Methods for promoting ocular nerve regeneration

Abstract

This disclosure features clusterin (CLU) as a multifunctional biotherapeutic for promoting ocular nerve regeneration for various ocular conditions. This disclosure features methods of using CLU for treating a subject with an ocular disorder, condition, or disease (e.g., an ocular surface disease, corneal disease or retinal disease). The methods described herein can be used for the treatment of ocular inflammatory conditions involving barrier disruption and nerve loss due to its ability to protect and promote the regeneration of ocular nerves.

Description

[0001] PATENT

[0002] Attorney Docket Number: 51907-002W02

[0003] METHODS FOR PROMOTING OCULAR NERVE REGENERATION

[0004] Sequence Listing

[0005] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on December 16, 2025, is named “51907-002W02_Sequence_Listing_12_16_25.xml” and is 5,799 bytes in size.

[0006] Statement Regarding Federally Sponsored Research

[0007] This invention was made with government support under R41 EY034396 / EY / NEI awarded by the National Institute of Health. The government has certain rights in the invention.

[0008] Background of the Disclosure

[0009] Numerous ocular diseases or disorders are characterized by inflammation, reduced epithelial or endothelial barrier function, or loss of ocular nerves. These ocular diseases or disorders include chronic ocular surface diseases, corneal diseases, and secondary ocular disorders that result from other diseases (e.g., diabetes) or surgical complications. Approved treatments for inflammatory ocular diseases, such as dry eye, include artificial tears which provide only temporary symptomatic relief, and anti-inflammatory drugs, which often yield suboptimal results and are associated with significant limitations, including notable side effects. Currently, there is no approved therapy that promotes ocular nerve regeneration for chronic ocular surface diseases, corneal diseases, or retinal diseases. Managing ocular symptoms in these patients remains a significant challenge. Accordingly, there exists a clinical need for new methods and compositions to treat neurosensory damage and promote ophthalmologic barrier function in subjects with ocular diseases.

[0010] Summary of the Disclosure

[0011] The present disclosure relates to methods of using clusterin (CLU) for promoting ocular nerve regeneration and treatment of ocular disorders, such as in treatment of dry eye disease.

[0012] In an aspect, this disclosure features a method of treating corneal neuropathy in a subject in need thereof, the method involving administering to the subject a therapeutically effective amount of an isolated clusterin (CLU) or a functional fragment thereof. In some embodiments, the subject suffers from neurogenic dry eye disease. In some embodiments, the subject suffers from moderate to severe dry eye disease. In some embodiments, the subject suffers from Sjogren's disease-associated keratoconjunctivitis. In some embodiments, the subject suffers from stage I neurotrophic keratopathy (NK), stage II NK, or stage III NK. In some embodiments, the subject suffers from genetic limbal stem cell deficiency, acquired limbal stem cell deficiency, or idiopathic limbal stem cell deficiency. In some embodiments, the subject suffers from keratitis, blepharitis, surgical injury, corneal ectasia, mustard gas keratopathy, or conjunctivitis. In some embodiments, the subject suffers from keratitis selected from diabetic keratitis, metabolic keratitis, herpetic keratitis, bacterial keratitis, and interstitial keratitis. In some embodiments, the subject suffers from surgical injury selected from refractive surgery, cataract surgery, or corneal transplantation surgery. In some embodiments, the subject suffers from conjunctivitis selected from allergic conjunctivitis, irritant conjunctivitis, and infectious conjunctivitis. In some embodiments, the PATENT

[0013] Attorney Docket Number: 51907-002W02 subject suffers from infectious conjunctivitis selected from acute bacterial conjunctivitis, subacute bacterial conjunctivitis, fungal conjunctivitis, acute viral follicular conjunctivitis, chronic viral follicular conjunctivitis, and viral blepharoconjunctivitis. In some embodiments, the subject suffers from blepharitis selected from anterior blepharitis or posterior blepharitis. In some embodiments, the subject is experiencing corneal neuropathic pain.

[0014] In an aspect, this disclosure features a method of treating neurogenic dry eye disease in a subject in need thereof, the method involving administering to the subject a therapeutically effective amount of an isolated clusterin (CLU) or a functional fragment thereof.

[0015] In an aspect, this disclosure features a method of treating moderate to severe dry eye disease in a subject in need thereof, the method involving administering to the subject a therapeutically effective amount of an isolated clusterin (CLU) or a functional fragment thereof. In some embodiments, the dry eye disease is severe.

[0016] In an aspect, this disclosure features a method of treating Sjogren's disease-associated keratoconjunctivitis in a subject in need thereof, the method involving administering to the subject a therapeutically effective amount of an isolated clusterin (CLU) or a functional fragment thereof.

[0017] In an aspect, this disclosure features a method of treating mustard gas keratopathy in a subject in need thereof, the method involving administering to the subject a therapeutically effective amount of an isolated clusterin (CLU) or a functional fragment thereof.

[0018] In an aspect, this disclosure features a method of treating neurotrophic keratopathy (NK) in a subject in need thereof, the method involving administering to the subject a therapeutically effective amount of an isolated clusterin (CLU) or a functional fragment thereof. In some embodiments, the NK is selected from stage I NK, stage II NK, and stage III NK.

[0019] In an aspect, this disclosure features a method of treating limbal stem cell deficiency in a subject in need thereof, the method involving administering to the subject a therapeutically effective amount of an isolated clusterin (CLU) or a functional fragment thereof. In some embodiments, the limbal stem cell deficiency is selected from genetic limbal stem cell deficiency, acquired limbal stem cell deficiency, and idiopathic limbal stem cell deficiency.

[0020] In an aspect, this disclosure features a method of treating an ocular condition selected from inherited retinopathy, iatrogenic retinopathy, traumatic retinopathy, and toxic retinopathy in a subject in need thereof, the method involving administering to the subject a therapeutically effective amount of an isolated clusterin (CLU) or a functional fragment thereof.

[0021] In an aspect, this disclosure features a method of treating retinal detachment in a subject in need thereof, the method involving administering to the subject a therapeutically effective amount of an isolated clusterin (CLU) or a functional fragment thereof.

[0022] In an aspect, this disclosure features a method of treating diabetic retinopathy (DR) in a subject in need thereof, the method involving administering to the subject a therapeutically effective amount of an isolated clusterin (CLU) or a functional fragment thereof.

[0023] In an aspect, this disclosure features a method of treating an ocular condition selected from keratitis, blepharitis, surgical injury, corneal ectasia, and conjunctivitis in a subject in need thereof, the method involving administering to the subject a therapeutically effective amount of an isolated clusterin (CLU) or a functional fragment thereof. PATENT

[0024] Attorney Docket Number: 51907-002W02

[0025] In some embodiments, the subject suffers from keratitis selected from diabetic keratitis, metabolic keratitis, herpetic keratitis, bacterial keratitis, and interstitial keratitis. In some embodiments, the subject suffers from surgical injury selected from refractive surgery, cataract surgery, or corneal transplantation surgery. In some embodiments, the subject suffers from conjunctivitis selected from allergic conjunctivitis, irritant conjunctivitis, and infectious conjunctivitis. In some embodiments, the infectious conjunctivitis is bacterial conjunctivitis, fungal conjunctivitis, and viral conjunctivitis.

[0026] In some embodiments, the subject suffers from blepharitis selected from anterior blepharitis or posterior blepharitis. In some embodiments, treatment results in regeneration of corneal nerves. In some embodiments, the treatment results in increased corneal nerve density.

[0027] In an aspect, this disclosure features a method of treating retinal neuropathy in a subject in need thereof, the method involving administering to the subject a therapeutically effective amount of an isolated clusterin (CLU) or a functional fragment thereof.

[0028] In some embodiments, the subject suffers from retinopathy. In some embodiments, the retinopathy is selected from inherited retinopathy, iatrogenic retinopathy, traumatic retinopathy, and toxic retinopathy.

[0029] In some embodiments, the subject suffers from retinopathy selected from nonproliferative diabetic retinopathy (DR), moderate nonproliferative DR, severe nonproliferative DR, or diabetic macular edema.

[0030] In some embodiments, the subject suffers from retinopathy selected from moderate to late-stage age-related macular degeneration (AMD). In some embodiments, the subject suffers from moderate to late-stage AMD selected from dry AMD or wet AMD.

[0031] In some embodiments, treatment results in regeneration of retinal nerves. In some embodiments, the treatment results in increased retinal nerve density. In some embodiments, treatment results in a reduction in retinal atrophy. In some embodiments, the reduction in retinal atrophy is measured by optical coherence tomography (OCT).

[0032] In some embodiments, the subject is a human patient. In some embodiments, the subject is administered the isolated clusterin (CLU) topically by eyedrop. In some embodiments, the subject is administered the isolated clusterin (CLU) by injection. In some embodiments, the injection is an intraocular or a periocular injection.

[0033] In some embodiments, the subject exhibits improvement in visual impairment. In some embodiments, the improvement in visual impairment results in improvement in color vision, reading speed or dynamic visual acuity. In some embodiments, the improvement in visual impairment is measured by the multi-luminance mobility test. In some embodiments, the improvement in visual impairment results in improvement in visual acuity. In some embodiments, the improvement in visual acuity is measured by a clinical evaluation scale selected from the group consisting of the Snellen eye test, the dynamic visual acuity test, and the pinhole visual acuity test.

[0034] Brief Description of the Figures

[0035] The features of the present disclosure are set forth with particularity in the claims appended. A better understanding of the features and advantages of the present will be obtained by reference to the PATENT

[0036] Attorney Docket Number: 51907-002WG2 following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings of which:

[0037] FIGs. 1 A-1 E show that the topical application of clusterin (CLU) improves corneal epithelial barrier in a preclinical mouse model of Sjogren's disease. FIG. 1A is a schematic illustrating the timeline of the double-blinded CLU study. 12 weeks-old Thrombospondin-1 (Thbsl) deficient mice were used as a preclinical model of chronic ocular surface inflammation associated with Sjogren's disease. Mice (n=5 per group) were treated bilaterally, twice daily, with vehicle, recombinant human CLU (rhCLU) (1 pg / ml or 50 pg / ml), human plasma-derived CLU (pCLU) (50pg / ml), or 0.1% dexamethasone (positive control) for 3 weeks. Disease progression was monitored before starting (baseline) and after 1 , 2 and 3 weeks of treatment by assessing corneal epitheliopathy with corneal fluorescein staining (CFS). Normalized weekly CFS scores of Thbs / -deficient mice with indicated treatments for male and female mice are quantified in FIG. 1 B and 1C, respectively. FIG. 1D shows quantitative analysis of CFS, relative to the baseline, with each treatment group after 3 weeks of treatment. FIG. 1E are representative images of CFS (green staining) taken at the endpoint of the study. Error bars represent ± standard error of the mean (SEM). (ns > 0.05, *p < 0.05, **p < 0.01 , ***p < 0.001 , ****p < 0.0001).

[0038] FIGs. 2A-2H shows that the nerve density and proportion of calcitonin gene-related peptide (CGRP)-positive nerves are reduced in the central and peripheral corneas of mice with Sjogren's disease. Corneal nerve density and CGRP-positive nerves in peripheral and central cornea of wild-type (WT) and Thbs / -deficient mice were evaluated by confocal microscopy in wholemount corneas immunostained with antibodies against a pan-neuronal marker, beta-3-tubulin, and the neurotransmitter, CGRP. FIG. 2A is a schematic illustrating the peripheral and central areas of the flattened wholemount corneas. Z scans from peripheral and central areas of flattened wholemount corneas (wild-type (WT) n= 5 and Thbs / -deficient n= 5) were analyzed. FIG. 2B are representative confocal images of staining for beta-3-tubulin in the central cornea of WT and Thbs / -deficient mice. FIG. 2D are representative confocal images of staining for beta-3-tubulin in the peripheral cornea of WT and Thbs / -deficient mice. FIG. 2C and FIG. 2E show the quantitative analysis of the images for the central and peripheral cornea, respectively. Nerve density represents the percentage of the total image area occupied by positively stained nerves. FIG. 2F are representative confocal images from the peripheral cornea of WT and Thbsl- deficient mice immunostained for CGRP and beta-3-tubulin. The corresponding quantitative analysis of CGRP-expressing nerves in WT (n= 3) and Thbs / -deficient mice (n= 5), are shown in FIG. 2G and FIG. 2H, respectively. Percentage of CGRP positive nerves was determined based on the ratio of CGRP positive nerve length to total beta-3-tubulin positive nerve length in each image. Arrows in merged images indicate CGRP positive nerve fibers. Error bars indicate ± SEM (*p <0.05, ***p < 0.001 , ****p < 0.0001).

[0039] FIGs. 3A-3E shows that topically applied CLU improves corneal nerve density in mice with Sjogren's disease. Corneas harvested at the study end point (3 weeks) were immunostained with anti- beta-3-tubulin. FIG. 3A and 3B are representative confocal microscopy images from indicated treatment groups and corresponding quantitative analysis of peripheral and central corneal nerves, as shown in FIG. 3C and 3D, respectively. Nerve density was analyzed as the percentage of beta-3-tubulin positive nerves relative to the total image area (n= 5 mice / group). Corneal epithelial barrier improvement correlates with the enhancement in corneal nerve density, as shown in FIG. 3E. Corneal Fluorescein Staining score (CFS) for each mouse was plotted against the corresponding corneal nerve density in PATENT

[0040] Attorney Docket Number: 51907-002WG2 central and peripheral cornea (n= 20 mice). Spearman correlation (r) analysis revealed a significant negative correlation between CFS and nerve density. Error bars represent ± SEM (ns > 0.05, **p < 0.01 ,***p < 0.001 , ****p < 0.0001).

[0041] FIGs. 4A-4C shows that topical CLU increases the proportion of CGRP-positive corneal nerves. FIG. 4A are representative immunostainings showing anti-CGRP and anti-beta-3-tubulin in peripheral cornea from mice with indicated treatments (Vehicle, recombinant human CLU (rhCLU), and dexamethasone (0.1%) after 3-weeks of treatment. FIG. 4B and 4C are the quantitative analysis of CGRP-expressing nerves in the peripheral and central corneas, respectively (n= 5 mice / group). Arrows in merged images indicate CGRP positive nerve fibers. Error bars represent ± SEM (ns > 0.05, *p < 0.05, **p < 0.01 , ***p < 0.001).

[0042] FIGs. 5A-5C show that CLU treatment promotes regeneration of conjunctival Goblet cells, correlating with reduced conjunctival tumor necrosis factor (Tnfa) expression levels in mice with Sjogren's disease. FIG. 5A shows a representative alcian blue-stained conjuctiva tissue sections of Thbs / -deficient mice from groups with indicated treatments. Cropped images represent only marked areas in overviews of conjunctiva that are representative of each group. Blue-stained goblet cells are indicated by arrows.

