunit beta-3, laminin subunit gamma-2, and / or any of these functional fragments in chronic and / or acute wound sites of a subject.

[0066] The expression levels (e.g., mRNA levels) of laminin subunit genes (e.g., LAMA3, LAMB3, LAMC2) are measured by any standard method for measuring gene mRNA levels, including quantitative RT-PCR, Northern blotting, and sequential gene expression analysis (SAGE). The expression levels of laminin subunits (e.g., laminin subunit alpha-3, laminin subunit beta-3, laminin subunit gamma-2) are measured by any standard method for measuring protein levels, including Western blotting, ELISA, and dot blotting.

[0067] The composition may be homogeneous or heterogeneous with respect to the subject. The subject may be any animal that has or is suspected of having EB. In certain embodiments, the subject is human. For example, the method of the present disclosure involves treating a human subject that has or is at risk of developing EB, and includes administering to the subject a therapeutically effective amount of a pharmaceutical composition containing human amniotic fluid that is not or substantially free of endogenous cells.

[0068] In some embodiments, the compositions of the present disclosure supplement or replace one or more proteins that are deficient or absent in the target cells, tissues, or organs having EB, in addition to increasing the production of COL7A1 mRNA, collagen alpha-1(VII) chain, type VII collagen, collagen, LAMA3 mRNA, LAMB3 mRNA, LAMC2 mRNA, laminin subunit alpha-3, laminin subunit beta-3, laminin subunit gamma-2, laminin 332, laminin, and / or any of their functional fragments. The one or more proteins supplemented or replaced may be one or more of type VII collagen, keratin, laminin, decorin, or neurotrophin. Similarly, in some embodiments, the compositions of the present disclosure supplement or replace mRNA that is deficient or absent in the target cells, tissues, or organs having EB, in addition to increasing the production of COL7A1 mRNA, collagen alpha-1(VII) chain, type VII collagen, and / or collagen. The mRNA may be one or more mRNAs of COL7A1, COL17A, COL17A1, KRT5, KRT14, KLHL24, PLEC, DST, EXPH5, ​​CD151, LAMA3, LAMB3, LAMC2, ITGA3, ITGA6, ITGB4, FERMT1, DCN, or their variants. In some embodiments, the composition comprises therapeutic cell-free mRNA which is taken up by a target cell having EB for de novo production of the protein that is deficient or missing in EB, i.e., to cause the cell to produce a replacement protein for the deficient or missing protein.

[0069] In some embodiments, cell-free amniotic fluid compositions may modulate TGF signaling pathways in the subject, resulting in increased or decreased levels of TGF-α, TGF-β, and / or other TGF-related proteins in the subject's cells, tissues, fluids, or organs. In some EB patients, the TGF-β pathway is activated (e.g., upregulated). While we do not wish to be bound by theory, activation of the TGF-β pathway is an independent regulator (exacerbating factor) of the clinical severity of EB, independently of, for example, the patient's type VII collagen levels (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 the cell-free amniotic fluid composition provided herein downregulates (e.g., attenuation, reduction) the TGF signaling pathway compared to a control without administration of the cell-free amniotic fluid composition. The effect of cell-free amniotic fluid on the TGF signaling pathway can be evaluated 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 confluent. The medium is then replaced with test medium containing the cell-free amniotic fluid composition at various dilutions in PBS (gradually increasing from a 1% composition containing 99% PBS to a 99% composition containing 1% PBS), and the cells are incubated at 37°C for 0, 3, 6, 12, 24, and 48 hours. After the incubation period, TGF-α, TGF-β, ACTA2 / α-SMA, SERPINE1 / PAI-1, BMP2K, decorin, TGF-β R2, ZEB1, IL7, MMP3, DKK2, tenascin-C, and / or other TGF-related proteins in the whole cell lysate can be measured by standard methods such as ELISA or Western blotting, or in the supernatant.The amounts of phosphorylated Smad3, phosphorylated Smad2, phosphorylated p38, phosphorylated ERK1 / 2, and phosphorylated AKT, and their ratios to their non-phosphorylated counterparts, can be measured in whole cell lysates by immunoblotting, quantified by densitometry, and normalized to GAPDH as indicators of TGF-β pathway activity. Furthermore, the expression of TGF-α, TGF-β, ACTA2 / α-SMA, SERPINE1 / PAI-1, BMP2K, decorin, TGF-β 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 TGF-β receptor I inhibitor SB431542 can be included in the assay as a control.

[0070] The compositions of this disclosure can be used, in accordance with the methods of this disclosure, to provide preventive, mitigating, or therapeutic relief of signs or symptoms of EB. In some embodiments, the methods of this disclosure prevent, mitigate, or treat one or more signs, symptoms, or conditions associated with EB. Accordingly, the methods provided herein, but not limited to, can be used to provide preventive, mitigating, or therapeutic relief of signs or symptoms of EB, including, but not limited to, pain, pruritus, vesicles, keratosis, granulation, erosions, ulceration, pseudosyndactyly, open wounds, tissue scarring, histofibrosis, corneal scarring, blepharitis, ectropion, blepharoplasty, pterygium, visual impairment, caries, dilated cardiomyopathy, hypoalbuminemia, dysplasia, muscular dystrophy, osteopenia, osteoporosis, and post-streptococcal glomerulonephritis. In some embodiments, the compositions and methods of the Disclosure prevent, alleviate, or treat one or more ocular manifestations of EB, such as corneal opacity, corneal scarring, corneal ulceration, corneal abrasion, blepharitis, ectropion, blepharoplasty, pterygium, and visual impairment. Additionally or alternatively, the compositions and methods of the Disclosure can prevent, alleviate, or treat one or more cutaneous manifestations of EB, such as vesicles, keratosis, granulation, erosion, ulceration, pseudosyndactyly, open wounds, tissue scarring, and histofibrosis. Additionally or alternatively, the compositions and methods of the Disclosure can prevent, alleviate, or treat one or more neurological manifestations of EB, such as neuropathic pain, pruritus, and muscle weakness.

