In-vitro method of identifying biomarkers associated with ocular surface disease

Abstract

Disclosed is in-vitro method of identifying at least one biomarker associated with one or more ocular surface diseases, wherein at least one biomarker is expressed in tear fluid, cornea, corneal epithelium, conjunctival epithelium, or ocular surface tissues In-vitro method comprises processing ocular sample to quantify expression level of at least one biomarker therein; comparing quantified expression level of at least one biomarker with expression level of corresponding at least one biomarker in reference samples; and identifying at least one biomarker, having altered expression level, to be associated with one or more ocular surface diseases. Disclosed also is point-of-care device for identifying at least one biomarker associated with one or more ocular surface diseases. Point-of-care device comprises: panel comprising genomic regions of reference genome corresponding to at least one biomarker associated with ocular surface disease; and processor, communicably coupled with panel, configured to perform one or more steps of aforementioned method.

Description

[0001] IN-VITRO METHOD OF IDENTIFYING BIOMARKERS ASSOCIATED WITH OCULAR SURFACE DISEASES

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to identifying pathological markers of ocular surface diseases. The present invention particularly relates to an in-vitro method of identifying at least one biomarker associated with one or more ocular surface diseases, such as for determining one or more ocular surface diseases, in a subject. Moreover, the present invention also particularly relates to a point-of-care device for identifying at least one biomarker associated with one or more ocular surface diseases.

[0004] BACKGROUND OF THE INVENTION

[0005] Ocular surface diseases typically are a group of eye conditions that primarily affect the outermost layers of the eye, including the cornea and the conjunctiva. Ocular surface diseases lead to discomfort, visual disturbances, and potential damage to the ocular structures. Therefore, various technologies and approaches are employed for checking the same.

[0006] Current methods for ocular surface diseases rely on patient-reported symptoms, clinical observations, and specialized tests such as tear film analysis, corneal topography, and meibomian gland imaging. While these techniques provide valuable clinical information, they often lack sensitivity and specificity, leading to challenges in accurate differentiation between various ocular surface conditions, thus resulting in delayed or incorrect follow ups, impacting patient management and result outcomes. Moreover, current options for ocular surface diseases encompass artificial tears, anti-inflammatory medications, and surgical interventions in severe cases. While these options offer symptomatic relief, they often fail to target the underlying molecular mechanisms driving the diseases. Additionally, individual response to therapy varies, necessitating the development of personalized treatment strategies. In this regard, the emerging therapies, including immunomodulators, growth factors, and regenerative medicine approaches, have been gaining a lot of attention in mitigating the limitations of the aforementioned diagnostic techniques. However, the development of targeted therapies is hindered by the limited understanding of the specific molecular pathways involved and the absence of reliable markers to identify suitable candidates for treatment.

[0007] Furthermore, biomarkers, including cytokines, chemokines, and interleukins, among others, are employed monitoring progression and prognosis. However, these biomarkers have limitations in terms of their specificity, variability in expression levels, and lack of correlation with severity cases, thereby posing challenges in accurate determinations. FIG. 1, 2 and 3 (Prior Art) are graphical representations of existing databases, i.e., the GTEx (Genotype-Tissue Expression) database and the Human Protein Atlas, that do not identify certain biomarkers in ocular region in a subject, thereby not accounting as a comprehensive database for all potential biomarkers.

[0008] Therefore, in light of the foregoing discussion, there exists a need to overcome the aforementioned drawbacks associated with conventional techniques of alleviating symptoms associated with ocular surface diseases.

[0009] SUMMARY OF THE INVENTION

[0010] The present disclosure seeks to provide an in-vitro method of identifying at least one biomarker associated with one or more ocular surface diseases for determining one or more ocular surface diseases. The present disclosure also seeks to provide a point-of-care device for identifying at least one biomarker associated with one or more ocular surface diseases for determining one or more ocular surface diseases.

[0011] The principal object of the present invention is to provide a solution to the existing problem of inaccurate determination (for the purposes of diagnosis, monitoring and management) of one or more ocular surface diseases by providing a novel in-vitro method for a spectrum of one or more ocular surface diseases, spanning a diverse array of conditions impacting the ocular surface, including, but not restricted to, keratoconus (KC), vernal keratoconjunctivitis (VKC), KC with VKC, KC with allergies, dry eye syndrome, conjunctivitis, keratitis, corneal dystrophies, and ocular surface tumors, by detecting and quantifying expression levels of 12 proteins, including PLA2G2A, MUC13, TFPI2, LOC122319436, KCNB2, RERG, GALNT14, SLC40A1, SEMA3E, and NRIP2, AQP1, AQP5, particularly PLA2G2A, within ocular samples, as diagnostic markers and therapeutic targets for one or more ocular surface diseases.