[0043] FIG. 5B represents the goblet cell densities as number of goblet cells counted in different treatment groups. (n= 3 mice / group). FIG. 5C show conjunctival Tnfa expression levels after three weeks of CLU treatment were determined from conjunctival RNA of indicated treatment groups using the SYBR™ Green real-time PCR method. Dexamethasone (Dex.) served as positive control with established anti-inflammatory effects. Error bars represent ± SEM (ns > 0.05, *p < 0.05, **p < 0.01 , ***p < 0.001).

[0044] FIGs. 6A and 6B show that topical CLU inhibits corneal ADAM metallopeptidase domain 17 (ADAM17) expression levels in a mouse model of Sjogren’s disease. Eyes harvested from Thbsl- deficient mice treated with vehicle or 50 pg CLU (plasma derived CLU (pCLU) or rhCLU) or 0.1 % dexamethasone, as a positive control, for up to 3 weeks (5 days / week) were harvested, and parafin- embedded sections were immunostained for ADAM 17 as described in the methods. FIG. 6A shows representative immunofluorescence images of ADAM 17 expression (red) in the corneal Epithelium (E) from the indicated treatment groups are shown. FIG. 6B shows the quantitative analysis of ADAM17 staining within the corneal epithelium presented as the ratio of ADAM17 (red) to DAPI (blue) staining. Data presented as mean ± SEM (**p < 0.01).

[0045] FIGs. 7A and 7B show that corneal and conjunctival tissue morphology in Hematoxylin and Eosin (H&E) staining remains unchanged after topical application of CLU. Representative H&E images (20x magnification) of corneal and conjunctival tissues, shown in FIG. 7A and 7B, respectively, from indicated treatment groups showed no changes in tissue morphology and no inflammatory infiltrates.

[0046] Definitions

[0047] It is to be understood that this invention is not limited to the particular methodology, protocols, and reagents described, as these may vary. The use of "or" means "and / or" unless stated otherwise. The use of the term "including," as well as other forms, such as “includes” and “included,” is not limiting.

[0048] As used herein, the term “administering” means to provide a compound, biologic (e.g., polypeptide or polynucleotide), or other therapy, remedy, or treatment such that an individual internalizes PATENT

[0049] Attorney Docket Number: 51907-002W02 the compound or therapy. Administering a compound is the process by which a subject becomes exposed to a therapeutic compound or composition of the disclosure. By way of example and without limitation, the compositions disclosed herein can be administered topically, or by injection and / or infusion, where exemplary routes of injection and / or infusion include intravitreal, subretinal, subconjunctival, intracameral, retrobulbar, suprachoroidal, retro-orbital, and any combination thereof. The constructs of the disclosure can be administered once or more than once (e.g., once, twice, three, four or more times) to the subject (e.g., a human patient in need).

[0050] "Carriers" as used herein include pharmaceutically acceptable carriers, excipients, or stabilizers which are nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed. Often the physiologically acceptable carrier is an aqueous pH buffered solution. Examples of physiologically acceptable carriers include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptide; proteins, other proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants such as TWEEN™, polyethylene glycol (PEG), and PLURONICS9™.

[0051] As used herein, the terms “clusterin gene” and “CLU gene” refer to a region of genomic DNA that encodes a clusterin polypeptide or functional fragment thereof. In some instances, the clusterin polypeptide may have at least 95% (e.g., at least 95%, 96%, 97%, 98%, 99%, 99.9%) sequence identity to Homo sapiens CLU isoform 1 preproprotein (NP_001822.3) (SEQ ID NO: 2) or a functional fragment thereof. In some instances, the clusterin polypeptide may have the sequence of Homo sapiens CLU isoform 1 preproprotein (NP_001822.3) (SEQ ID NO: 2) or a functional fragment thereof. Alternate terms known in the art for the CLU gene include apolipoprotein J (APO J), testosterone-repressed prostate message-2 (TRPM2), and sulphate glycoprotein-2 (SGP-2).

[0052] The terms “clusterin’’ or “clusterin polypeptide” optionally encompass non-peptidic components, such as carbohydrate groups or any other non-peptidic substituents that may be added to clusterin by a cell in which the protein is produced and may vary with the type of cell.

[0053] An "effective amount" of an isolated CLU polypeptide or functional fragment thereof is an amount sufficient to obtain at least one beneficial or desirable result in a subject (e.g., a human subject), or to ameliorate a symptom of disease. Thus, an effective amount may be determined empirically and in a routine manner in relation to the stated purpose. For example, an effective amount of CLU in treatment of ocular surface disease (OSDs) would be an amount of CLU sufficient to cause any beneficial therapeutic results, e.g., regeneration of retinal nerves, regeneration of ocular nerves, reduction in corneal epitheliopathy, reduced pain or discomfort, conjunctival goblet cell regeneration, or improved corneal barrier integrity.

[0054] As used herein, an “ocular disorder” encompasses any disease or condition affecting the eye. As used herein, an ocular disorder additionally encompasses any disease or condition affecting the areas directly around the eye that impact eye function, such as eye muscles, eyelids, tear glands, and eye socket. Ocular disorders may be acute (e.g., due to surgical injury) or chronic (e.g., due to chronic ocular PATENT

[0055] Attorney Docket Number: 51907-002W02 surface diseases or diabetic retinopathy). Ocular disorders may be caused by one or more direct or indirect causes, including but not limited to, a genetic condition, environmental exposure (e.g., to ultraviolet light), an infectious disease (e.g., bacteria, virus, parasite, or fungi), an autoimmune disorder (e.g., Sjogren's disease (SjD)) , a metabolic disorder (e.g., diabetes), or injury (e.g., due to surgery). An ocular disorder may result in visual impairment or blindness. Visual impairment may be measured qualitatively or quantitatively with methods known in the art, including but not limited to behavioral assessments and clinical tests. An exemplary assessment is the multi-luminance mobility test (MLMT), which assesses the effects of illumination levels on an individual's mobility performance. Other exemplary assessments include visual acuity tests, such as the Snellen eye test, the dynamic visual acuity test, and the pinhole visual acuity test. Visual impairment encompasses loss of visual acuity (i.e., the relative ability of the eye to resolve detail), contrast sensitivity, color sensitivity, depth perception, and / or motion / dynamic processing. Examples of visual impairment include blurred vision, tunnel vision, diplopia. In some embodiments, ocular disorders may result in symptoms impacting how the subject’s eyes feel, including but not limited to, experiencing pain, discomfort, irritation, eye tiredness, or strain. An ocular disorder may result in changes to areas directly around the eye that impact eye function (e.g., epiphora, changes to tear composition or production, or changes in blinking rate). An ocular disorder may result in changes to the appearance of the eye, including but not limited to, changes to the sclera (e.g., scleral icterus or red eye), pupil (e.g., miosis), or iris. In addition, an ocular disorder may cause changes in eye movement or alignment, such as exotropia or esotropia.

[0056] As used herein, the term “ocular neuropathy” refers to damage, disease, or dysfunction of one or more nerves (e.g., corneal nerves or retinal nerves) or nervous structures (e.g., the cornea or retina) of the eye. Exemplary symptoms of ocular neuropathy include stinging, burning, irritation, eye discharge (e.g., mucus), photophobia, redness, foreign body sensation, and blurry vision. Ocular neuropathy may also result in visual impairment, reduction in eye lubrication (e.g., dry eye disease), and / or reduction in rate of wound healing (e.g., of the ocular surface). As used herein, ocular neuropathy encompasses damage, disease or dysfunction of nerves or nervous structures due to ocular neurotrophic disorders, such as neurotrophic keratopathy (NK). As used herein, a “neurotrophic disorder” refers to any disorder or disease that relates to dysfunction or abnormal levels of neurotrophic factors (i.e., any molecules that enhance the growth and / or survival of neurons).

[0057] As used herein, the term “corneal neuropathy” refers to refers to damage, disease, or dysfunction of one or more nerves or nerve structure of the cornea (e.g., as evidenced by decreased corneal nerve density). Corneal neuropathy may result in decreased corneal sensitivity or corneal reflexes. Corneal neuropathy may be clinically measured with methods known in the art, including methods described herein. As used herein, the term “retinal neuropathy” refers to refers to damage, disease, or dysfunction of one or more nerves or nervous structures of the retina (e.g., as evidenced by decreased retinal nerve density). Retinal neuropathy may be clinically measured with methods known in the art, including methods described herein.

[0058] As used herein, the terms "isolated," "purified," or "biologically pure" refer to material that is free to varying degrees from components which normally accompany it as found in its native state. "Isolate" denotes a degree of separation from original source or surroundings. "Purify" denotes a degree of separation that is higher than isolation. A "purified" or "biologically pure" protein is sufficiently free of other PATENT

[0059] Attorney Docket Number: 51907-002W02 materials such that any impurities do not materially affect the biological properties of the protein or cause other adverse consequences. For example, a polynucleotide or polypeptide of this invention is purified if it is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. Purity and homogeneity are typically determined using analytical chemistry techniques, for example, polyacrylamide gel electrophoresis or high-performance liquid chromatography.

[0060] A "protein" is a macromolecule comprising one or more polypeptide chains. A protein may also comprise non-peptidic components, such as carbohydrate groups. Carbohydrates and other non-peptidic substituents may be added to a protein by the cell in which the protein is produced, and will vary with the type of cell. Proteins are defined herein in terms of their amino acid backbone structures; substituents such as carbohydrate groups are generally not specified, but may be present nonetheless.

[0061] As used herein, “treatment” and “treating” refer to an approach for obtaining at least one of the following beneficial or desired results in a subject (e.g., a human subject): slowing or inhibiting the progression of ocular disease in a subject identified as having ocular disease; reversing the progression of ocular disease in a subject identified as having ocular disease; promoting the maintenance of ocular disease in a subject identified as having ocular disease; alleviating or ameliorating one or more symptoms of ocular disease in a subject identified as having ocular disease; reducing one or more clinical manifestations of ocular disease (e.g., pain in eyes, sensitivity to light, blurred vision, loss of visual acuity, blindness). “Alleviating,” “ameliorating,” “reducing,” “reversing,” or “slowing” means that the extent of the disease, clinical manifestations of the disease, or symptoms of the disease are lessened such that a time course of disease progression is slowed, as compared to the absence of treatment. “Treatment” can also mean prolonging ocular function of the subject, as compared to not receiving treatment.

[0062] Detailed Description of the Invention

[0063] This disclosure features clusterin (CLU) as a multifunctional biotherapeutic for a widespread range of ocular conditions. This disclosure features methods of using CLU for treating a subject with an ocular disorder, condition, or disease (e.g., an ocular surface disease or corneal disease). In addition, the methods described herein can be used for the treatment of ocular inflammatory conditions involving barrier disruption and nerve loss due to its ability to protect and promote the regeneration of ocular nerves.

[0064] At the ocular surface, CLU is expressed by epithelial cells and in the lacrimal gland and CLU protein can be detected in tears. CLU binds to damaged or denatured proteins, protecting cells, sealing the ocular surface or blood retinal barrier, and promotes healing.

[0065] The cornea plays a critical role in maintaining ocular health, serving as a transparent, densely innervated outer layer that protects internal structures and contributes significantly to the eye’s refractive power. As non-limiting examples, dry eye disease, trauma, infections or decreased tear production can lead to disruptions in the epithelial barrier, also known as ocular epitheliopathy, that are associated with ocular neuropathy.

[0066] This disclosure features CLU as a biologic for treating a wide range of ocular diseases and disorders, which may involve corneal epitheliopathy and nerve loss, such as dry eye disease, laser refractive surgeries, erosions, trauma, corneal diseases, retinal diseases, and neurotrophic keratopathy. PATENT

[0067] Attorney Docket Number: 51907-002W02

[0068] Pharmaceutical Compositions

[0069] This disclosure features pharmaceutical compositions containing clusterin (CLU) or a functional fragment thereof. CLU or functional may be formulated into various compositions (e.g., a pharmaceutical composition) for administration to a subject in a biologically compatible form suitable for administration in vivo.

[0070] CLU is an evolutionarily conserved, homeostatic, glycoprotein which is expressed and secreted by mucosal epithelia at fluid-tissue interfaces. As disclosed herein, CLU may be produced recombinantly (e.g., in bacteria, in yeast, in mammalian cells) or isolated from a human biological sample, such as plasma. Methods to produce recombinant human CLU (rhCLU) are known in the art, e.g., in Dabbs, R. A. & Wilson, M. R. Expression and purification of chaperone-active recombinant clusterin. PLoS One 9, e86989. https: / / doi.org / 10.1371 / journal.pone.0086989 (2014). and Satapathy, S., Dabbs, R. A. & Wilson, M. R. Rapid high-yield expression and purification of fully post-translationally modified recombinant clusterin and mutants. Sci Rep 10, 14243., the disclosure of which is incorporated herein by reference.

[0071] As an exemplary CLU, human CLU is composed of two disu Ifide-linked alpha (34-36 kDa) and beta (36-39 kDa) subunits derived from a single amino acid chain (449 amino acids) that become glycosylated in the endoplasmic reticulum and Golgi bodies. The glycosylated human CLU undergoes intramolecular cleavage and dimerization before secretion (e.g., secretion into body fluids, such as tears). The first 22 amino acids are the secretory signal sequence. The cleavage site between the alpha and beta chains is between amino acids 227 and 228. Table 1 lists exemplary CLU sequences of the disclosure: PATENT

[0072] Attorney Docket Number: 51907-002W02

[0073] For example, the agents described herein may be administered in a suitable diluent, carrier, stabilizer, or excipient, and may contain a preservative, e.g., to prevent the growth of microorganisms. Conventional procedures and ingredients for the selection and preparation of suitable compositions are described, for example, in Remington, J.P. The Science and Practice of Pharmacy, Easton, PA. Mack Publishers, 2012, 22nd ed. And in The United States Pharmacopeial Convention, The National Formulary, United States Pharmacopeial, 2015, USP 38 NF 33.

[0074] Mixtures of agents described herein may be prepared in water suitably mixed with one or more excipients, carriers, or diluents. Dispersions may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations may contain a preservative to prevent the growth of microorganisms. The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions (described in US 5,466,468, the disclosure of which is incorporated herein by reference). In any case the formulation may be sterile and may be fluid to the extent that easy syringability exists. Formulations may be stable under the conditions of manufacture and storage and may be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and / or vegetable oils. Proper fluidity may be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin. PATENT

[0075] Attorney Docket Number: 51907-002W02

[0076] For example, a solution containing a pharmaceutical composition described herein may be suitably buffered, if necessary, and the liquid diluent first rendered isotonic with sufficient saline or glucose. In this connection, sterile aqueous media that can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage may be dissolved in 1 mL of isotonic NaCI solution and either added to 1 ,000 mL of hypodermoclysis fluid or injected at the proposed site of infusion. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Moreover, for human administration, preparations may meet sterility, pyrogenicity, general safety, and purity standards as required by FDA Office of Biologies standards.