[0071] In certain embodiments, the compositions and methods of this disclosure promote wound healing in a subject (e.g., an EB subject), such as wound healing on the skin, cornea, or mucous membrane surface. Wound healing, the replacement of damaged or destroyed tissue with newly produced tissue or cells, is a biological process in living organisms such as humans, achieved through 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 US healthcare system $10 billion to $20 billion annually (Sen et al., 2009 Wound Repair Regen. 17, 763-771). Non-healing wounds cause significant morbidity and mortality, and their burden has been compared to that of cancer (Armstrong et al., 2007 Int.Wound J. 4, 286-287). Chronic wounds result from the disruption of normal acute wound healing into one of the four organizing stages described above: hemostasis, inflammation, proliferation, and remodeling (Sorg et al., 2017 Eur.Surg.Res. 58, 81-94). Dysregulation of any of these processes leads to non-healing ulcers or excessive scarring. Delayed, dysregulated, or impaired wound healing, chronic wounds, and / or non-healing wounds are associated with EB and subjects with EB. The compositions and methods provided herein can promote (e.g., increase) wound healing compared to the healing of a control wound not administered with the cell-free amniotic fluid compositions of this disclosure. Wound healing can be measured by any standard method for evaluating wound healing in vitro, ex vivo, or in vivo, which includes, but is not limited to, scratch assays, cell proliferation assays, cell migration assays, cell detachment assays, cell adhesion assays, collagen lattice contraction assays, or functional evaluation of the repaired tissue (e.g., tissue integrity). This disclosure provides exemplary evaluation methods that can be used.

[0072] The compositions and methods of this disclosure can also promote cell adhesion and / or attachment in a subject (e.g., an EB subject). The methods may include administering a therapeutically effective amount of a pharmaceutical composition to a subject, comprising amniotic fluid substantially free of endogenous cells. The therapeutically effective amount of the pharmaceutical composition may be an amount that enhances cell adhesion and attachment without altering cell proliferation. The methods provided herein can increase cell adhesion and / or attachment independently of cell proliferation (e.g., without affecting cell proliferation).

[0073] The compositions and methods of this disclosure can also increase corneal nerve regeneration in subjects (e.g., EB subjects or non-EB subjects). While we do not wish to be bound by theory, administration of amniotic fluid substantially free of endogenous cells to a subject, for example at a corneal injury site, may increase TUBB3 expression. TUBB3 is a marker of corneal nerves.

[0074] In some embodiments, the compositions of the present disclosure, namely cell-free 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 cell-free amniotic fluid compositions may be administered before, after, or concurrently with the second therapy.

[0075] The second therapy may be an additional therapeutic agent. The cell-free amniotic fluid composition and the additional therapeutic agent can be administered together in the same composition or simultaneously as separate compositions. In some embodiments, the additional therapeutic agent is an anti-inflammatory agent, an analgesic, or an antibiotic. The cell-free amniotic fluid composition can be administered in conjunction with the second therapy. Exemplary combination therapies include debridement, skin grafting, and gene therapy. [Examples]

[0076] The following examples are provided as illustrations, not as limitations.

[0077] Example 1: Concentrations of type VII collagen and decorin in cell-free amniotic fluid samples Cell-free amniotic fluid was obtained from five human donors who did not have EB. The concentrations of type VII collagen and decorin in the five cell-free amniotic fluid samples were measured using a commercially available ELISA kit according to the manufacturer's instructions.

[0078] As shown in Table 1, the concentration of type VII collagen in cell-free amniotic fluid samples ranged from approximately 77 ng / mL to approximately 987 ng / mL, with an average concentration of 335 ng / mL. Dystrophy of epidermolysis bullosa (DEB) is caused by mutations in the gene encoding type VII collagen protein. The data demonstrate that cell-free amniotic fluid compositions can be used to supplement therapeutic proteins, including type VII collagen, in DEB patients. The concentrations of other deficient or missing proteins in EB patients, such as laminin and keratin, can also be measured in cell-free amniotic fluid.

[0079] Furthermore, as shown in Table 1, the decorin concentration in cell-free amniotic fluid samples ranged from approximately 9 ng / mL to approximately 58 ng / mL, with an average concentration of 27 ng / mL. Decorin is known to modulate the TGF signaling pathway, among other things, and may play a therapeutic role in EB. The data demonstrate that therapeutic proteins containing decorin can be provided to EB patients using cell-free amniotic fluid compositions. JPEG2026515750000001.jpg46145

[0080] Example 2: mRNA levels in cell-free amniotic fluid samples The levels of mutated or deficient cell-free mRNAs involved in the pathogenesis of EB, such as COL7A1, LAMA3, LAMB3, and LAMC2, were analyzed based on data from 98 cell-free amniotic fluid samples collected from subjects without EB.

[0081] As shown in Table 2, higher levels of COL7A1, LAMA3, LAMB3, and LAMC2 mRNA (detected using gene-specific probes) were present in cell-free amniotic fluid samples compared to control mRNA (detected using all gene-related probes). Levels of cell-free mRNA for other types of EBs, such as COL17A, COL17A1, KRT5, KRT14, KLHL24, PLEC, DST, EXPH5, ​​CD151, ITGA3, ITGA6, ITGB4, and FERMT1, can also be analyzed.