[0012] Another principal object of the present invention is to provide an in-vitro method for monitoring the effectiveness of therapeutic interventions in one or more ocular surface diseases.

[0013] Yet another principal object of the present invention is to provide a therapeutic composition or formulation, namely, corresponding modulators of the at least one biomarker, for treating one or more ocular surface diseases, encompassing a wide spectrum of conditions affecting the ocular surface.

[0014] Still another principal object of the present invention is to provide a personalized therapeutic approach for one or more ocular surface diseases, spanning a diverse array of conditions affecting the ocular surface.

[0015] In a first aspect, an embodiment of the present disclosure provides an in-vitro method for identifying at least one biomarker associated with one or more ocular surface diseases, wherein said at least one biomarker is expressed in tear fluid, cornea, corneal epithelium, conjunctival epithelium, or ocular surface tissues, said in-vitro method comprising: processing an ocular sample to quantify an expression level of said at least one biomarker therein; comparing said quantified expression level of said at least one biomarker with an expression level of a corresponding at least one biomarker in reference samples; and identifying said at least one biomarker, having an altered expression level, to be associated with said one or more ocular surface diseases. Beneficially, the present disclosure effectively introduces a set of proteins as novel predictive markers and therapeutic targets for said one or more ocular surface diseases. By incorporating these proteins as biomarkers, the precision and the monitoring of therapeutic interventions is significantly enhanced, as substantiated by the below-provided experimental data. Furthermore, the development of targeted therapies directed at modulating the activity of these set of proteins introduces a paradigm shift in the realm of personalized treatment strategies for said one or more ocular surface diseases. This approach holds immense potential to yield improved outcomes for patients ultimately enhancing the quality of care and life, as demonstrated by the below-provided experimental data.

[0016] Notably, a biomarker is a measurable biological molecule or characteristic that indicates the presence of a specific physiological or pathological process, condition, or disease. In the context of the eye, biomarkers can provide valuable information about the health of various ocular tissues. The one or more ocular surface diseases refer to a group of eye conditions that primarily affect the outermost layers of the eye, including the cornea (clear, dome-shaped, front surface of the eye) and the conjunctiva (clear, thin membrane that covers the front surface of the eye (the sclera) and lines the inside of the eyelids). These conditions can lead to discomfort, visual disturbances, and potential damage to the ocular structures.

[0017] Optionally, said one or more ocular surface disease selected from a group comprising at least one of: keratoconus (KC), vernal keratoconjunctivitis (VKC), KC with VKC, dry eye syndrome, conjunctivitis, keratitis, corneal dystrophies, and ocular surface tumors. Keratoconus (KC) is an eye disorder that affects the cornea such that the cornea gradually thins and bulges outward into an abnormal cone-like shape, leading to distorted vision, astigmatism, myopia (near-sightedness). Vernal keratoconjunctivitis (VKC) is a chronic allergic inflammation of the eyes that affects the conjunctiva and can also involve the cornea with intense itching, redness, watering of the eyes, and light sensitivity. In case of KC with VKC, i.e., KC and VKC occurring together in the same subject, unique challenges in managing both conditions simultaneously may be presented to the subject. Dry eye syndrome occurs when the eye doesn't produce enough tears or when the tears, namely tear fluid, evaporate too quickly, resulting from various factors, including age, hormonal changes, medications, and environmental conditions, and leading to discomfort, redness, itching, and a gritty sensation in the eyes. Conjunctivitis, also known as "pink eye," is an inflammation of the conjunctiva, the thin membrane that covers the front surface of the eye and lines the inside of the eyelids, caused by infections (viral, bacterial, or fungal) or non-infectious factors such as allergies or irritants. Optionally, the Conjunctivitis may be an allergic conjunctivitis resulting from allergic reaction of the conjunctiva to allergens such as pollen, dust, or pet dander. It leads to redness, itching, tearing, and discomfort. Corneal Erosion can affect the cornea's integrity, such as recurrent corneal erosions, where the outermost layers of the cornea detach from the underlying tissue, causing pain and sensitivity to light. Corneal dystrophies are genetic disorders that can affect the transparency and structure of the cornea. Infections of the cornea, such as bacterial, viral (herpes simplex virus), and fungal infections, can lead to corneal ulceration, pain, redness, and vision impairment. Ocular surface tumors, or pinguecula and pterygium, are growths that develop on the conjunctiva, typically due to exposure to UV light and environmental factors. While pinguecula is benign, a pterygium can extend onto the cornea and affect vision if it becomes large enough.