[0077] Although the descriptions of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions which are suitable for administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to any other animal, e.g., to non-human animals, e.g., non-human mammals, such as mice. Modification of pharmaceutical compositions suitable for administration to humans to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and / or perform such modification with merely ordinary, if any, experimentation. Subjects to which administration of the pharmaceutical compositions are contemplated include, but are not limited to, humans and / or other primates and mammals, such as rodents.

[0078] The pharmaceutical compositions disclosed herein may adjunct components conventionally found in pharmaceutical compositions or secondary active agents. For example, the pharmaceutical compositions disclosed herein may contain antipruritic, astringents, local anesthetics or anti-inflammatory agents. In addition, the pharmaceutical compositions may contain additional materials useful in physically formulating various dosage forms of the compositions of the present invention, such as dyes, preservatives, antioxidants, opacifiers, thickening agents and stabilizers. However, such materials, when added, should not unduly interfere with the biological activities of the components of the compositions of the present invention.

[0079] Pharmaceutical compositions disclosed herein include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions may be generated from a variety of components that include, but are not limited to, preformed liquids, self-emulsifying solids and selfemulsifying semisolids. The pharmaceutical compositions disclosed herein may be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, lotions, oils, creams, and drops. The compositions of the present invention can also be formulated as suspensions in aqueous, non-aqueous or mixed media. Aqueous suspensions can further contain substances that increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and / or dextran. The suspension can also contain stabilizers.

[0080] Dosage and Administration

[0081] The methods herein include administering to the subject (e.g., a human subject with an ocular disease or disorder) an effective amount of a clusterin (CLU) composition described herein. Identifying a subject in need of such treatment can be in the judgment of a subject or a health care professional and can be subjective (e.g. opinion) or objective (e.g. measurable by a test or diagnostic method). PATENT

[0082] Attorney Docket Number: 51907-002W02

[0083] The dose when using the CLU compositions of the disclosure can vary within limits and as is customary and is known to the practitioner (e.g., a physician), it is to be tailored to the individual conditions in each individual case. The actual dosage amount of a composition of the present disclosure administered to a patient may be determined by physical and physiological factors such as body weight, severity of condition, previous or concurrent therapeutic interventions, idiopathy of the patient and on the route of administration (e.g., topical administration or by injection). Depending upon the dosage and the route of administration, the number of administrations of a preferred dosage and / or an effective amount may vary according to the response of the subject. The practitioner responsible for administration will, in any event, determine the concentration of active ingredient(s) in a composition and appropriate dose(s) for the individual subject. Administration may occur any suitable number of times per day, week, month or year, and for as long as necessary. Subjects may be adult or pediatric (e.g., humans), with or without a comorbid condition. In addition to the methods of treatment described herein, the subject may be treated with an additional intervention or therapy.

[0084] In some embodiments, the pharmaceutical composition includes CLU produced recombinantly (e.g., by a mammalian cell line, such human embryonic kidney (HEK) or Chinese hamster ovary (CHO) cells). In some embodiments, the pharmaceutical composition includes CLU isolated from a natural source (e.g., from blood plasma).

[0085] Administration can be topical (e.g., by eye drop, gel, cream, or lotion). For example, the pharmaceutical composition for use in the treatment of ocular surface disease may be administered topically, such that CLU is combined with a liquid carrier, and administered to the subject’s eye. An exemplary dosage form is a sterile solution for topical use, such as use as drops. For example, the CLU may be combined with a liquid carrier and administered in the form of an eye drop by contacting the pharmaceutical composition to the surface of an eye of the patient. The concentration of CLU in the resulting liquid pharmaceutical should be sufficiently high that when administered to the patient, there is a sufficient concentration of CLU to reduce the activity sufficient to produce the required result.

[0086] The pharmaceutical compositions disclosed herein may be formulated for infusion and / or injection (e.g., intravitreal, subretinal, subconjunctival, intracameral, retrobulbar, suprachoroidal, retro- orbital, and any combination thereof). Exemplary injectable pharmaceutical compositions are sterile aqueous solutions that may also contain buffers, diluents and other suitable additives such as, but not limited to, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients.

[0087] Dosages and desired drug concentrations of pharmaceutical compositions of the present invention may vary depending on the particular use envisioned. The determination of the appropriate dosage or route of administration is well within the skill of an ordinary physician. Animal experiments provide reliable guidance for the determination of effective doses for human therapy. Interspecies scaling of effective doses can be performed following the principles laid down by Mordenti, J. and Chappell, W. "The use of interspecies scaling in toxicokinetics" In Toxicokinetics and New Drug Development, Yacobi et al., Eds., Pergamon Press, New York 1989, pp. 42-96. PATENT

[0088] Attorney Docket Number: 51907-002W02

[0089] Methods of Treatment

[0090] The methods using clusterin (CLU) disclosed herein may be used to treat a wide range of ocular disorders or diseases, including, but not limited to, ocular diseases characterized by inflammation, reduced epithelial or endothelial barrier function and / or loss of nerves. A subject can be a mammal (e.g., a human). In some embodiments, the subject is a patient (e.g., a clinical patient).

[0091] Chronic inflammation plays a central role in the development and persistence of ocular surface disease, and the inflammatory cascade can cause tissue damage and corneal nerve sensitization, effectively reducing pain threshold to perpetuate the dry eye disease cycle in the corneal and conjunctival tissues. As a result, currently approved treatments for many chronic ocular surface diseases (e.g., dry eye) primarily target inflammation. However, current treatments offer limited efficacy in repairing corneal epithelial damage and typically provide only short-term symptomatic relief.

[0092] This disclosure features methods of using CLU to treat ocular neuropathy (e.g., corneal neuropathy or retinal neuropathy) in a subject in need thereof. In some embodiments, this disclosure features methods of using CLU to treat corneal neuropathy (e.g., resulting from a chronic ocular surface disease or corneal disease). In some embodiments, this disclosure features methods of using CLU to treat retinal neuropathy (e.g., resulting from a retinopathy). In addition, this disclosure features methods of using CLU to treat chronic ocular surface diseases and corneal diseases (e.g., a chronic ocular surface disease or corneal disease that results in corneal neuropathy). This disclosure features methods of using CLU to treat retinopathies (e.g., a retinopathy that results in retinal neuropathy).

[0093] Identification of a Subject Having Corneal Neuropathy

[0094] A subject that has been identified as having corneal neuropathy can be treated with an isolated clusterin (CLU) in accordance with the methods described herein. In some embodiments, the CLU is administered topically. Corneal neuropathy may be caused by any etiology that causes damage directly or indirectly to the nerves of the cornea, including ocular disorders described herein (e.g., chronic ocular surface disease or corneal diseases, such as keratitis, neurotrophic keratopathy, surgical injury, or corneal ectasia).

[0095] Diagnosis of corneal neuropathy may be performed by any method or technique known in the art, including the exemplary methods described herein. Diagnosis and assessment of corneal neuropathy may be direct (e.g., through assessments of the corneal nerves structure or function) or indirect (e.g., assessment of the causative ocular surface disease or corneal disease). Diagnostic methods may be used to initially diagnosis corneal neuropathy (e.g., due to an ocular surface disease or corneal disease) or to assess the severity of corneal neuropathy in a subject. A person of skill in the art (e.g., a physician or clinician) may utilize multiple diagnostic methods (e.g., methods known in the art or disclosed herein) to initially diagnose a subject or evaluate the disease severity in a subject. Diagnosis may be made based on one or more clinical tests (e.g., corneal aesthesiometry or in vivo corneal confocal microscopy), signs (e.g., reduced blinking rate or tear production) and symptoms (e.g., eye dryness, burning / stinging, itching, foreign body sensation, eye discomfort, photophobia and pain), in the context of a subject’s clinical history.

[0096] An exemplary diagnostic method of corneal neuropathy is corneal esthesiometry, also known as corneal aesthesiometry, which is a qualitative or quantitative measurement of corneal sensation. For PATENT

[0097] Attorney Docket Number: 51907-002WQ2 example, a common qualitative method used in clinical practice is the contacting of a subject’s eye with a cotton-tipped applicator. Another exemplary quantitative corneal esthesiometry test is the is the handheld esthesiometer, often termed the Cochet-Bonnet test, which evaluates the ophthalmic branch of the fifth cranial nerve (trigeminal).

[0098] Another exemplary clinical measurement of corneal neuropathy is in vivo confocal microscopy (IVCM), which is a non-invasive imaging technique that allows for direct visualization and quantitative analysis of corneal nerve fibers, revealing changes in their density, tortuosity, and morphology. IVCM enables real-time, unparalleled visualization of cellular and subcellular corneal structures, facilitating early diagnosis and monitoring of corneal neuropathy resulting from ocular disorders (e.g., such as the ocular disorders described herein, such as keratitis, corneal dystrophies, and nerve injury following refractive or corneal transplant surgeries). Corneal neuropathy may disrupt ocular surface homeostasis, which may result in epithelial compromise and inflammation. IVCM may be used to detect anatomical or functional changes caused by corneal neuropathy, such as by the identification of the four main plexiform nerve layers based on corneal involvement: mid-stroma, anterior-mid, anterior, and subbasal epithelial layers.

[0099] Other exemplary diagnostic methods of corneal neuropathy include questionnaires that gauge a patient’s subjective experience of ocular symptoms to produce more objective and reproducible data relating to disease severity (e.g., the visual analogue scale (VAS), the National Eye Institute Visual Function Questionnaire-25 (NEI-VFQ25), the Ocular Surface Disease Index (OSDI), Standard Patient Evaluation of Eye Dryness Questionnaire (SPEED), Dry Eye Questionnaire (DEQ), and Symptom Assessment In Dry Eye (SANDE)).

[0100] In some embodiments, the improvement in corneal neuropathy is measured by a clinical measurement selected from the group consisting of a tear volume assessment, ocular surface staining, a tear osmolarity test, a matrix metalloproteinase-9 (MMP-9) test, a lactoferrin test, or interferometry.

[0101] In some embodiments, the methods of treatment described herein result in a regeneration of corneal nerves. In some embodiments, the methods of treatment described herein result in increased corneal nerve density.

[0102] Identification of a Subject Having Retinal Neuropathy

[0103] A subject that has been identified as having retinal neuropathy can be treated with clusterin (CLU) in accordance with the methods described herein. Retinal neuropathy may be caused by any etiology that causes damage directly or indirectly to the nerves of the retinal, which include ocular disorders described herein (e.g., diabetic retinopathy (DR), retinitis pigmentosa, diabetic macular edema, chemotherapy induced retinopathy, or age-related macular degeneration). Diagnosis of retinal neuropathy may be performed by any method or technique known in the art or by a method described herein. Diagnosis may be made based on one or more clinical tests (e.g., optical coherence tomography (OCT) or fundus autofluorescence (FAF)) , signs, and symptoms (e.g., visual impairment or changes in vision), in the context of a subject’s clinical history.

[0104] Exemplary measurements include but are not limited to the Amsler grid test, OCT, FAF, dye tests (e.g., fluorescein angiography or indocyanine green angiography), ultrasound, and scanning laser polarimetry (SLP). The Amsler grid is a grid of straight lines, with a reference dot in the center. The Amsler grid may be used to detect vision problems resulting from retinal neuropathy, particularly damage PATENT

[0105] Attorney Docket Number: 51907-002W02 to the macula (i.e. , the central part of the retina), or damage to the optic nerve. The subject (e.g., a human patient, such as a patient diagnosed with retinopathy) reports visual distortions, such as wavy or blurred lines, dark regions, missing area, or changes to the size or shape of the squares.

[0106] Other exemplary measurements include OCT and optical coherence tomography angiography (OCTA), which are non-invasive imaging tests that utilize light waves to take cross-section pictures of your retina. OCT and OCTA can be used to diagnose and assess the severity of many ocular disorders (e.g., ocular disorders that may cause retinal neuropathy) and retinal abnormalities, including but not limited to, diabetic retinopathy, diabetic macular edema, macular irregularities (e.g., macular hole, macular pucker, macular edema), age-related macular degeneration (e.g., wet age-related macular degeneration or dry age-related macular degeneration), geographic atrophy, glaucoma, central serous retinopathy, vitreous traction, abnormal blood vessels, blood vessel blockage, and drusen.

[0107] Fundus autofluorescence (FAF) is a non-invasive imaging modality that may be utilized in both research and clinical settings due to its ability to map naturally and pathologically occurring fluorophores in the posterior pole. FAF highlights a retinal pigment, called lipofuscin, that increases with retinal damage or dysfunction. FAF can be used to diagnose and assess the severity of retinal disorders (e.g., retinal disorders that result in retinal neuropathy), including but not limited to, diabetic retinopathy, diabetic macular edema, and macular irregularities. In a normal fundus without retinal pathology, blood vessels will appear dark since blood is able to strongly absorb the blue (~488nm) or green (~514nm) light that is typically used in FAF imaging. Abnormal regions of hyper-autofluoresence (AF) are a result of increased levels of lipofuscin / compounds with similar autofluorescent spectra, or increased transmission of fluorescence. Exemplary causes of hyper-AF include increased retinal pigment epithelium (RPE) lipofuscin secondary to RPE dysfunction, presence of subretinal autofluorescent material (eg. vitelliform lesions, blood breakdown products, certain types of drusen, subretinal fluid in central serous chorioretinopathy), optic disc drusen, and loss of macular photopigment and photoreceptor attenuation. Abnormal regions of hypo-autofluorescence (AF) are a result of decreased levels of lipofuscin, decreased RPE density, or blockage of fluorescence. Exemplary causes of hypo-AF include decreased RPE lipofuscin (e.g., RPE atrophy or RPE tears), presence of naturally occurring macular pigments, acute intraretinal or subretinal hemorrhage, fibrosis / scar tissue, Media opacities (e.g., cataract, vitreous hemorrhage, posterior capsular opacification, asteroid hyalosis, vitritis).