[0082] The data in Table 2 demonstrate that cell-free mRNA, which is deficient or missing in EB subjects, is present at high concentrations in cell-free amniotic fluid. Cell-free mRNA can be incorporated into EB subject cells and lead to de novo production of the protein that is deficient or missing in EB. JPEG2026515750000002.jpg89154

[0083] Example 3: Cell-free amniotic fluid composition promotes wound healing Experimental method Based on informed consent, amniotic fluid (crude amniotic fluid) was obtained from a healthy donor during a cesarean section. After irradiating the crude amniotic fluid with gamma rays, it was centrifuged at 1400 × g for 15 minutes at 4°C. The supernatant was collected, the pH was adjusted to 7.4, and then sequentially filtered through a 40 μm filter and a 0.2 μm filter to obtain sterile acAF.

[0084] To investigate the effect of cell-free amniotic fluid compositions ("acAF compositions") on cell migration, an in vitro scratch assay was performed as described in Pitzurra 2020 J.Peridont.Res.55:287-295. Briefly, fibroblasts derived from patients with recessive dystrophic epidermolysis bullosa (RDEB) ("EB-fibroblasts") were seeded at an optimal density for confluence onto special mobile silicone inserts in serum-free medium, with or without the acAF composition (25% acAF in serum-free medium), and incubated overnight (two groups per group). To segment the region of interest, binary masks were created from raw phase-contrast images using ImageJ software, and gap / wound closure percentages were quantified as described in Youssefian et al., 2021 J.Invest.Dermatol.141(7):1754-1764. Swamp trajectory and gap closure percentage were measured 24, 48, and 120 hours after treatment.

[0085] result As shown in Figures 1, 2A, and 2B, the gap region remained clearly detectable at 120 hours in untreated control cells. In contrast, closure occurred between 48 and 120 hours in acAF composition-treated fibroblasts. These data demonstrate the ability of the acAF composition to promote fibroblast migration and accelerate wound healing.

[0086] 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 the acAF composition on the TGF signaling pathway was evaluated by standard methods, such as those described in Odorisio et al. 2014 Human Mol. Genet. 23:15;3907-3922. Briefly, EB-fibroblasts derived from RDEB patients as described in Example 3, and control fibroblasts derived 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 after 48 hours. To evaluate the activation of the TGF-β signaling pathway, Smad3 phosphorylation was quantified by immunoblotting of whole cell lysates. The intensity and ratio of phosphorylated and unphosphorylated Smad3 were evaluated.

[0087] result Increased TGF-β signaling, determined by increased phosphorylation of Smad3, a downstream target of TGF-β, was evident in primary EB fibroblasts (RDEB fibroblasts) (Figure 3, lane 1). Addition of recombinant decorin ("r-decorin"), a TGF-β inhibitor, resulted in a moderate decrease in smad3 phosphorylation (Figure 3, lane 2). Importantly, 24-hour treatment of EB fibroblasts (RDEB primary fibroblasts) with a 25% acAF composition showed a significant decrease in Smad3 phosphorylation (Figure 3, lane 3). Undetectable phosphorylated Smad3 was observed in control fibroblasts with or without treatment with decorin and / or the acAF composition. These data demonstrate that the acAF composition of this disclosure can downregulate the TGF signaling pathway. Downregulation of the TGF pathway can treat EB and improve EB-related signs and symptoms in the target tissue, regardless of the amount of collagen in the target tissue.

[0088] Example 5: Functional in vitro assay Proper cell proliferation, migration, and adhesion are crucial cellular functions for proper tissue homeostasis. These cellular functions are typically altered in EB patients, leading to excessive blistering and improper wound healing. Cells derived from EB subjects, such as fibroblasts, keratinocytes, or ophthalmic epithelial cells, exhibit similar altered cellular functions in vitro.

[0089] Exemplary in vitro models of EB (e.g., EB cells or EB cell lines) are established as follows: Cells such as fibroblasts, keratinocytes, and ophthalmic epithelial cells are collected from human and non-human subjects who have EB or EB-causing mutations and cultured in vitro. EB cells are also produced by knocking in or knocking down 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, and FERMT1. Primary cell cultures are established from these EB cells and selectively passaged. Immortalized EB cell lines have also been established. The therapeutic effects of the compositions of this disclosure are evaluated by cell assays including the following:

[0090] 1. Cell proliferation assay EB cells and control cells are cultured with compositions of the present disclosure, comprising various concentrations or amounts of therapeutic molecules, such as cell-free amniotic fluid containing proteins or mRNA of the gene lacking EB. For example, cells are cultured in serum-free cell culture media containing cell-free amniotic fluid at various dilutions, e.g., 100%, 75%, 50%, 25%, 10%, 1%, or 0.3%. Cell proliferation is measured by methods known to those skilled in the art, including, among others, DNA synthesis cell proliferation assays, metabolic cell proliferation assays (e.g., MTT assays), and detection of proliferation markers.

[0091] 2. Cell migration assay EB cells are cultured with compositions of this disclosure, which include various concentrations or amounts of therapeutic molecules, such as cell-free amniotic fluid containing proteins or mRNA of the gene lacking EB. For example, cells are cultured in serum-free cell culture medium containing cell-free amniotic fluid at various dilutions, e.g., 100%, 75%, 50%, 25%, 10%, or 1%. Cell migration characteristics are studied by methods known in the art, particularly Boyden chambers, cell culture wounds, and scratch assays.