[0108] Angiography (e.g., fluorescein angiography or Indocyanine green angiography) are tests that utilize a dye that show retinal blood vessels and the deeper, harder-to-see blood vessels behind the retina (i.e., the choroid). For a fluorescein angiograph, results are typically described in terms of relative fluorescence (i.e., relative to the fluorescence from a healthy eye). Hypofluorescence is a reduction from the normal expected fluorescence and hyperfluorescence refers to an increased or abnormal fluorescence. Hypofluorescence can occur secondary to a blocking effect or because of a vascular filling defect. Hyperfluorescence can occur because of fluorescein leakage, staining, pooling or by transmission defects and autofluorescence. An angiograph may be used to diagnose and assess the severity of retinal disorders (e.g., retinal disorders that result in retinal neuropathy), including but not limited to, diabetic retinopathy, diabetic macular edema, retinitis pigmentosa, cystoid macular edema, Age-related macular degeneration and macular irregularities. PATENT

[0109] Attorney Docket Number: 51907-002W02

[0110] Chronic Ocular Surface Disease (OSD) and Corneal Disease

[0111] The methods described herein may be used for treating chronic ocular surface diseases (OSDs), including dry eye, Sjogren's disease-associated keratoconjunctivitis, mustard gas keratopathy, blepharitis, eye allergies, keratitis, conjunctivitis, limbal stem cell deficiency, neurotrophic keratopathy, or any combination thereof. The methods described herein may be used to treat loss of nerves due to ocular surgery, such as refractive surgery or cataract surgery. The methods described herein may be useful for treating corneal disease, including diabetic keratopathy, keratitis due to contact lens use, corneal ectasia, herpes simplex keratitis, interstitial keratitis, or any combination thereof. In some embodiments, the subject suffers from keratitis (e.g., from diabetic keratitis, metabolic keratitis, herpetic keratitis, bacterial keratitis, and interstitial keratitis). In some embodiments, the subject suffers from surgical injury (e.g., from refractive surgery, cataract surgery, or corneal transplantation surgery). In some embodiments, the subject suffers from conjunctivitis including, but not limited to, allergic conjunctivitis, irritant conjunctivitis, and infectious conjunctivitis (e.g., bacterial conjunctivitis, fungal conjunctivitis, viral conjunctivitis). In some embodiments, the subject suffers from blepharitis (e.g., anterior blepharitis or posterior blepharitis). In some embodiments, the subject is experiencing corneal neuropathic pain.

[0112] Exemplary methods to assess or diagnose chronic ocular surface disease (OSD) or corneal disease severity include questionnaires that gauge a patient’s subjective experience of ocular symptoms to produce more objective and reproducible data relating to disease severity. Different disease severity assessment methods may use different numerical values to express disease severity, but these scales will be readily interpretable by a person of skill in the art (e.g., a physician or clinician). For example, the National Eye Institute Visual Function Questionnaire-25 (NEI-VFQ25), has numerical scale from 0-100, with a score of 0 representing extremely severe disease, and a score of 100 indicating that a subject has no vision problems. Another exemplary scale, the Ocular Surface Disease Index (OSDI), utilizes a numerical scale from 0-100, with scores 0 to 12 representing normal, 13 to 22 representing mild disease, 23 to 32 representing moderate disease, and greater than 33 representing severe disease. A person of skill in the art (e.g., a physician or clinician) may readily interpret values taken by these questionnaires or other comparable methodologies (e.g., dry eye disease questionnaires) to assess disease severity. Other exemplary questionnaires include the Standard Patient Evaluation of Eye Dryness Questionnaire (SPEED), Dry Eye Questionnaire (DEQ), and Symptom Assessment In Dry Eye (SANDE).

[0113] In some embodiments, the improvement in chronic ocular surface disease (OSD) or corneal disease is measured by a clinical measurement selected from the group consisting of a tear volume assessment, ocular surface staining, a tear osmolarity test, a matrix metalloproteinase-9 (MMP-9) test, a lactoferrin test, or interferometry. Exemplary tear volume assessments include the Schirmer's test and tear meniscus assessment. For example, the Schirmer's Test utilizes a small paper strip with rulers printed along their length. This strip is placed over the temporal one-third of the lower lid margin inserting the folded end inside the inferior conjunctival fornices. The strips are removed after 5 minutes and the tear volume produced in that time is measured by reading the wetting length in millimeters, where less than 10mm of tear production in 5 minutes is suggestive of some form of ocular surface disease (e.g., dry eye).

[0114] Another exemplary measure used to assess chronic ocular surface disease (OSD) or corneal disease severity is a MMP-9 test, which measures MMP-9 levels in tears. Elevated MMP-9 levels (e.g., PATENT

[0115] Attorney Docket Number: 51907-002W02 measured with a commercial kit, such as InflammaDry®) is indicative of the loss of ocular surface barrier function and found to be elevated in patients with ocular surface disease (e.g., dry eye). Another exemplary test is a lactoferrin test (e.g., measured with a commercial device, such as the TearScan® 270 MicroAssay System), which measures lactoferrin produced by the acinar cells of the lacrimal gland and can be detected in tears. Lactoferrin has established antimicrobial and anti-inflammatory properties, and lower concentrations of lactoferrin are present in patients with ocular surface disease. Thickness of the lipid layer on the ocular surface is also indicative of ocular surface disease severity, where thinner lipid layers are indicative of increased ocular surface disease severity. Lipid layer thickness can be quantified by interferometry (e.g., LipiView®). Interferometers may also provide information on blink dynamics.

[0116] In some embodiments, the improvement in chronic ocular surface disease (OSD) or corneal disease severity is measured by ocular surface staining (e.g., corneal staining). Ocular surface staining may be done as part of a routine eye exam. The ocular surface is typically stained with fluorescein dye. The fluorescein is applied on a moistened sterile strip of paper to the inner lining of the lower eyelid or applied in an eye drop mixed with a topical anesthetic. When abnormal or missing epithelial cells are stained with fluorescein and observed under the cobalt blue light, they appear bright green. Bright green areas of the cornea may indicate a corneal disease or ocular surface disease, such as dry eye. Other exemplary stains include lissamine green and rose bengal.

[0117] Dry Eye

[0118] Epithelial disease caused by ocular surface desiccation due to tear dysfunction is referred to collectively as dry eye, an affliction that affects 5% to 34% of all people globally. Dry eye disease, trauma, infections, or decreased tear production can lead to those disruptions in the epithelial barrier, also known as ocular epitheliopathy. Epitheliopathy in dry eye is routinely assessed by staining with a clinical dye, e.g., fluorescein or rose bengal and, along with symptom assessment, which serve as a primary endpoint in clinical trials for new drugs. However, the pharmaceuticals currently approved for dry eye by the U.S. Food and Drug Administration (FDA), such as Cyclosporine A (Restasis; Allergan, Dublin, Ireland) and Lifitegrast (Xiidra; Shire, Lexington, MA), are not effective in reversing epitheliopathy in clinical trials. Managing ocular symptoms in these patients remains a significant challenge. Currently approved treatments for dry eye include artificial tears, which provide only temporary symptomatic relief, and anti-inflammatory drugs, which often yield suboptimal results and are associated with significant limitations, including notable side effects. Importantly, no approved treatment specifically addresses the neurosensory damage of the ocular surface that contributes to dry eye symptoms. Therefore, finding new, targeted and specific therapeutic alternatives to treat neurosensory damage in dry eye is of great clinical interest.

[0119] The two main types of dry eye are evaporative and aqueous-deficient. Evaporative dry eye occurs when the oily layer of tears, which slow their evaporation rate, are deficient or poor quality. Evaporative dry eye can be due to intrinsic factors such as meibomian gland dysfunction, disorders of lid aperture, or a low blink rate. Extrinsic factors causing evaporative dry eye include ocular surface diseases (OSDs) and as a reaction to drugs. Exemplary systemic drugs known to cause dry eye include antihistamines, acutance, beta-blockers, antispasmodics, diuretics, and certain psychotropic drugs. PATENT

[0120] Attorney Docket Number: 51907-002W02

[0121] Aqueous-deficient dry eye occurs when the lacrimal glands fail to produce enough of the water components of tears, known as the aqueous layer. Aqueous-deficient dry eye can be due to a deficiency in lacrimal glands, lacrimal gland duct obstruction, lack of a tear formation reflex, or systemic drugs that inhibit lacrimal gland production. Aqueous-deficient dry eye can also be due to Sjogren's disease (SjD) . Other risk factors for aqueous-deficient dry eye include occupation, chronic exposure to high wind velocity and / or low relative humidity, aging, hormonal imbalances or reduced androgen levels, and diabetes.

[0122] In some embodiments, this disclosure features methods of treating corneal neuropathy that results from dry eye disease, where the dry eye disease is moderate to most severe dry eye disease. Dry eye disease severity (e.g., mild dry eye disease, moderate dry disease, severe dry eye disease, most severe dry eye disease) may be clinically evaluated by methods known in the art, including exemplary methods disclosed herein. Dry eye disease severity may be ranked qualitatively (e.g., mild dry eye disease, moderate dry disease, severe dry eye disease, or most-severe dry eye disease) or quantitatively (e.g., stage I dry eye disease, stage II dry eye disease, stage III dry eye disease, or stage IV dry eye disease) depending on the assessment method.

[0123] Diagnosis of dry eye disease may be performed by any method or technique known in the art, including the exemplary methods described herein. Diagnostic methods may be used to initially diagnosis dry eye disease or to assess dry eye disease severity in a subject. A person of skill in the art (e.g., a physician or clinician) may utilize multiple diagnostic methods (e.g., methods known in the art or disclosed herein) to initially diagnose a subject or evaluate the disease severity in a subject. Diagnosis may be made based on one or more clinical tests (e.g., corneal aesthesiometry or in vivo corneal confocal microscopy), signs (e.g., reduced blinking rate or tear production) and symptoms (e.g., eye dryness, burning / stinging, itching, foreign body sensation, eye discomfort, photophobia and pain), in the context of a subject’s clinical history.

[0124] For example, dry eye may be diagnosed by a subject meeting one or more of the following criteria: a Schirmer tear test (without anesthesia) >1 and <10 mm, corneal fluorescein staining score >2.0 (0-4 scale), eye dryness score (EDS) >40 (0-100 visual analogue scale [VAS]), and history of artificial tear use within 30 days of the clinical evaluation.

[0125] In some embodiments, CLU may be used to treat dry eye in a subject in need. In some embodiments, CLU may be administered topically (e.g., by eye drop or ointment) to the ocular surface.

[0126] Sjogren's disease-associated keratoconjunctivitis

[0127] Sjogren's disease (SjD) is a chronic autoimmune disorder characterized by inflammatory destruction and functional loss of the lacrimal glands, leading to reduced tear production and subsequent dry eye disease. This tear deficiency results in ocular surface inflammation, including epithelial defects in both the cornea and conjunctiva, a condition clinically termed as keratoconjunctivitis sicca. Patients often experience burning, stinging and itching sensations in the eyes, along with blurred vision. These symptoms correlate with the altered corneal nerve morphology and reduced nerve density observed in SjD patients.

[0128] SjD is classified into two main types: primary SjD and secondary SjD. Primary SjD refers to instances where SjD develops on its own and is not caused by another health condition. Secondary SjD PATENT

[0129] Attorney Docket Number: 51907-002W02 refers to when SjD is triggered or caused by another disease, such as hepatitis C, cytomegalovirus (CMV), Epstein-Barr virus, human T-lymphotropic virus 1 , coronavirus disease 2019 (COVID-19), lupus, psoriatic arthritis, rheumatoid arthritis.

[0130] Exemplary methods to diagnose SjD include diagnostic blood tests (e.g., the Sjo® test) that measure key biomarkers associated with SjD disease pathology (e.g., with an antibody panel of antibodies to detect SjD specific markers, such as anti-SjD-A / Ro, anti-SjD-B / La, anti-rheumatoid factor, or anti-nuclear antibodies). Dry eye caused by SjD can be diagnosed using methods known in the art, including those disclosed herein (e.g., by a clinical measurement selected from the group consisting of a tear volume assessment, ocular surface staining (e.g., of the cornea and / or conjunctiva), a tear osmolarity test, a matrix metalloproteinase-9 (MMP-9) test, a lactoferrin test, or interferometry).

[0131] Other exemplary tests to diagnose or assess SjD disease severity include patient questionnaires, such as the American College of Rheumatology (ACR) / European League Against Rheumatism (EULAR) classification criteria ACR / EULAR diagnostic criteria. A salivary gland biopsy may also be utilized to diagnose oral and ocular signs involvement in patients with SjD.

[0132] In some embodiments, CLU may be used to treat SjD-associated keratoconjunctivitis in a subject in need. In some embodiments, CLU may be administered topically (e.g., by eye drop or ointment) to the ocular surface.

[0133] Blepharitis

[0134] Blepharitis, an inflammatory condition of the eyelid margin, is a common cause of ocular discomfort and irritation across all age and ethnic groups. While generally not sight-threatening, it can lead to permanent alterations in the eyelid margin or vision loss from superficial keratopathy, corneal neovascularization, and ulceration.

[0135] Blepharitis can be divided into anterior and posterior according to anatomic location, although there is considerable overlap and both are often present. Anterior blepharitis affects the eyelid skin, base of the eyelashes, and the eyelash follicles and includes the traditional classifications of staphylococcal and seborrheic blepharitis. Posterior blepharitis affects the meibomian glands and gland orifices and has a range of potential etiologies, the primary cause being meibomian gland dysfunction (MGD).

[0136] Currently, long-term management of symptoms may include daily eyelid cleansing routines and the use of therapeutic agents that reduce infection and inflammation, however these provide only shortterm symptomatic relief.

[0137] In some embodiments, CLU may be used to treat blepharitis in a subject in need. In some embodiments, CLU may be administered topically (e.g., by eye drop or ointment) to the ocular surface, eyelid skin, or base of the eyelids.

[0138] Conjunctivitis

[0139] Inflammation of the conjunctiva is known as conjunctivitis and is characterized by dilation of the conjunctival vessels, resulting in hyperemia and edema of the conjunctiva, typically with associated discharge. The prevalence of conjunctivitis varies according to the underlying cause, which may be influenced by the patient’s age, as well as the season of the year. In some instances, conjunctivitis can result from an allergic ophthalmological reaction. Treatment of conjunctivitis generally depends on it’s PATENT

[0140] Attorney Docket Number: 51907-002W02 etiology. For bacterial conjunctivitis, an antibacterial or antibiotic topical treatment may be used. For certain types of viral conjunctivitis (e.g., adenoviral conjunctivitis) or allergic conjunctivitis, treatment is limited to the use of artificial tears and / or anti-inflammatory medications (e.g., topical antihistamines) to limit the symptoms.

[0141] Infectious conjunctivitis can be caused by numerous bacteria, including but not limited to Acute and subacute bacterial strains such as, Neisseria gonorrhoeae, Neisseria meningitides, Streptococcus pneumoniae, and Haemophilus influenzae, chronic bacterial strains such as, Staphylococcus aureus, and Moraxella lacunata, and less common bacterial causes such as, Streptococcus, Moraxella catarrhalis, Corynebacterium diphtheriae, Mycobacterium tuberculosis, Rickettsia and Chlamydia trachomatis. Infectious conjunctivitis can also be caused by fungus, parasites, and a range of viral infections. Viral conjunctivitis can be categorized into acute viral follicular conjunctivitis, chronic viral follicular conjunctivitis, viral blepharoconjunctivitis.

[0142] In some embodiments, CLU may be used to treat conjunctivitis. In some embodiments, CLU may be administered topically (e.g., by eye drop or ointment) to the ocular surface.

[0143] Limbal stem cell deficiency

[0144] The corneal epithelium is a stratified squamous epithelium from which superficial terminal cells are naturally shed. Limbal stem cell deficiency (LSCD) is characterized by a loss or deficiency of the stem cells in the limbus that are vital for the re-population of the corneal epithelium and to the barrier function of the limbus. When these stem cells are lost, the corneal epithelium is unable to repair and renew itself. This results in epithelial breakdown and persistent epithelial defects, corneal conjunctivalization and neovascularization, corneal scarring, and chronic inflammation. All of these contribute to loss of corneal clarity, potential vision loss, chronic pain, photophobia, and failure of keratoplasty if done before addressing this.

[0145] Eye pain and blurry vision are common complaints in this disease as the epithelial surface breaks down. Eye irritation, contact lens intolerance, and blurred or decreased vision were the most common symptoms in one study. Management is typically symptom driven at the early stages of the disease. When limbal stem cell injury is transient, sometimes termed limbal stem cell disease or limbal stem cell distress, conservative medical measures as above may be sufficient. However, total LSCD must be surgically managed.

[0146] The etiology of LSCD can be genetic, acquired, or idiopathic. Genetic LSCD has been associated with numerous mutations, such as PAX6 gene mutations, which are also implicated in aniridia and Peter’s Anomaly. Other genetic disorders that have been reported with LSCD include ectrodactyly-ectodermal- dysplasia-clefting syndrome, keratitis-ichthyosis-deafness (KID) Syndrome, xeroderma pigmentosum, dominantly inherited keratitis, turner syndrome, and dyskeratosis congenita. Acquired LSCD can be due to infections, trauma, tumors, or an inflammatory response to another disease / disorder. Exemplary diseases and disorders known to trigger acquired LSCD include Steven-Johnsons Syndrome, ocular cicatricial pemphigoid, graft versus host disease, chronic ocular allergy (e.g., vernal keratoconjunctivitis), neuronal neurotrophic keratopathy, ischemic neurotrophic keratopathy, and bullous keratopathy. Any corneal infection, such as herpes keratitis and trachoma, can predispose an individual to acquired LSCD. Acquired causes of LSCD also include chemical or thermal burns and prior ocular surgeries or PATENT

[0147] Attorney Docket Number: 51907-002W02 cryotherapies performed around the limbus. Radiation and chemotherapy are other potential causes, and systemic as well as topical chemotherapeutic medications may cause acquired LSCD. LSCD has also been seen with benzalkonium chloride toxicity with glaucoma medications. Inappropriate contact lens use with consequent hypoxia and ocular irritation with the destruction of the limbus may also contribute to both focal and total acquired LSCD. Ocular surface tumors are a known cause of LSCD. Pterygium may also cause a focal acquired absence of limbal stem cells.

[0148] LSCD progresses through several stages, characterized by increasing severity of symptoms and ocular surface changes, ultimately leading to significant vision impairment if untreated. Patients with early-stage LSCD may experience mild symptoms, such as blurry vision, foreign body sensation, and photophobia. At the early stages of LSCD the ocular surface may appear relatively stable, but there may be subtle signs of epithelial irregularity. Clinical examination of patients with early-stage LSCD may exhibit dull corneal reflexes and slight conjunctivalization of the cornea. Patients may present with nonspecific ocular signs and symptoms that may be mistaken for other ocular surface disorders.

[0149] Subjects with intermediate-stage LSCD may experience more pronounced symptoms including increased eye pain, tearing, persistent blurry or obscured vision. Patient may have an increasing intolerance to contact lens use. Intermediate-stage LSCD patients may exhibit “whirled” epithelium during slit-lamp examination. They may also exhibit loss of limbal palisades of Vogt, and fluorescein staining may show a stippled pattern due to epithelial defects.

[0150] Subjects with advanced-stage LSCD may experience chronic pain, significant vision loss, and severe photophobia. The quality of life can be greatly affected due to persistent discomfort and visual impairment. Advanced LSCD typically requires surgical interventions to restore the ocular surface and / or improve vision.

[0151] In some embodiments, CLU may be used to treat LSCD. In some embodiments, CLU may be administered topically (e.g., by eye drop or ointment) to the ocular surface.

[0152] Surgical complications

[0153] Surgical complications for cataract surgery or refractive eye surgery can range from immediate to delayed complications following the procedure but can include ocular neuropathy. Refractive eye surgery is a set of surgical procedures designed to correct vision problems like myopia, hyperopia, and / or astigmatism. Exemplary refractive eye surgery includes surgical remodeling of the cornea (keratomileusis), lens implantation or lens replacement. The most common refractive eye surgery techniques use excimer lasers to reshape the curvature of the cornea.

[0154] The current goal of cataract surgery is to remove the cataract and replace it with an intraocular lens, which is typically placed in the capsular bag of the posterior chamber.

[0155] In some embodiments, CLU may be used to treat ocular neuropathy that result due to cataract surgery. In some embodiments, CLU may be administered topically (e.g., by eye drop or ointment) to the ocular surface. In some embodiments, CLU may be administered by injection (e.g., intravitreal injection).

[0156] Neurotrophic keratopathy (NK)

[0157] Neurotrophic keratopathy (NK) is a corneal degenerative disease characterized by a reduction or absence of corneal sensitivity. In NK, corneal innervation by the trigeminal nerve is impaired. Partial or PATENT

[0158] Attorney Docket Number: 51907-002W02 complete loss of corneal sensation may result in epithelial keratopathy, epithelial defect, stromal ulceration, and eventually corneal perforation. Most common ocular conditions associated with NK are herpetic keratitis (zoster and simplex), topical anesthetic abuse, chemical and thermal burns, contact lens abuse, topical drug toxicity, irradiation to eye or adnexa, and corneal surgery. Histological alterations may be seen in corneas of subjects with NK, including thinning / disruption of the epithelial layer, cytoplasmic swelling of epithelial cells, loss of microvilli, disorganization of Bowman’s membrane, stromal melting / scarring, and corneal neovascularization. The conjunctiva is also involved with a reduction in goblet cell density and in cell-surface microplicae.

[0159] Every ocular or systemic condition altering corneal sensory innervation — which runs from the cornea itself to the pontine trigeminal nucleus — can result in NK. Exemplary ocular conditions associated with NK are herpetic keratitis (zoster and simplex), topical anesthetic abuse, chemical and thermal burns, contact lens abuse, topical drug toxicity, irradiation to eye or adnexa, and corneal surgery. Chronic use of topical medications containing benzalkonium chloride (BAK) may reduce corneal sensation via neuropathy and impair corneal epithelial healing.

[0160] With regards to corneal procedures, corneal refractive procedures, such as laser in situ keratomileusis (LASIK) and photorefractive keratectomy (PRK), have been linked to NK. The incidence of transient neuropathy, however, seems to be significantly higher in LASIK than in PRK. Corneal transplantation surgery, and specifically penetrating keratoplasty (PK) and deep anterior lamellar keratoplasty (DALK), can result in reduced central corneal sensitivity up to 12 months after surgical approach.

[0161] NK is usually graded in three different stages according to the “Mackie classification”. Therapy of NK depends on the disease stage. Stage I is characterized by epithelial irregularity most commonly in the form of punctate keratopathy without epithelial defect. For stage I, the therapeutic goal is to improve the quality and transparency of epithelium and to avoid epithelial breakdown. At this stage, frequent application of preservative-free artificial tear eye drops and lubricant ointments is suggested. Punctal occlusion may be beneficial at this stage. In cases of persistent keratopathy, autologous serum tears could be considered. Also, therapeutic soft contact lens application could improve the quality of vision in some cases.

[0162] Stage II is defined by recurrent or persistent epithelial defects (PED) without stromal involvement. The PED is usually oval in shape and its margins are characteristically smooth and rolled due to impaired epithelial healing. Descemet's folds and anterior chamber inflammation may be observed. For stage II, the aim of the therapy is to promote PED healing and prevent the development of a corneal ulcer. Treatment includes (i) the use of unpreserved artificial tears, lubricant ointments, (ii) therapeutic soft contact lenses or patching, (iii) topical autologous serum application, (iv) amniotic membrane grafting either in-office using a self-retaining cryopreserved amniotic membrane (PROKERA®, Bio-Tissue, Miami, FL) or in the operating room, (v) tarsorrhaphy or botulinum induced ptosis, and (vi) topical rhNGF treatment. Antibiotic eye drops can be prescribed to prevent bacterial infections. Topical corticosteroids can be administered to control inflammation cautiously, as they could induce stromal melting.

[0163] Stage III is characterized by stromal involvement leading to corneal ulcer, melting, and perforation. For Stage III, the therapy focuses on ulcer healing and prevention of corneal perforation. In addition to the therapy suggested for stages I and II, N-acetylcysteine, oral tetracycline, and PATENT

[0164] Attorney Docket Number: 51907-002WG2 medroxyprogesterone can be prescribed in case of stromal melting. Vitamin C supplementation can be helpful to prevent collagen degradation.

[0165] Keratitis

[0166] Keratitis, also known as keratopathy, refers to inflammation of the cornea, the clear dome-shaped tissue on the front of the eye. It can be caused by infections (infectious keratitis) or non-infectious factors such as injuries or prolonged contact lens wear. Common symptoms include eye pain, redness, light sensitivity, and impaired vision. Treatment options vary depending on the cause and may include antibiotic eye drops for infections or anti-inflammatory medications for non-infectious cases.

[0167] Diabetic keratitis, also known as diabetic keratopathy, is an ocular complication of diabetic mellitus and is the most common clinical condition affecting the human cornea. Diabetic keratopathy exhibits several clinical manifestations, including persistent corneal epithelial erosion, superficial punctate keratopathy, delayed epithelial regeneration, and decreased corneal sensitivity that may lead to compromised visual acuity or permanent vision loss. Standard treatments for diabetic keratopathy include the use of topical lubricants, topical antibiotic ointments, patching, bandage soft contact lenses (BSCLs), tarsorrhaphy, and / or corneal transplants. In some embodiments, CLU may be used to treat diabetic keratopathy. In some embodiments, CLU may be administered topically (e.g., by eye drop or ointment) to the ocular surface. In some embodiments, CLU may be administered by injection (e.g., intravitreal injection).

[0168] Other exemplary metabolic causes of keratitis include lysosomal storage disorder diseases. Exemplary lysosomal storage disorder diseases known to cause keratitis include Mucopolysaccharidoses (MPS), such as Hunter's syndrome, Sanfilippo syndrome, Morquio syndrome, Maroteaux-Lamy syndrome. Other exemplary storage disorder diseases known to cause keratitis include lipidoses, such as generalized gangliosidosis (GM) 1 , GM 2 gangliosidosis (Tay-Sachs), GM 2 gangliosidosis (Sandhoff), GM2 activator deficiency, Fabry Disease, Multiple sulfatase deficiency, Farber lipogranulomatosis, Gaucher disease, and Niemann-Pick A & B, and mucolipidoses, such as sialidosis I, pseudo-Hurler, ML II, and ML IV. Other exemplary storage disorder diseases known to cause keratitis include galactosialidosis and cystinosis.

[0169] Metabolic syndromes associated with lipid metabolism can cause keratitis. Exemplary disorders of lipid & lipid metabolism known to cause keratitis include hyperlipoproteinemias such as hyperchylomicronemia, hyperbetalipoproteinemia, familial dysbetalipoproteinemia, hyperprebetalipoproteinemia, hyperprebetalipoproteinemia and hyperchylomicronemia. Other exemplary disorders of lipid & lipid metabolism known to cause keratitis include Schnyder corneal dystrophy and Hypolipoproteinemias such as, Lecithin-cholesterol acyltransferase (LCAT) deficiency, Tangier disease (familial HDL deficiency), Fish eye disease, Familial hypobetalipoproteinemia, and Bassen-Bronzweig. disease.

[0170] Metabolic syndromes associated with amino acid, nucleic acid, and / or protein metabolism can cause keratitis. Exemplary disorders known to cause keratitis, include tyrosinemia, alkaptonuria, amyloidosis (e.g., primary cornea amyloidosis, systemic primary amyloidosis, localized secondary corneal amyloidosis, secondary systemic amyloidosis with corneal manifestations), gout, and porphyria (e.g., porphyria cutanea tarda, congenital erythropoietic porphyria. PATENT

[0171] Attorney Docket Number: 51907-002W02

[0172] Herpetic keratitis can be unilateral or, more rarely, bilateral. The latter is more common in patients with atopy, due to proposed immune dysregulation that increases susceptibility to viral infections. The incidence of bilateral involvement ranges from 1 .3% to 12%. HSV can affect all layers of the cornea, and may be accompanied by a blepharoconjunctivitis, which may result in lesions of the eyelids and a follicular conjunctivitis. Characteristically, HSV epithelial keratitis presents with classic dendritic lesions with terminal bulbs. Recurrent activations within the sensory ganglion can result in cornea scarring, necrosis, and decreased corneal sensation (neurotrophic cornea), all of which can be vision threatening.

[0173] Other types of infectious keratitis include, but are not limited to, Pythium keratitis, Acanthamoeba keratitis, and bacterial keratitis. Bacterial keratitis can be caused, for example, by Staphylococcus, Streptococcus, Pseudomonas, Neisseria, Corynebacterium, Shigella, and Listeria species.

[0174] Interstitial keratitis is any non-ulcerating inflammation of the corneal stroma without the involvement of either the epithelium or endothelium. Practically, however, the term refers to a common endpoint for several diseases which primarily manifest as inflammation and vascularization of the corneal stroma with minimal loss of tissue. Diseases that also involve other layers of the cornea secondarily are also included if the stroma is the predominant layer affected. The underlying causes of interstitial keratitis can broadly be either infectious or immune-mediated. The stroma constitutes the greatest thickness of all the layers of the cornea and is made up of specially arranged collagen fibrils that allow clear transmission of light. The inflammation and blood vessel invasion characteristic of interstitial keratitis can result in scarring of this layer resulting in decreased vision.

[0175] The primary goals in the treatment of interstitial keratitis are to control local inflammation to prevent pain and visually significant scarring and to identify an underlying cause to reduce systemic sequelae of the disease.

[0176] The methods disclosed herein may be used to treat keratitis, including but not limited to herpes simplex keratitis, interstitial keratitis and keratitis due to contact lens use. In some embodiments, CLU may be administered topically (e.g., by eye drop or ointment) to the ocular surface.

[0177] Mustard Gas Keratopathy

[0178] Mustard gas keratopathy, also known as mustard gas keratitis, refers to an ocular surface disease that occurs following exposure to mustard gas. Mustard gas is a potent blistering and alkylating agent. The mustard gas agent has two exemplary chemical forms: the sulfur analog (sulfur mustard or 2,2-dichlorodiethyl sulfide) typically applied as a chemical weapon, and the nitrogen analogs.