[0092] 3. Cell exfoliation assay EB cells are cultured with the compositions of this disclosure, which include cell-free amniotic fluid containing various concentrations or amounts of therapeutic molecules, such as proteins or mRNA of the gene lacking EB. For example, cells are cultured in serum-free cell culture medium containing cell-free amniotic fluid at various dilutions, e.g., 100%, 75%, 50%, 25%, 10%, or 1%. 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.

[0093] For example, fibroblasts or corneal epithelial cells are seeded on a cell culture plate and cultured for 24 hours. The cells are then washed with phosphate-buffered saline (PBS) and treated with trypsin / EDTA (0.05 / 0.02%) for 10, 6, 4, 2, 1, and 0 minutes, followed by another PBS wash. Adherent cells are stained with 0.5% crystal violet in distilled water for 30 minutes, lysed with 1% sodium dodecyl sulfate (SDS), and the percentage of adherent cells can be determined by measuring absorbance at 540, 590, or 595 nm using a spectrophotometer. The results are expressed as a percentage relative to 0 minutes (untreated with trypsin).

[0094] In addition to or instead of the above, a centrifugal assay is performed. Briefly, a cell culture plate is coated overnight with the composition of the present disclosure, and EB cells are seeded for a period ranging from 10 minutes to several hours. Subsequently, the cell culture plate is centrifuged under different 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.

[0095] 4. Cell adhesion assay EB cells are cultured with the compositions of this disclosure, which include cell-free amniotic fluid containing various concentrations or amounts of therapeutic molecules, such as proteins or mRNA of the gene lacking EB. For example, cells are cultured in serum-free cell culture medium containing cell-free amniotic fluid at various dilutions, e.g., 100%, 75%, 50%, 25%, 10%, 1%, or 0%. Cell adhesion properties are measured by standard methods, including those described in Chen et al. 1999 Experim. Cell Res. 249(2):231-239. Briefly, a cell culture plate is coated overnight with the composition. Fibroblasts or corneal epithelial cells are added and allowed to adhere for a period such as 1.5 hours at 37°C. Subsequently, unbound cells are removed by washing with PBS. Adherent cells are stained with 0.5% crystal violet for 15 minutes, washed extensively with distilled water, solubilized in 1% SDS, and quantified by measuring absorbance.

[0096] 5. Collagen lattice contraction assay EB cells are cultured with the compositions of this disclosure, which include cell-free amniotic fluid containing various concentrations or amounts of therapeutic molecules, such as proteins or mRNA of the gene lacking EB. For example, cells are cultured in serum-free cell culture medium containing cell-free amniotic fluid at various dilutions, e.g., 100%, 75%, 50%, 25%, 10%, 1%, or 0%. Collagen lattice contraction properties were measured by standard methods, including those described in Odorisio et al. 2014 Human Mol. Genet. 23:15;3907-3922. Briefly, a collagen solution was prepared by mixing 3 mg / ml of acid-soluble type I collagen (Symatese Biomateriaux, Chaponost, France), 5-fold concentration DMEM, and buffer solution (0.05 M NaOH, 2.2% NaHCO3, 200 mM HEPES) in a ratio of 7:2:1. The collagen solution is mixed with the cell suspension in serum-free medium and seeded into a 6-well cell culture cluster (Costar; Corning, New York, USA), and gelled at 37°C for 30 minutes. The final collagen concentration may be 2.1 mg / ml. Serum-free DMEM is poured over the gel to prevent surface dehydration. After 12 hours of incubation, the gel is detached from each well and left suspended. The surface area of ​​the gel sample is measured at detachment (time 0) and at 24 and 48 hours. Gel shrinkage can be expressed as a percentage of the initial grid area according to the following formula: A2 / A1 × 100, where A1 is the initial gel area and A2 is the area at the observed interval. Three culture plates are used for each experimental group. The assay was performed in the following experimental groups: (1) gelation using 0.25 ng / ml recombinant human TGF-β1 (R&D Systems) (contraction-positive control); (2) gelation using 200 nM recombinant human DCN (R&D Systems) and / or in the absence of TGF-β1 (0.25 ng / ml) (negative control); and (3) gelation using 0%, 50%, or 100% acAF compositions (experimental group).

[0097] Example 6: Cell-free amniotic fluid increases the production of therapeutic proteins in vitro. EB cells are cultured at various dilutions using the composition of this disclosure, which includes cell-free amniotic fluid. For example, cells are cultured for incubation times (0, 12, 24, 48, 72 hours or more) in serum or serum-free cell culture medium containing cell-free amniotic fluid at various dilutions, e.g., 100%, 75%, 50%, 25%, 10%, 1%, or 0%. After incubation, the cells are washed with serum or serum-free medium and cultured for a certain period (0, 12, 24, 48 hours or more). At the end of the study, cells and / or culture media are collected for qPCR, Western blotting (WB), and immunofluorescence or immunohistochemical staining to quantify the mRNA and / or protein expression of molecules involved in EB pathology, such as type VII collagen, collagen alpha-1 (VII) chain protein, Col7A1, laminin 332, laminin subunit alpha-3, laminin subunit beta-3, laminin subunit gamma-2, LAMA3, LAMB3, LAMC2, TGF-β, α-SMA, decorin, Ki67, MMP9, tenascin-C, and / or beta-III tubulin. Techniques used by those skilled in the art can be used to distinguish between the levels of endogenous and exogenous molecules involved in EB pathology. Incubation with acAF compositions can increase the expression levels of type VII collagen, collagen alpha-1(VII) chain protein, Col7A1 gene, laminin 332, laminin subunit alpha-3, laminin subunit beta-3, laminin subunit gamma-2, LAMA3 gene, LAMB3 gene, and / or LAMC2 gene in EB cells. The production of type VII collagen, laminin 332, and / or their functional fragments, such as the transcription and / or translation of collagen alpha-1(VII) chain protein, Col7A1 gene, laminin subunit alpha-3, laminin subunit beta-3, laminin subunit gamma-2, LAMA3 gene, LAMB3 gene, LAMC2 gene, and / or their functional fragments, can be increased in EB cells treated with acAF compositions.