[0179] Mustard gas is highly lipophilic and quickly penetrates the epithelial lining of tissues. Damage to the cell membrane, DNA cross-linking, the release of inflammatory cytokines, and oxidative stress result in various acute and chronic ocular, cutaneous and respiratory injuries. The cornea is 10-fold more susceptible to mustard gas-related injury than the skin and the lungs due to its exposure to the environment and high turnover of epithelial cells. The dosage and duration of exposure, in addition to factors related to the host, may determine the severity of the damage. There are a wide range of ocular surface complications resulting in vision loss, broadly referred to as mustard gas keratopathy.

[0180] Most victims exposed to mustard gas will exhibit signs or symptoms of mustard gas keratopathy. Generally, there is a latent period (e.g., approximately 1-6 hours) before developing ocular symptoms PATENT

[0181] Attorney Docket Number: 51907-002W02 and signs following mustard gas exposure. The duration and level of exposure to mustard gas are determining factors of severity in the acute stage.

[0182] The primary lesions, also known as the acute phase of mustard gas keratopathy, are subdivided into three groups based on the severity: mild, moderate, and severe. Exemplary signs and symptoms of mild acute disease are eyelid swelling, eye redness, and ocular discomfort, which typically begin to result 4-24 hours following exposure. Exemplary signs and symptoms of moderate acute disease are photophobia, transient loss of vision, chemosis, blepharitis, pain, blepharospasm, edematous cornea and corneal bullae formation. Exemplary signs and symptoms of severe acute disease are uveitis, iris vasodilation, miosis, conjunctival and limbal necrosis, eyelid necrosis, adhesion between eyelids and globe, corneal ulceration, corneal neovascularization, cataract, and permanent blindness.

[0183] The chronic and delayed symptoms of mustard gas keratopathy are subdivided based on the severity: mild, moderate, and severe. Exemplary signs of chronic mild disease are meibomian gland dysfunction, blepharitis, shortened tear meniscus, telangiectatic conjunctival vessels, comma-shaped vascular tortuosity in the palpebral fissure area, subconjunctival fibrosis, hemorrhage and scarring, and punctate epithelial erosions. Exemplary symptoms of chronic mild disease are photophobia, foreign body sensation, dry eye, lacrimation, and mild eye redness. Exemplary signs of chronic moderate disease, include all the signs of chronic mild disease, and include corneal irregular astigmatism, mild to moderate limbal ischemia, corneal irregularity, peripheral corneal thinning, lipoid and amyloid deposits in the corneal periphery, neovascularization of peripheral cornea, peripheral stromal scars and corneal opacity, peripheral intra-stromal hemorrhage, and decreased corneal sensation. Exemplary symptoms of chronic moderate disease, include all the symptoms of chronic mild disease, and include decreased visual acuity, remarkable redness, itching, and pain. Exemplary signs of chronic severe disease, include all the signs of chronic mild to moderate disease, and include severe limbal ischemia and limbal stem cell deficiency, corneal thinning and opacity, central and peripheral corneal vascularization and conjunctivalization, central and peripheral stromal band keratopathy and scars, central and peripheral intra-stromal hemorrhage, descemetocele, corneal ulcer, and corneal melting / perforation. Exemplary symptoms of severe disease, include all the symptoms of chronic mild to moderate disease, and include severe photophobia, severe vision loss, and severe pain. One common exemplary feature of mustard gas keratopathy is limbal stem cell deficiency (LSCD). Mustard gas keratopathy may also cause dry eye disease.

[0184] The methods disclosed herein may be used to treat mustard gas keratopathy. In some embodiments, CLU may be administered topically (e.g., by eye drop or ointment) to the ocular surface.

[0185] Corneal ectasia

[0186] Corneal ectasia is a group of conditions that cause your cornea to thin and bulge outward. Ectasia can happen naturally or as a result of certain surgeries, such as refractive eye surgery. Corneal ectasia results in progressive loss of vision. Current treatment options involve use of glasses and contacts improve vision, and surgery to slow or stop the progressive vision loss. The methods disclosed herein may be used to treat corneal ectasia. In some embodiments, CLU may be administered topically (e.g., by eye drop or ointment) to the ocular surface. PATENT

[0187] Attorney Docket Number: 51907-002WG2

[0188] Retinopathy

[0189] The methods described herein may be used for treating retinopathies, such as retinopathies that cause retinal neuropathy. In some embodiments, the methods described herein may be used to treat inherited retinopathy, iatrogenic retinopathy, traumatic retinopathy, and toxic retinopathy in a subject in need thereof. The methods described herein may be used to treat ocular complications of diabetes mellitus, such as diabetic retinopathy and / or diabetic macular edema. In some embodiments, the methods described herein may be used to treat retinal detachment.

[0190] Retinopathy, also known as retinal disease, encompasses any disease or condition affecting the retina (e.g., the structure or function of the retina). Retinal diseases may be caused by numerous etiologies, including but not limited to, genetic causes, iatrogenic causes, trauma, or environmental toxicity. For example, toxic retinal diseases may be caused by exposure to pesticides (e.g, exposure to one or more organophosphate, pyrethroid, benzoylurea, neonicotinoid, organotin, organosulfur, benzimidazoles compounds), natural products, drugs (e.g., retroviral drugs, fibroblast growth factor receptor inhibitors, or chemotherapeutic agents, such as cisplatin). Exemplary retinal diseases include diabetic retinopathy, diabetic macular edema, retinal detachment, retinal perforation, retinal vein occlusion (e.g., central retinal vein occlusion and / or branch retinal vein occlusion), infectious retinitis, central serous retinopathy, birdshot chorioretinopathy, and other inflammatory diseases. Retinal diseases also encompass diseases that affect your macula, such as macular pucker (i.e., epiretinal membrane), macular hole, age-related macular degeneration (e.g., wet or dry age-related macular degeneration), macular edema.

[0191] Exemplary inherited retinal disease include, but are not limited to retinitis pigmentosa, Usher syndrome, Stargardt disease, and retinoschisis (e.g., X-linked retinoschisis), Best disease, PRPH2- associated pattern dystrophy, Sorsby fundus dystrophy, autosomal dominant drusen (EFEMP1 ), cone and cone-rod dystrophies (e.g., due to disease-causing mutations in GUCA1 A, PRPH2, ABCA4, KCNV2 and RPGR), enhanced S-Cone syndrome (NR2E3), Bietti crystalline corneoretinal dystrophy (CYP4V2), Leber congenital amaurosis / early-onset severe retinal dystrophy (e.g., due to disease-causing mutations in GUCY2D, CEP290, CRB1 , RDH12, RPE65, TULP1 , AIPL1 and NMNAT1 ), achromatopsia (e.g., due to disease-causing mutations in CNGA3, CNGB3, PDE6C, PDE6H, GNAT2, ATF6), Bornholm disease, oligocone trichromacy, and blue-cone monochromatism.

[0192] In some embodiments, CLU may be used to treat retinal diseases, such as the retinal diseases described herein. In some embodiments, CLU may be administered by intraocular (e.g., intravitreal or subretinal injection) or periocular injection.

[0193] Retinal detachment

[0194] Retinal detachment occurs when subretinal fluid accumulates between the neurosensory retina and the retinal pigment epithelium, resulting in disruption of photoreceptor function and consequential vision loss. The retina is composed of multiple neuronal layers responsible for phototransduction. The outermost layer, the retinal pigment epithelium (RPE) layer, is essential for maintaining photoreceptor metabolism, adhesion, and fluid transport. Retinal detachment occurs when subretinal fluid accumulates between the neurosensory retina and the RPE layer, thus overwhelming these adhesive mechanisms and PATENT

[0195] Attorney Docket Number: 51907-002W02 disrupting the retinal interface. Oxygen and nutrient diffusion are then impaired, resulting in rapid photoreceptor degeneration.

[0196] Exemplary types of retinal detachment include rhegmatogenous retinal detachment, traction retinal detachment, and exudative (serous) retinal detachment combined detachment. Once a retinal detachment has been identified, one must determine what type of detachment is present. The goal of treatment for retinal detachment is anatomic reattachment of the neurosensory retina and restoration of visual function. Management depends on the type and extent of detachment, macular involvement, lens status, and surgeon experience. Surgical management is indicated for rhegmatogenous and tractional detachments.

[0197] In some embodiments, CLU may be used to treat retinal diseases, such as retinal detachment. In some embodiments, CLU may be administered by intraocular (e.g., intravitreal or subretinal injection) or periocular injection.

[0198] Diabetic retinopathy

[0199] Diabetic retinopathy (DR) and diabetic macular edema represent microvascular end-organ damage due to diabetes. Diabetic retinopathy progresses through four stages: mild nonproliferative DR, Moderate nonproliferative DR, Severe nonproliferative DR, and proliferative DR. As the disease progresses, diabetic macular edema may also become apparent. Mild nonproliferative DR is the earliest stage, characterized by the presence of microaneurysms, which are small bulges in the retinal blood vessels. Typically, there are no noticeable symptoms at this stage, and vision may remain unaffected.

[0200] Moderate nonproliferative DR is characterized by the blockage of blood vessels, leading to reduced blood flow to the retina, which may cause changes in the retina's structure. Patients with moderate nonproliferative DR are still often asymptomatic, but some patients may begin to experience slight vision changes.

[0201] Severe nonproliferative DR is characterized by numerous blocked blood vessels, depriving areas of the retina of necessary blood supply. The retina sends signals to the body to grow new blood vessels in response to this deprivation. Vision may start to deteriorate, and patients may experience more noticeable changes in their sight.

[0202] Proliferative DR is an advanced stage involving the growth of new, abnormal blood vessels in the retina and along the vitreous gel inside the eye, which are fragile and prone to leaking. Patients may experience significant vision problems, including blurriness, reduced field of vision, and even blindness if left untreated.

[0203] Among patients aged 25-74, diabetic retinopathy and diabetic macular edema are leading causes of vision loss worldwide. By 2030, an estimated 191 million people globally will have diabetic retinopathy, and approximately 56.3 million will have vision-threatening diabetic retinopathy. The Wisconsin Epidemiologic Study of Diabetic Retinopathy (WESDR) cohort showed that after 20 years of diabetes mellitus, 99% of patients with type 1 and 60% of patients with type 2 show some degree of retinopathy. There are several other key risk factors for the development of diabetic retinopathy beyond years since diagnosis and type of diabetes. Additionally, elevated hemoglobin A1 c (HbA1 c) levels and blood pressure are associated with increased risk of diabetic retinopathy. PATENT

[0204] Attorney Docket Number: 51907-002W02

[0205] In some embodiments, CLU may be used to treat diabetic retinopathy and diabetic macular edema. In some embodiments, CLU may be administered by injection (e.g., intravitreal injection).

[0206] Ocular Neuropathic Pain

[0207] Ocular neuropathic pain, also referred to as corneal neuropathic pain, is a condition where corneal pain is seen in response to normally non-painful stimuli. This results from repeated direct damage to corneal nerves. Aberrant regeneration with upregulation of nociceptors responsible for processing of painful stimuli leads to hyper-responsivity and increased perception of pain in response to even normally unpainful stimuli. The distorted neuronal excitability which persists even after the tissue has healed is the basis of symptoms of self-sustained chronic corneal pain persisting even in the absence of stimuli and clinical signs. This condition is the ocular analogue of systemic neuropathic pain, complex regional pain syndrome or reflex sympathetic dystrophy (RSD). Ocular or corneal neuropathic pain, corneal neuropathy, corneal neuralgia, and corneal allodynia are all terms that are used to describe the same disease entity.

[0208] Ocular neuropathic pain may be triggered by numerous underlying causes e.g., by surgery (e.g., cataract surgery, corneal transplant surgy, refractive surgery), chronic ocular surface diseases (e.g., dry eye or ocular surface neoplasia), infections, trauma, exposure to radiation and / or UV light, and / or systemic neuropathies (e.g., diabetes, small fiber neuropathy, multiple sclerosis).

[0209] In some embodiments, CLU may be used to treat ocular neuropathic pain. In some embodiments, CLU may be administered by injection (e.g., intravitreal injection).

[0210] Examples

[0211] The following examples are put forth so as to provide those of ordinary skill in the art with a description of how the methods described herein may be used and evaluated and are intended to be purely exemplary of the invention and are not intended to limit the scope of what the inventors regard as their invention.

[0212] Example 1. Treatment of Ocular Surface Diseases with Clusterin (CLU)

[0213] Objective

[0214] This disclosure features clusterin (CLU) as a multifunctional biotherapeutic for a widespread range of ocular conditions, such as ocular surface diseases. An exemplary ocular surface disease is Sjogren's disease (SjD)-associated keratoconjunctivitis, which is chronic autoimmune disorder characterized by inflammatory destruction and functional loss of the lacrimal glands, leading to reduced tear production and subsequent dry eye disease. This tear deficiency results in ocular surface inflammation, including epithelial defects in both the cornea and conjunctiva, a condition clinically termed as keratoconjunctivitis sicca. Patients often experience burning, stinging and itching sensations in the eyes, along with blurred vision. These symptoms correlate with the altered corneal nerve morphology and reduced nerve density observed in SjD patients. Managing ocular symptoms in these patients remains a significant challenge. Currently approved treatments for dry eye include artificial tears, which provide only temporary symptomatic relief, and anti-inflammatory drugs, which often yield suboptimal results and are associated with significant limitations, including notable side effects. Importantly, no approved treatment PATENT

[0215] Attorney Docket Number: 51907-002W02 specifically addresses the neurosensory damage of the ocular surface that contributes to dry eye symptoms. Therefore, finding new, targeted and specific therapeutic alternatives to treat neurosensory damage in ocular surface diseases is of great clinical interest.

[0216] In this study, the therapeutic efficacy of topically applied CLU in thrombospondin 1 (Thbsl)- deficient mice was investigated for treatment of established ocular surface disease. Recombinant human CLU (rhCLU), produced in-house with recombinant DNA methodologies at two different doses, was compared to human plasma-derived CLU (pCLU) and to 0.1% dexamethasone, a standard of care.