[0098] Example 6-1: Cell-free amniotic fluid increases laminin expression in EB fibroblasts. Experimental method The following primary fibroblasts, obtained from patients with epidermolysis bullosa (EB), were purchased from the Coriell Institute Cell Bank: GM10245 (hereinafter referred to as cell 45) is a primary dystrophy EB (DEB) fibroblast obtained from a 12-year-old Italian woman;

[0099] GM02857 (hereinafter referred to as cell 57) is a primary DEB fibroblast obtained from a 5-year-old Puerto Rican;

[0100] GM10319 (hereinafter referred to as cell 19) is a primary junctional EB (JEB) fibroblast derived from a 5-year-old Caucasian male infant; and

[0101] GM09590 (hereinafter referred to as cell 90) is a primary JEB fibroblast obtained from a 21-year-old Belizean woman.

[0102] All cells were received in passage 3 and cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin (Pen / Strep). This combination was hereafter referred to as the total medium. Cell proliferation and subsequent passages were carried out using methods known to those skilled in the art.

[0103] Cells 19 and 90 were seeded in 6-well plates and allowed to adhere overnight. The cells were then treated with cell-free amniotic fluid (acAF) at various concentrations for 48 hours. The acAF concentrations were applied sequentially from left to right: 0%, 5%, 10%, 15%, 20%, 25%, 40%, and 60%.

[0104] After incubation, the culture medium was discarded, the cells were rinsed with PBS, and then lysed for protein extraction using RIPA buffer with standard techniques well known to those skilled in the art. Protein concentrations were determined by BCA (bicinchoninic acid) protein assay. Subsequently, equal volumes of protein were loaded onto 4-12% Tris-glycine gradient gels in 1× Tris-glycine solution using techniques well known to those skilled in the art of Western blotting.

[0105] The gel was immersed in deionized water and gently stirred to release excess water. Then, using iBlot2 Transfer Stacks and an iBlot2 instrument, it was transferred onto a nitrocellulose membrane at 25V and room temperature for 10 minutes. Subsequently, the membrane was blocked at room temperature for 1 hour with 5% milk (RPI) in either PBS or Tris-type physiological saline (TBS) containing Tween-20 (PBST / TBST; prepared by combining 100 ml of 10×PBS or TBS, 50 ml of 20×Tween-20, and 850 ml of deionized water), while gently stirring. Next, it was washed three times in PBST / TBST at room temperature for 10 minutes, similarly while gently stirring. The membrane was incubated overnight at 4°C with a 1:1,000 dilution of primary LAMC2 antibody in 5% BSA in PBST / TBST, while shaking. The membranes were washed three times with PBST / TBST for 10 minutes at room temperature, gently shaken, and then incubated with a 1:4,000 dilution of secondary anti-mouse-HRP antibody at room temperature for 1 hour with gentle agitation. For the cell lysates, the membranes were cut to the indicated ladder size, and the lower halves were treated with a 1:2,000 dilution of primary GAPDH antibody and a 1:5,000 dilution of secondary anti-rabbit HRP antibody. The membranes were then washed three times with PBST / TBST at room temperature with gentle shaking, and subsequently colored with a chemiluminescent substrate for approximately 30 seconds. Imaging was performed using the Chemi Blot setting of an iBright imager for optimal exposure time. Band intensities were analyzed in ImageJ, and LAMC2 band intensities were normalized to GAPDH-loaded controls to evaluate relative protein expression across different conditions.

[0106] result Typically, JEB cells do not express LAMC2. However, as shown in Figure 4, treatment with acAF resulted in a dose-dependent increase in LAMC2 expression.

[0107] Example 6-2: Analysis of cell-free amniotic fluid fraction Ultracentrifugation at high radial centrifugal force (RCF) is a commonly used method for separating exosomes from solution. Using an MX120+ ultracentrifuge (ThermoFisher), 7 mL of acAF was ultracentrifuged at 53,500 rpm for 24 hours at 4 degrees Celsius, corresponding to 141,000 RCF (xg). This process yielded distinct fractions: one free of exosomes and another rich in exosomes.

[0108] After ultracentrifugation, 1 mL of the supernatant was carefully extracted to establish supernatant fraction 1 (S1). This procedure was repeated for the next 1 mL of supernatant to generate sequential fractions S2 to S7. Therefore, the fractions were named S1 to S7, with S1 representing the uppermost layer of the supernatant and S7 representing the layer closest to the bottom. Next, the residual pellet identified as P was resuspended in 1 to 2 mL of DMEM (hereinafter referred to as serum-free medium) supplemented with 1% penicillin / streptomycin. As shown in Figure 5, after ultracentrifugation at 4°C for 24 hours at 53,500 rpm / 141,000 RCF (xg), a clear brown pellet was evident at the bottom.

[0109] Cluster differentiation 24 (CD24) is a widely recognized exosome marker in human amniotic fluid. The presence of exosomes in fractions S1-S7 and pellet (P) was determined by Western blot analysis targeting CD24. As shown in Figure 6, fraction S1 did not show the presence of exosomes, as indicated by the absence of CD24 in Western blot analysis, but exosomes were clearly identified in pellet fraction (P).