[0217] Results

[0218] Topical application of clusterin (CLU) improves corneal epithelial barrier in a preclinical mouse model of Sjogren's disease

[0219] To assess the efficacy of CLU in repairing corneal epithelial damage associated with SjD, Thbsl- deficient mice were used, which are a well-established preclinical model of SjD-related ocular surface disease. In Thbs / -deficient mice, corneal epithelial damage becomes fully evident by 12 weeks of age. Therefore, the double-blinded preclinical study began when the Thbs / -deficient mice were 12-weeks-old. Different groups of male and female Thbs / -deficient mice were topically treated twice a day, five days per week (skipping weekends), for three weeks with either recombinant human CLU (rhCLU) (1 pg / ml or 50 pg / ml), pCLU (50 pg / ml), or vehicle control (PBS). These treatments were compared to treatment with dexamethasone 0.1%, the current standard of care, which served as a positive control due to its well- documented anti-inflammatory effects. Corneal epithelial damage was assessed before treatment initiation (baseline), once per week, and at the study endpoint using corneal fluorescein staining (CFS) (FIG. 1A). Normalized CFS scores were compared across different treatment groups (FIG. 1 B, 1C). In vehicle treated mice, CFS scores progressively increased each week, consistent with the age-related disease progression. Conversely, in mice treated with 1 pg / ml rhCLU, the weekly CFS scores remained largely stable, showing neither progression nor improvement (FIG. 1 B). However, at higher concentration of 50 pg / ml, both rhCLU and pCLU treatments led to a significant epithelial barrier improvement, as evidenced by a significant decrease in CFS. This improvement was already detectable after one week of treatment and continued to progress until the end of the study. A similar pattern of corneal epithelial improvement was observed in both male and female mice treated with 50 pg / ml of either rhCLU or pCLU (FIG. 1 B, 1C). When comparing overall treatment efficacy in repairing corneal epithelial damage, both rhCLU and pCLU demonstrated improvements comparable to those achieved with 0.1% dexamethasone, irrespective of sex (FIG. 1D, 1E). Finally, whether CLU treatment induced any toxic morphological changes or inflammatory cell infiltrations in the cornea and conjunctiva was investigated. Hematoxylin and Eosin (H&E) staining revealed no morphological alterations or inflammatory infiltrates in these tissues following CLU application (FIG. 7).

[0220] Taken together, these results demonstrate that the previously reported corneal epithelial sealing properties of CLU effectively improve corneal barrier integrity, even under chronic inflammatory conditions, in Thbs / -deficient mice. PATENT

[0221] Attorney Docket Number: 51907-002WQ2

[0222] Corneal nerve density is reduced in Thbsl -deficient mice, as seen in SjD patients

[0223] Significant reduction in corneal nerve density has been previously observed in SjD patients. A similar change was also reported in 12-week-old Thbs / -deficient mice. Here, we confirmed reduced corneal nerve density in 15-week-old Thbs / -deficient mice, which matched the age of the mice used at the endpoint of this study. Corneas from 15-week-old Thbs / -deficient and wild-type (WT) mice were harvested and immunostained for beta-3-tubulin, a component of the neuronal tubulin cytoskeleton, as well as for the neurotransmitter calcitonin gene-related peptide (CGRP). Central and peripheral corneas have different patterns of subbasal nerve organization. Therefore, to gain a deeper understanding of nerve morphology changes, we analyzed the central and peripheral corneas separately (FIG. 2A). As shown in FIG. 2B, the localization of subbasal nerves in the central cornea exhibited a typical densely- packed, vortex-like pattern. Quantitative assessment and comparison of beta-3-tubulin-stained nerve densities between WT and Thbs / -deficient mice revealed a significant reduction in nerve density in Thbs / -deficient mice (FIG. 2C). In contrast, in the periphery, corneal nerves predominantly ran parallel to each other (FIG. 2D), forming a complex, well-organized network extending to the limbal area. Both nerve density and organization were significantly disrupted in peripheral corneas of Thbs / -deficient mice, as evidenced by large nerve-free gaps in the whole mount images and the loss of fine nerve structures (FIG. 2D). Quantitative analysis of the nerve density, both in the central and peripheral regions, confirmed a significant reduction in Thbs / -deficient mice compared to WT (FIG. 2E). Interestingly, the decline in peripheral corneal nerve density in Thbs / -deficient mice was nearly five times greater than that observed in the central cornea. In addition, the relative level of immunoreactive CGRP was notably reduced in both the central and peripheral corneas of Thbs / -deficient mice compared to WT mice (FIG. 2F-2H). These results further confirm corneal nerve abnormalities in Thbs / -deficient mice, mirroring the findings in SjD patients and supporting the use of this mouse model to evaluate effects of topically delivered CLU on corneal nerve alterations and related biology.

[0224] Topically applied CLU improves corneal nerve density in mice with Sjogren's disease.

[0225] To evaluate whether CLU can also mediate corneal nerve regeneration, corneal nerve densities in Thbs / -deficient mice were analyzed following treatment. At the study endpoint, corneas from groups treated with vehicle, 1 pg / ml of rhCLU, 50 pg / ml of rhCLU, pCLU or 0.1% dexamethasone were harvested and immunostained for beta- 3-tubulin and CGRP. Quantitative analysis was then performed on beta-3-tubulin-stained corneal nerves, as well as the proportion of CGRP-positive nerves within the corneal nerve population. Representative immunofluorescence images of stained corneas indicated that, compared to vehicle-treated mice, those treated with rhCLU (50 pg / ml) or 0.1% dexamethasone displayed improvements in nerve densities in both the peripheral and central corneas (FIG. 3A, 3B). Quantitative analyses confirmed that nerve densities in the peripheral cornea of mice treated with 50 pg / ml of either rhCLU or pCLU - but not 1 pg / ml of rhCLU - were significantly improved compared to vehicle-treated mice (FIG. 3C). Treatment with 0.1% dexamethasone also significantly improved corneal nerve density. Treatment with either rhCLU or pCLU at 50 pg / ml resulted in a significantly greater improvement in peripheral corneal nerve density than dexamethasone (FIG. 3C). In contrast, in the central cornea, only pCLU (50 pg / ml) treatment led to a significant improvement in nerve density compared to either vehicle or dexamethasone treatment (FIG. 3D). Although treatment with 50 pg / ml of PATENT

[0226] Attorney Docket Number: 51907-002WG2 rhCLU showed a trend toward improved central corneal nerve density, quantitative analysis did not reveal a statistically significant change (FIG. 3D). Taken together, in comparison to the vehicle-treated group, nerve density improvements were notably more prominent in the peripheral cornea than in the central cornea, while the most improvement was achieved by CLU treatment.

[0227] Additionally, potential correlation between corneal epithelial integrity, as reflected by CFS scores, and corneal nerve density was assessed. As shown in FIG. 3E, a significant negative correlation was found between CFS scores and corneal nerve density in both the peripheral and central corneal regions. Specifically, lower CFS scores (indicating improved epithelial barrier) are associated with higher nerve density in mice with SjD-associated keratoconjunctivitis. This correlation is consistent with the known reciprocal trophism between the corneal epithelium and subbasal nerves in patients with SjD neurotrophic keratopathy.

[0228] Topical CLU increases the proportion of CGRP-positive corneal nerves

[0229] A reduced proportion of CGRP-positive corneal nerves in Thbs / -deficient mice is consistent with the reported wound-healing and anti-inflammatory properties of this neuropeptide. To determine if CLU- driven repair of corneal epithelial damage also helps restore CGRP-positive corneal nerves, further quantification of immunoreactive CGRP in beta-3-tubulin-stained corneal nerves was performed. This analysis revealed an increased proportion of beta-3-tubulin-stained nerves that were also stained positively for CGRP in mice treated with rhCLU (50 pg / ml) or 0.1% dexamethasone, as indicated by confocal imaging (FIG. 4A) and the corresponding quantitative data. Enhanced CGRP positivity was observed in both the peripheral and central regions of the cornea (FIG. 4B, 4C). In conclusion, our results demonstrate that, in addition to repairing corneal epithelial damage and improving corneal nerve density, CLU also enhances the proportion of neuropeptide CGRP-positive corneal nerves.

[0230] CLU treatment helps counter SjD-associated conjunctival inflammation

[0231] As reported in humans, chronic dry eye associated with SjD in Thbs / -deficient mice is characterized by a substantial loss of conjunctival goblet cells, driven by local inflammation. To determine whether topically applied CLU also helps mitigate conjunctival inflammation and aids in the recovery of goblet cell density, mucin-filled goblet cells stained with Alcian Blue in histological sections of the conjunctiva were enumerated. Tissues harvested at the study endpoint from the various treatment groups were examined. Overall, increased numbers of goblet cells were detectable in mice treated with rhCLU, pCLU or dexamethasone, as compared to vehicle-treated control mice (FIG. 5A). Quantitative analysis of goblet cells confirmed significantly increased numbers in mice treated with 0.1% dexamethasone (FIG. 5B), as reported previously. In addition, treatment with rhCLU reached statistical significance (FIG. 5B).

[0232] We have previously reported that inflammatory cytokines induce conjunctival goblet cell apoptosis and contribute to their loss in Thbs / -deficient mice. To determine if increased goblet cell numbers correlate with reduced inflammation, the expression of Tnfa as a marker of inflammation was evaluated by RT-PCR, using RNA isolated from conjunctival tissues harvested from different treatment groups at the study endpoint. Consistent with its known anti-inflammatory effect, dexamethasone treatment resulted in significantly reduced expression of Tnfa as compared to vehicle treatment (FIG. 5C). While treatment with 1 pg / ml of rhCLU showed no significant difference, treatment with 50 pg / ml of PATENT

[0233] Attorney Docket Number: 51907-002W02 rhCLU resulted in significantly reduced Tnfa expression levels as compared to the vehicle control (FIG. 5C). Together, these results highlight the potential of CLU to mitigate conjunctival inflammation.

[0234] CLU treatment reduces corneal inflammation associated with Sjogren's disease

[0235] Since ocular surface disease-related inflammation affects both cornea and conjunctiva, the effect of CLU on corneal inflammation was investigated to gain a deeper understanding of its anti-inflammatory potential. Corneal inflammation in Thbs / -deficient mice is associated with an increased expression of Tnfa, and release of this cytokine from the surface of the cells as a mature protein is mediated by the action of the metalloproteinase ADAM17 (also known as TN Fa-converting enzyme or TACE). Recently, increased expression of ADAM17 was reported in corneal epithelium of Thbs / -deficient mice. To evaluate the effect of topically administered CLU on corneal inflammation ADAM 17 expression was examined in the corneal epithelium of Thbs / -deficient mice from each treatment group. As shown in FIG. 6A, ADAM17 immunostaining, indicated by the intense red fluorescence, was detected in the corneal epithelium of vehicle-treated mice, while the intensity of this staining was reduced in mice treated with CLU (50 pg / ml pCLU or rhCLU) or 0.1% dexamethasone (positive control).

[0236] Quantitative analysis (FIG. 6B) confirmed a significant reduction in the amount of immunoreactive ADAM17 in the corneal epithelium of CLU (66% and 60% decline in pCLU and rhCLU-treated mice, respectively) and dexamethasone-treated mice (57%), as compared to vehicle-treated mice.

[0237] In summary, our results demonstrate that CLU (pCLU and rhCLU) effectively reduces ADAM17 immunoreactivity in the corneal epithelium and reduces Tnfa expression in the conjunctiva, supporting its anti-inflammatory effect in the treatment of chronic ocular surface disease.

[0238] Materials and Methods

[0239] Animals

[0240] Twelve-week-old, homozygous thrombospondin-1 (Thbsl) deficient mice (B6.129S2- Thbs1tm1i^n / J, cat. # 6141) and the corresponding wild-type (WT) mice (C57BL / 6J, cat. # 664) were obtained from Jackson Laboratories (Bar Harbor, Maine, USA). All experimental procedures were conducted in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and were approved by the Institutional Animal Care and Use Committee (protocol TR201900023) at Boston University School of Medicine (Boston, MA, USA). Throughout the study, animals were housed in a pathogen-free environment with veterinary care provided by the animal facility at Boston University School of Medicine. After reaching the experimental endpoint, mice were euthanized with CO2 gas according to the American Veterinary Medical Association Guidelines for the Euthanasia of Animals.

[0241] Preclinical study design

[0242] The therapeutic efficacy of CLU in resolving chronic ocular surface inflammation was evaluated in a double-blinded study using male and female Thbs / -deficient mice. These mice spontaneously develop chronic ocular surface inflammation resembling SjD, which affects the lacrimal gland, cornea, and conjunctiva, with symptoms fully established by 12 weeks of age. Consequently, 12-week-old Thbsl- deficient mice were selected for this preclinical study. A total of seven groups of 12-week-old Thbsl- PATENT

[0243] Attorney Docket Number: 51907-002W02 deficient mice (n = 5 per group) were included. Five groups of male mice received topical treatments with 5 pl of the respective therapeutic eyedrop per eye, administered twice daily (b.i.d.) for five days per week over a three-week period. The treatment groups included: (1) vehicle (PBS), (2) rhCLU, 1 pg / ml, (3) pCLU, 50 pg / ml, (4) rhCLU, 50 pg / ml, and (5) 0.1% dexamethasone (Sigma-Aldrich, St. Louis, MO, USA), as a positive control. pCLU was purified from human plasma using immunoaffinity chromatography, as described by, while rhCLU was generated in our laboratory according to methods known in the art, e.g., e.g., in Dabbs, R. A. & Wilson, M. R. Expression and purification of chaperone-active recombinant clusterin. PLoS One 9, e86989. https: / / doi.org / 10.1371 / journal.pone.0086989 (2014). and Satapathy, S., Dabbs, R. A. & Wilson, M. R. Rapid high-yield expression and purification of fully post-translationally modified recombinant clusterin and mutants. Sci Rep 10, 14243., the disclosure of which is incorporated herein by reference. Corneal epithelial barrier integrity was assessed prior to initiating treatment (baseline) and monitored weekly throughout the study. At the study endpoint, mice were euthanized, and various tissues, including whole eyes with eyelids, corneas and conjunctiva, were harvested for further analyses.

[0244] Corneal fluorescein staining

[0245] Corneal epithelial barrier integrity was performed using corneal fluorescein staining (CFS). Briefly, 3 pl of 1% sodium fluorescein (Sigma-Aldrich, St. Louis, MO, USA) was applied to both eyes of isoflurane-anesthetized mice for 1 minute, followed by a brief wash to remove excess dye. The staining of the corneal epithelium was then evaluated using a slit-lamp microscope (Haag-Streit, Koniz, Switzerland) equipped with a cobalt blue light. Punctate fluorescein staining was graded weekly for both eyes of each mouse using the standardized National Eye Institute (NEI) grading system of 0-3 for each of the five areas of the cornea. Finally, CFS scores were normalized to the baseline mean of each treatment group for comparison.