[0110] Example 6-3: S1 and P fractions of cell-free amniotic fluid increase collagen expression in EB fibroblasts. Experimental method Cells 45 and 57 were seeded in 6-well plates and allowed to adhere overnight. The following day, the cells were treated for 24 hours under one of the following conditions: serum-free medium (SF), 25% S1 fraction in serum-free medium (S1), 25% pellet in serum-free medium (P), or 25% unfractionated acAF in serum-free medium. After this treatment period, the medium was removed, the cells were washed, and cell lysates were prepared as previously described for Western blot analysis targeting collagen 7 and GAPDH. Briefly, the membrane was divided into upper and lower segments for incubation: the upper segment was treated with a 1:1,000 dilution of primary anti-collagen 7 antibody, and the lower segment was treated with a 1:2,000 dilution of primary anti-GAPDH antibody, each treated in a solution of 5% BSA in PBST / TBST. This incubation was carried out overnight at 4°C with gentle agitation. The membrane was then washed and treated with the corresponding secondary antibody. After a series of three washes with PBST / TBST at room temperature with gentle agitation, the film was developed for approximately 30 seconds using a chemiluminescent substrate. Imaging was performed using an iBright imager to capture the bands at appropriate exposure levels.

[0111] result Western blot analysis demonstrated that treatment with any fraction of acAF (S1, P, acAF) significantly enhanced collagen 7 expression in EB fibroblasts. Notably, the exosome-deficient S1 fraction, the exosome-rich pellet fraction, and the entire unfractionated acAF sample all successfully increased collagen 7 expression in EB cells after a 24-hour incubation period. The results indicate that the enhancement of collagen 7 expression by acAF occurs independently of exosomes and is not related to exosome activity.

[0112] Example 7: Cell-free amniotic fluid increases the adhesion and attachment of EB fibroblasts. Experimental method Cells 45 and 57 were seeded in a 96-well plate containing complete medium and allowed to adhere overnight. The following day, the medium was removed, the cells were washed with pre-warmed sterile PBS, and then cultured for 48 hours in serum-free medium containing 0% or 25% acAF. After this period, the medium was replaced with pre-warmed sterile PBS. The plate was then tightly sealed with Parafilm, inverted, and centrifuged at 1,000 g for 10 minutes to apply centrifugation force for cell detachment. Subsequently, the PBS was replaced with pre-warmed serum-free medium. In parallel, a second plate was prepared but not subjected to centrifugation. This served as a reference to evaluate cell proliferation and establish a baseline of cell volume before centrifugation. Cell viability was evaluated using a luminescence cell viability assay (Promega CellTiter-Glo) according to the manufacturer's guidelines. Relative luminescence units (RLU) were measured using a GloMax plate reader.

[0113] result Maintaining proper adhesion is a critical challenge for EB cells. As shown in Figure 8A, acAF significantly improves the ability of EB cells to resist centripetal forces, thereby maintaining their adhesion to culture plates. A comparison of RLU values ​​of centrifuged plates with those of uncentrifuged control plates demonstrates that the effectiveness of acAF in enhancing and maintaining EB cell adhesion (Figure 8A) is independent of cell proliferation (Figure 8B).

[0114] Example 8: Cell-free amniotic fluid increases the production of therapeutic proteins and promotes wound healing in an animal model of epidermolysis bullosa. Experimental method The compositions of this disclosure, including cell-free amniotic fluid, are administered to animal models of EB, and their therapeutic effects are tested. Several EB animal models are available in various species, including cattle, dogs, horses, sheep, cats, rats, and mice. Exemplary EB animal models are described, for example, Bruckner-Tuderman et al. 2010 J.Invest.Dermatol. 130:1485-1488, which are incorporated herein by reference in their entirety. EB animal models include knockout models, conditional knockout models, and down-expression models.

[0115] Amniotic fluid and / or placental material is provided by a healthy mother during a routine cesarean section. The collection of amniotic fluid or placental material is harmless to the mother or newborn and does not require termination of pregnancy induction. Each donor has been tested using FDA-approved methods and found to be non-reactive to hepatitis B, hepatitis C, human immunodeficiency virus types 1 and 2, human T lymphotropic virus types 1 and 2, syphilis, West Nile virus, and Zika. The acAF composition is prepared as provided in this disclosure.

[0116] The composition is administered to EB subjects and control subjects as eye drops. Additionally or alternatively, the composition is delivered to EB subjects and control subjects by topical, subcutaneous, intradermal, intravenous, intracorneal, or intraocular administration. Additionally or alternatively, the composition is formulated as a gel or ointment, with permeability enhancers added, and administered to EB subjects and control subjects. In some embodiments, the composition is combined with pharmaceuticals such as drugs for managing pain or inflammation and administered simultaneously to EB subjects and control subjects.