[0246] Immunohistochemistry

[0247] At the study endpoint, one eyeball from each mouse was enucleated and corneas were dissected and immediately fixed in 2% paraformaldehyde for 1 hour on ice. Radial incisions were made in fixed corneas to allow creating flat mounts. Fixation was followed by washes in phosphate buffered saline (PBS) and permeabilization in PBS / 1 % Triton ™ X-100 for 30 minutes and blocking with a solution of 1 % bovine serum albumin (BSA), 0.3% Triton ™ X-100 and 10% donkey serum for 1 hour. Corneas were incubated with primary antibodies for beta-3-tubulin for 24 hours at 4°C or CGRP for 72 hours at 4°C prior to staining with anti-beta-3-tubulin antibody. Following washes with wash buffer corneas were incubated with fluorescence-conjugated secondary antibodies for 1 hour at room temperature (RT) each. After final washes, corneas were mounted in polyvinyl alcohol mounting medium (Sigma-Aldrich, St. Louis, MO, USA).

[0248] To visualize in situ ADAM17 immunoreactivity in the corneal epithelium, eyes harvested at the end of the study period were fixed in 4% paraformaldehyde before paraffin embedding. Sections 5 pm in thickness (n = 3 per group) were used for immunostaining. Sections were blocked at room temperature (RT) for 30 minutes with 10% goat serum in PBS, followed by 2% bovine serum albumin (BSA) (Sigma- Aldrich, St. Louis, MO, USA), 0.1% Triton ™ -X 100 in PBS for 1 hour (h), and incubated overnight at 4°C PATENT

[0249] Attorney Docket Number: 51907-002WG2 with primary anti-ADAM 17 antibody in PBS-BSA. Tissues were washed with PBS-0.05% Tween-20 ® and incubated for 1 hour at RT with fluorescence-conjugated secondary antibody. Tissues were further washed with PBS- 0.05% Tween-20 ® and counterstained with 4',6-diamidino-2-phenylindole (DAPI).

[0250] Primary antibodies were rabbit anti-TuJ1 (1 :500; Cat. # T2200, Sigma-Aldrich, St. Louis, MO, USA), mouse anti-CGRP (1 :200; Cat. # sc-57053, Santa Cruz Biotechnology, Dallas, USA) and rabbit anti-ADAM17 (1 :50; Cat. # sc-13973, Santa-Cruz Biotechnology, Dallas, USA). Secondary antibodies included Alexa Fluor-488-conjugated donkey anti-rabbit IgG (1 :1000; Cat.# A-21206, Invitrogen, Carlsbad, CA, USA), DyLight-650-conjugated donkey anti-mouse IgG (1 :200; Cat.# ab98769, Abeam, Cambridge, UK) and Alexa Fluor-568-conjugated goat anti-rabbit IgG (1 ;500, Cat.#A-11011 , Invitrogen, Carlsbad, CA, USA).

[0251] Microscopy and image analysis

[0252] To visualize immunostained corneal nerves, Z-stacks of images were acquired using a Zeiss laser scanning microscopy (LSM) 710-Live Duo Confocal microscope (Carl Zeiss, Oberkochen, Germany). Overview images of corneal nerve morphology in the peripheral cornea were acquired with a Zeiss Plan-Apochromat 10x / 0,45 M27 (0.6 zoom) objective (Carl Zeiss, Oberkochen, Germany) and corneal nerves in selected central and peripheral regions were imaged using a Plan-Apochromat 20x / 0,8 Ph2 M27 (Carl Zeiss, Oberkochen, Germany) objective. The same acquisition settings were consistently applied to all samples within the same biological replicate. Image processing, including brightness and contrast, were adjusted with Fiji Imaged. Corneal nerve density was quantified based on beta-3-tubulin immunostaining of wholemount corneas. For analysis, each cornea was divided into four central and four peripheral regions, as shown in FIG. 2A. The central region, characterized by a whorl-like nerve structure, was imaged using four adjacent Z-stack scans (425 pm x 425 pm each). Additionally, one Z-stack scan was randomly taken from each of the four peripheral regions. In total, 25 images from both the central and peripheral corneal regions were analyzed per treatment group. Maximum intensity projections of the subbasal corneal nerve zone were then used for nerve density measurements and nerves were segmented using the ‘Ridge detection’ plugin in Fiji Imaged. Consistent segmentation settings were applied across all treatment groups to ensure accurate comparisons. Finally, nerve density was calculated as the percentage of the area covered by nerves relative to the total image area. Quantification of CGRP-staining was performed in corneas immunostained for beta-3-tubulin and CGRP. First, the total length of beta-3-tubulin positive sub-basal corneal nerves was measured using the Imaged ’Ridge detection’ tool 90. Second, the length of CGRP-positive nerves was manually traced using the Fiji Imaged ’Neurond’ plugin 91 . The percentage of CGRP-positive nerve fibers was calculated relative to the total beta-3-tubulin-positive nerve length in each image.

[0253] Fluorescence staining of ADAM 17 in corneal epithelium was evaluated using a fluorescence microscope (Nikon Eclipse E800, Nikon, dapan) and images were captured with a MicroPublisher 6 camera. Images were further analyzed using Fiji Imaged software 89 to determine relative ADAM 17 staining (red) to nuclear DAPI (blue) staining. Fluorescence intensity values for both colors within the same region of interest (ROI) were used to calculate the ratio. PATENT

[0254] Attorney Docket Number: 51907-002WG2

[0255] Goblet cell density

[0256] At the end of the 3-week study period, one whole eye with lid was harvested from three mice per treatment group, fixed in 4% paraformaldehyde and embedded in paraffin. Sagittal sections (5 pm) from the center of the eye were cut, stained with alcian blue / PAS solution (Vector Laboratories, CA, USA) and mounted in Permount™ medium. Representative sections of the bulbar and palpebral conjunctiva, showing filled goblet cells stained blue, were captured using a Plan Fluor 10x / 0.30 DIC WD objective (Nikon, Tokyo, Japan). The mean goblet cell number in both bulbar and palpebral conjunctiva were determined for two sections per eyeball.

[0257] Real-time PCR

[0258] At the end of the study period (3 weeks), conjunctiva collected from mice in each treatment group was pooled and total RNA was isolated using TRIzol™ Reagent. RNA concentration and quality were assessed with a NanoDrop device. cDNA synthesis was performed using the Superscript™ VILO™ cDNA synthesis kit (Thermo Fisher Scientific, Waltham, USA) according to the manufacturer's instructions. 1 pg of RNA per group was subjected to reverse transcription under the following thermal conditions: 25°C for 10 minutes, 50°C for 10 minutes, and 85°C for 5 minutes. For amplification of Tnfa and Gapdh gene transcripts, a semi-quantitative PCR was conducted using diluted cDNA (1 :10), gene-specific primers (Tnfa F-5’-GGCCTCCCTCTCATCAGTTCTATG-3’, R-5’-GTTTGCTACGACGTGGGCTACA-3’; Gapdh: F- 5’-CGAGAATGGGAAGCTTGTCA-3’, R-5’-AGACACCAGTAGACTCCACGACAT-3’) and the SYBR™ Green PCR Master Mix (Thermo Fisher Scientific, Waltham, USA) with the following thermal profile: 3 minutes at 95°C, followed by 40 cycles of 20 seconds at 95°C, 30 seconds at 55°C, and 40 seconds at 72°C. Fluorescence signals were recorded during each cycle and analyzed using StepOne Software v2.3 (Thermo Fisher Scientific, Waltham, USA). Melting curve analysis was performed to confirm the specificity of each RT-PCR reaction. Threshold cycle values were used to evaluate gene expression levels relative to the reference gene, Gapdh.

[0259] Statistics

[0260] Statistical analyses were performed by using the GraphPad PRISM software (GraphPad Software, version 10, La Jolla, USA). Normal distribution of the data was assessed using D’Agostino and Pearson test. For comparisons between two groups with normally distributed data, an unpaired two-tailed Student's t-test was performed. When data did not follow Gaussian distribution, the Mann-Whitney test was applied. For comparisons between more than two groups, a one-way ANOVA test was applied. Pearson’s correlation coefficient was used to evaluate correlation between corneal fluorescein staining score and corneal nerve density. Error bars show ± SEM and differences were considered significant when p < 0.05 (*p < 0.05; **p <0.01 ; ***p < 0.001 ; ****p < 0.0001).

[0261] Other Embodiments

[0262] All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each independent publication or patent application was specifically and individually indicated to be incorporated by reference. PATENT

[0263] Attorney Docket Number: 51907-002W02

[0264] While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the invention that come within known or customary practice within the art to which the invention pertains and may be applied to the essential features hereinbefore set forth, and follows in the scope of the claims.

[0265] Other embodiments are within the claims.

Claims

PATENTAttorney Docket Number: 51907-002W02What is claimed is:CLAIMS1 . A method of treating corneal neuropathy in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an isolated clusterin (CLU) or a functional fragment thereof.

2. The method of claim 1 , wherein the subject suffers from neurogenic dry eye disease.

3. The method of claim 1 , wherein the subject suffers from moderate to severe dry eye disease.

4. The method of claim 1 , wherein the subject suffers from Sjogren's disease-associated keratoconjunctivitis.

5. The method of claim 1 , wherein the subject suffers from stage I neurotrophic keratopathy (NK), stage II NK, or stage III NK.

6. The method of claim 1 , wherein the subject suffers from genetic limbal stem cell deficiency, acquired limbal stem cell deficiency, or idiopathic limbal stem cell deficiency.

7. The method of claim 1 , wherein the subject suffers from keratitis, blepharitis, surgical injury, corneal ectasia, mustard gas keratopathy, or conjunctivitis.

8. The method of claim 7, wherein the subject suffers from keratitis selected from diabetic keratitis, metabolic keratitis, herpetic keratitis, bacterial keratitis, and interstitial keratitis.

9. The method of claim 7, wherein the subject suffers from surgical injury selected from refractive surgery, cataract surgery, or corneal transplantation surgery.

10. The method of claim 7, wherein the subject suffers from conjunctivitis selected from allergic conjunctivitis, irritant conjunctivitis, and infectious conjunctivitis.11 . The method of claim 10, wherein the subject suffers from infectious conjunctivitis selected from bacterial conjunctivitis, fungal conjunctivitis, viral conjunctivitis.

12. The method of claim 7, wherein the subject suffers from blepharitis selected from anterior blepharitis or posterior blepharitis.

13. The method of any one of claims 1 -12, wherein the subject is experiencing corneal neuropathic pain.PATENTAttorney Docket Number: 51907-002W0214. A method of treating neurogenic dry eye disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an isolated clusterin (CLU) or a functional fragment thereof.

15. A method of treating moderate to severe dry eye disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an isolated clusterin (CLU) or a functional fragment thereof.

16. The method of claim 15, wherein the dry eye disease is severe.

17. A method of treating Sjogren's disease-associated keratoconjunctivitis in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an isolated clusterin (CLU) or a functional fragment thereof.

18. A method of treating mustard gas keratopathy in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an isolated clusterin (CLU) or a functional fragment thereof.

19. A method of treating neurotrophic keratopathy (NK) in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an isolated clusterin (CLU) or a functional fragment thereof.

20. The method of claim 19, wherein the NK is selected from stage I NK, stage II NK, and stage III NK.21 . A method of treating limbal stem cell deficiency in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an isolated clusterin (CLU) or a functional fragment thereof.

22. The method of claim 20, wherein the limbal stem cell deficiency is selected from genetic limbal stem cell deficiency, acquired limbal stem cell deficiency, and idiopathic limbal stem cell deficiency.

23. A method of treating an ocular condition selected from inherited retinopathy, iatrogenic retinopathy, traumatic retinopathy, and toxic retinopathy in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an isolated clusterin (CLU) or a functional fragment thereof.

24. A method of treating retinal detachment in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an isolated clusterin (CLU) or a functional fragment thereof.PATENTAttorney Docket Number: 51907-002W0225. A method of treating diabetic retinopathy (DR) in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an isolated clusterin (CLU) or a functional fragment thereof.

26. A method of treating an ocular condition selected from keratitis, blepharitis, surgical injury, corneal ectasia, and conjunctivitis in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an isolated clusterin (CLU) or a functional fragment thereof.

27. The method of claim 26, wherein the subject suffers from keratitis selected from diabetic keratitis, metabolic keratitis, herpetic keratitis, bacterial keratitis, and interstitial keratitis.

28. The method of claim 26, wherein the subject suffers from surgical injury selected from refractive surgery, cataract surgery, or corneal transplantation surgery.

29. The method of claim 26, wherein the subject suffers from conjunctivitis selected from allergic conjunctivitis, irritant conjunctivitis, and infectious conjunctivitis.

30. The method of claim 26, wherein the infectious conjunctivitis comprises bacterial conjunctivitis, fungal conjunctivitis, and viral conjunctivitis.31 . The method of claim 26, wherein the subject suffers from blepharitis selected from anterior blepharitis or posterior blepharitis.

32. The method of any one of claims 1 -31 , wherein the treatment results in :(a) regeneration of corneal nerves; and / or(b) increased corneal nerve density.

33. A method of treating retinal neuropathy in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an isolated clusterin (CLU) or a functional fragment thereof.

34. The method of claim 33, wherein the subject suffers from retinopathy.

35. The method of claim 34, wherein the retinopathy is selected from inherited retinopathy, iatrogenic retinopathy, traumatic retinopathy, and toxic retinopathy.

36. The method of claim 34, wherein the subject suffers from retinopathy selected from nonproliferative diabetic retinopathy (DR), moderate nonproliferative DR, severe nonproliferative DR, or diabetic macular edema.PATENTAttorney Docket Number: 51907-002W0237. The method of claim 34, wherein the subject suffers from retinopathy selected from moderate to latestage age-related macular degeneration (AMD).

38. The method of claim 37, wherein the subject suffers from moderate to late-stage AMD selected from dry AMD or wet AMD.

39. The method of any one of claims 1 -38, wherein treatment results in:(a) regeneration of retinal nerves; and / or(b) increased retinal nerve density.

40. The method of any one of claims 1 -39, wherein treatment results in a reduction in retinal atrophy.41 . The method of claim 40, wherein the reduction in retinal atrophy is measured by optical coherence tomography (OCT).

42. The method of any one of claims 1 -41 , wherein the subject is a human patient.

43. The method of any one of claims 1 -42, wherein the subject is administered the isolated clusterin (CLU) topically by eyedrop.

44. The method of any one of claims 1 -42, wherein the subject is administered the isolated clusterin (CLU) by injection.

45. The method of claim 44, wherein the injection is an intraocular or a periocular injection.

46. The method of any one of claims 1 -45, wherein the subject exhibits improvement in visual impairment.

47. The method of claim 46, wherein the improvement in visual impairment comprises improvement in color vision, reading speed or dynamic visual acuity.

48. The method of claim 46, wherein the improvement in visual impairment is measured by the multiluminance mobility test.

49. The method of claim 46, wherein the improvement in visual impairment comprises improvement in visual acuity.

50. The method of claim 49, wherein the improvement in visual acuity is measured by a clinical evaluation scale selected from the group consisting of the Snellen eye test, the dynamic visual acuity test, and the pinhole visual acuity test.

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