[0117] In one example, a mouse model of reduced expression producing approximately 10% of the wild-type type VII collagen (COL7A1) protein ("C7Hypo"), as described in Fritsch et al. 2008 J.Clin.Invest. 8;118(5):1669-1679, was used as the EB animal model. Briefly, 9-week-old and 13-week-old mice were sedated with inhaled isoflurane, and the pre-injury eye condition was recorded at 40x magnification using a Topcon slit-lamp camera. After administration of 0.5% propalacaine eye drops for anesthetic, controlled surface corneal abrasions were created using a 30G needle standardized to a 1.8 mm trefin, carefully avoiding the corneal margin region. Successful removal of the epithelial layer was confirmed using fluorescein eye drops visualized under cobalt blue light, and the dimensions of the abrasion were recorded via photographs before commencing the procedure. Subsequently, 5 μL of acAF was administered to the right eye, and the left eye was treated with 5 μL of basal saline (BSS, placebo). Excess fluid was rinsed with a single-use Weck Cel spear. The treatment was applied four times a day, every two hours, for 10 days or until complete healing occurred (whichever occurred first). Drops of 0.3% tobramycin eye drops were applied to each eye as a prophylactic antibiotic. The animals were housed separately throughout the study. At the end of the study, corneal samples were taken, and mRNA expression of COL7 and TUBB3 (corneal nerve markers) was measured. Real-time PCR analysis of COL7 was performed on corneal extracts using methods known to those skilled in the art with the following specific primers: forward TGA TGC TGA CAG ATG AGC TG (SEQ ID NO: 1) and reverse CTC TTA TCA AGT CGC TGT CTC A (SEQ ID NO: 2). Real-time PCR analysis of TUBB3 was performed on corneal extracts using the following specific primers: forward CCT CCG TAT AGT GCC CTT TG (SEQ ID NO: 3) and reverse GTG GAC TTG GAA CCT GGA AC (SEQ ID NO: 4).

[0118] Further qPCR, Western blotting (WB), and immunofluorescence or immunohistochemical staining are performed to quantify the mRNA and / or protein expression of molecules involved in EB pathology, such as type VII collagen, collagen alpha-1 (VII) chain protein, Col7A1, laminin 332, laminin subunit alpha-3, laminin subunit beta-3, laminin subunit gamma-2, LAMA3, LAMB3, LAMC2, TGF-β, α-SMA, decorin, Ki67, MMP9, tenascin-C, and / or beta-III tubulin. Techniques used by those skilled in the art can be used to distinguish between the levels of endogenous and exogenous molecules involved in EB pathology. The production of type VII collagen, laminin 332 and / or its functional fragments is measured, for example, the transcription and / or translation of collagen alpha-1(VII) chain protein, Col7A1 gene, laminin subunit alpha-3, laminin subunit beta-3, laminin subunit gamma-2, LAMA3 gene, LAMB3 gene, LAMC2 gene and / or any of their functional fragments.

[0119] result In a cohort of 9-week-old mice, acAF was tested in 6 C7 Hypo mice and 2 wild-type mice. Corneal abrasions healed rapidly in wild-type (WT) mice, reaching the healing endpoint within 24 hours of injury. Intraocular administration of acAF or placebo did not result in a significant difference in healing time in WT mice. In contrast, acAF significantly shortened the healing time in C7 Hypo mice compared to placebo, with corneal abrasions healing in 3 days (2.66 times faster than placebo's 8 days, p=0.019, Figure 9). acAF treatment also resulted in significantly lower corneal opacity, as measured by the modified Fantes score, compared to placebo treatment.

[0120] Similar results were obtained in a study of 13-week-old mice. In a cohort of 13-week-old mice, acAF was tested in 5 C7 Hypo animals and 2 wild-type mice. A similar trend was observed, with acAF promoting healing in C7 Hypo mice (healing in 4 days compared to 7 days for placebo-treated corneas, 1.75 times faster healing, p=0.002), while there was no significant effect on wild-type mice treated with acAF or placebo. These findings highlight the potential of acAF to specifically enhance corneal healing in EB-affected corneas without affecting healing time in the normal wild-type state.

[0121] As shown in Figure 10, real-time PCR analysis of collagen 7 revealed a statistically significant increase in collagen 7 expression in EB-damaged animals treated with acAF. In contrast, no significant changes in collagen 7 levels were detected in wild-type animals treated with or without acAF, highlighting the specificity of acAF's effect on EB-affected corneas.

[0122] As shown in Figure 11, real-time PCR analysis of the corneal nerve marker TUBB3 revealed statistically significant upregulation of TUBB3 in EB and wild-type animals treated with acAF. This indicates enhanced corneal nerve regeneration after acAF treatment and highlights the broad capacity of acAF to promote corneal nerve regeneration in both EB-affected and non-EB subjects.

[0123] Immunofluorescence staining was performed on corneal sections using standard techniques known to those skilled in the art. First, the slides were allowed to dry at room temperature (approximately 30 minutes), and then fixed in 4% paraformaldehyde (PFA) at room temperature for 15–20 minutes. This was followed by three PBS washes in the staining jar, after which a hydrophobic barrier was created around each sample on the slide using an Elite Mini PAP Pen. The slides were blocked at room temperature for 1 hour with freshly prepared 2% bovine serum albumin (BSA). Primary antibodies diluted in 1% BSA were applied, and the slides were incubated overnight at 4°C. Antibodies used included collagen 7 (Sigma, catalog no. ZRB1507, 1:100 dilution), tubulin 3 (Sigma, catalog no. AB15708A4, 1:100 dilution), and mouse decorin (R&D Systems, catalog no. AF1060, 1:100 dilution). The following day, the slides were washed three times with PBS, incubated with secondary antibody (dilution 1:2,000) at room temperature for 2 hours, followed by three more PBS washes, and finally, DAPI was mounted. Intensity analysis was performed at 10x magnification, and the results were normalized against disease-negative controls (DisNeg). As shown in Table 3, acAF treatment was observed to significantly enhance the expression of collagen 7, tubulin 3, and decorin, demonstrating the therapeutic efficacy of acAF. JPEG2026515750000003.jpg28152

[0124] In summary, the results demonstrate that the acAF composition increases the expression of proteins such as COL7, TUBB3, and decorin, and significantly shortens wound healing time at wound sites in animal models of EB.

[0125] For example, all citations to references, including patents, published patent applications, and citations in papers, are incorporated herein by reference in their entirety.

[0126] The section headings used herein are for structural purposes only and should not be construed as limiting the subject matter described herein.

Claims

1. A method for increasing the production of one or more therapeutic proteins in a subject having epidermolysis bullosa (EB), the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition containing amniotic fluid substantially free of endogenous cells in the amniotic fluid.

2. The method according to claim 1, wherein the one or more therapeutic proteins are selected from the group consisting of collagen, laminin, decorin and tubulin.

3. The method according to claim 2, wherein the collagen comprises type VII collagen or a fragment thereof, the laminin comprises laminin 332 or a fragment thereof, and / or the tubulin comprises tubulin beta 3 class III.

4. The method according to claim 3, wherein the production of COL7A1 mRNA, collagen alpha-1 (VII) chain protein, LAMA3 mRNA, LAMB3 mRNA, LAMC2 mRNA, laminin subunit alpha-3, laminin subunit beta-3, laminin subunit gamma-2, decorin, TUBB3 mRNA, tubulin beta-3 class III, and / or any of these functional fragments is increased in the subject.

5. The method according to claim 3, wherein the production of COL7A1 mRNA, collagen alpha-1 (VII) chain protein, LAMA3 mRNA, LAMB3 mRNA, LAMC2 mRNA, laminin subunit alpha-3, laminin subunit beta-3, laminin subunit gamma-2, decorin, TUBB3 mRNA, tubulin beta-3 class III, and / or any functional fragment thereof is increased at the site of chronic and / or acute wounds of the subject.

6. A method for increasing cell adhesion and / or attachment in a subject having epidermolysis bullosa (EB), the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition containing amniotic fluid substantially free of endogenous cells in the amniotic fluid.

7. The method according to claim 6, wherein the therapeutically effective amount of the pharmaceutical composition enhances cell adhesion and attachment without altering cell proliferation.

8. The method according to claim 6 or 7, wherein the method increases cell adhesion and / or attachment independently of cell proliferation.

9. A method for increasing corneal nerve regeneration of a target, the method comprising administering to the target a pharmaceutical composition containing a therapeutically effective amount of amniotic fluid substantially free of endogenous cells in the amniotic fluid.

10. The method according to claim 9, wherein the subject has epidermolysis bullosa (EB).

11. The method according to any one of claims 1 to 10, wherein the composition is substantially free of vellus hair and vernix caseosa.

12. The method according to any one of claims 1 to 11, wherein the composition is sterile or sterilized.

13. The method according to any one of claims 1 to 12, wherein the composition further comprises amniotic membrane and / or Howardon's jelly.

14. The method according to any one of claims 1 to 13, wherein the method comprises reconstituting the pharmaceutical composition from freeze-dried amniotic fluid, amniotic membrane, and / or Wharton's jelly.

15. The method according to any one of claims 1 to 14, wherein the composition comprises a therapeutically effective amount of protein.

16. The method according to claim 15, wherein the protein is one or more of type VII collagen, keratin, laminin, and decorin.

17. The method according to any one of claims 1 to 16, wherein the composition comprises a therapeutically effective amount of cell-free mRNA.

18. The method according to claim 17, wherein 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.

19. The method according to any one of claims 1 to 18, wherein the composition comprises a therapeutically effective amount of one or more neurotrophin proteins or mRNAs.

20. The method according to claim 19, wherein one or more neurotrophins are selected from the group consisting of nerve growth factor, brain-derived neurotrophic factor, and neurotrophin-3.

21. The method according to any one of claims 1 to 20, wherein the composition further comprises a penetration enhancer or is administered simultaneously therewith.

22. The method according to any one of claims 1 to 21, wherein the composition is of the same type as the subject.

23. The method according to any one of claims 1 to 22, wherein the composition is administered to the subject topically, subcutaneously, intradermally, intravenously, intracorneally, or intraocularly.

24. The method according to claim 22, wherein the subject has a corneal wound, and the composition is administered locally to the site of the corneal wound.

25. The method according to any one of claims 1 to 24, wherein the composition is formulated as eye drops and / or a skin gel.

26. The method according to any one of claims 1 to 25, wherein the composition modulates the transforming growth factor (TGF) signaling pathway.

27. The method according to any one of claims 1 to 26, wherein the composition promotes wound healing.

28. The method according to any one of claims 1 to 27, wherein the composition alleviates or treats one or more signs, symptoms, or conditions associated with epidermolysis bullosa in the subject.

29. The method according to claim 28, wherein one or more of the signs, symptoms, or conditions are selected from the group consisting of pain, pruritus, vesicles, keratosis, granulation tissue, erosion, ulceration, pseudosyndactyly, open wounds, tissue scarring, histofibrosis, corneal opacity, corneal ulceration, corneal abrasion, corneal scarring, blepharitis, ectropion, blepharoplasty, pterygium, visual impairment, caries, dilated cardiomyopathy, hypoalbuminemia, developmental delay, muscular dystrophy, osteopenia, osteoporosis, and post-streptococcal glomerulonephritis.

30. The method according to claim 28, wherein the composition alleviates or treats the corneal opacity of the subject.

31. A method for increasing the production of collagen and / or laminin in a subject having epidermolysis bullosa (EB), the method comprising administering to the subject a therapeutically effective amount of a sterile pharmaceutical composition containing cell-free amniotic fluid.

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