[0018] Therefore, it is important that proper determination and management of these conditions is carried out. Hence, biomarkers, expressed in tear fluid, cornea, corneal epithelium, conjunctival epithelium, or ocular surface tissues, that are associated with any of the above-stated or other potential eye diseases, are required to be identified. The corneal epithelium is the outermost layer of the cornea. The ocular surface tissues refer to the collective layers that cover the outermost part of the eye, including the cornea and conjunctiva. The expression of said at least one biomarker in these different tissues can have implications for various aspects of eye health, disease diagnosis, treatment response, and research.

[0019] Herein, the at least one biomarker may serve as a potential marker, and a therapeutic target for the one or more ocular surface diseases. Biomarkers can indicate the presence of a specific ocular surface disease or condition, resulting in earlier and more accurate determination (namely, presence or absence, and / or stage or status of the ocular surface disease or condition) for a timely treatment and management. As prognostic markers, the biomarkers aid in providing information about the likely course and outcome of an ocular surface disease, thereby, helping clinicians in predicting the severity of the disease, the risk of complications, and the likelihood of disease progression. Biomarkers serving as therapeutic targets enable exploring interventions that target these molecules or pathways corresponding to these biomolecules to develop new treatments against the ocular surface disease. For example, a biomarker that is found to be upregulated in corneal epithelium of a subject with a certain type of eye disease, such biomarker serves as a diagnostic marker for that eye disease, its expression levels (herein, upregulation) provide a likelihood of progression of the eye disease, hence serving as the prognostic marker, and such information provided by the biomarker enables researchers to develop therapies that directly target this biomarker to alleviate the symptoms and address the underlying cause of the eye disease, ultimately leading to better patient outcomes and quality of life.

[0020] Optionally, said at least one biomarker is selected by measurement of at least one of: a protein, a gene, ribonucleic acid (RNA). Notably, protein biomarkers indicate the presence or progression of diseases, since proteins are essential for various biological processes and functions in the body. Specifically, over-expression or under-expression of certain proteins in specific diseases makes them suitable as biomarkers for early detection, monitoring disease progression, or assessing treatment response. Biomarkers based on gene expression patterns can help identify genetic predispositions, predict disease risk, or indicate the presence of specific diseases, since genes contain the instructions for building proteins and variations or mutations in genes can influence the risk of developing certain diseases or conditions. Similar to genes (fragments of DNA), RNA governs protein synthesis by carrying genetic information from the DNA to ribosomes, where proteins are assembled. RNA can be used as a biomarker by assessing the levels of specific RNA molecules. For example, microRNA (miRNA) profiles can be indicative of certain diseases or conditions, making them potential biomarkers. In this regard, advanced techniques such as genomics, proteomics, and transcriptomics are employed to analyse these molecules to identify patterns and changes that can provide valuable insights into various medical conditions. Beneficially, biomarkers can have a significant impact on disease determination and research, potentially leading to more personalized and effective medical interventions.

[0021] Optionally, said at least one biomarker is selected from a group comprising at least one of: Phospholipase A2 Group IIA (PLA2G2A), Mucin 13 (MUC13), Tissue factor pathway inhibitor 2 (TFPI2 or Placental protein 5 (PP5)), RING-type domain-containing protein (LOC 122319436), Potassium voltage-gated channel subfamily B member 2 (KCNB2), Ras-related and estrogen-regulated growth inhibitor (RERG), Polypeptide N-acetylgalactosaminyl transferase 14 (GALNT14), Solute carrier family 40 member 1 (SLC40A1 or Ferroportin-1 or Iron-regulated transporter 1), Semaphorin-3E (SEMA3E), Nuclear receptorinteracting protein 2 (NRIP2), Aquaporin 1 (AQP1), Aquaporin 5 (AQP5).

[0022] Typically, phospholipase A2 Group IIA (PLA2G2A) is involved in the breakdown of phospholipids. In one or more ocular surface diseases, PLA2G2A plays a role in inflammation and tissue damage by disrupting the delicate balance of lipid molecules in tear film. Mucin 13 (MUC13) provides protective coatings on cell surfaces. In one or more ocular surface diseases, changes in the expression or function of MUC13 typically affects the stability of the tear film and the protection of the ocular surface, potentially leading to dryness and irritation. Tissue Factor Pathway Inhibitor 2 (TFPI2 or PP5) is associated with blood clotting regulation and cell growth. In one or more ocular surface diseases, changes in TFPI2 levels impacts wound healing and tissue remodeling. RING-Type Domain-Containing Protein (FOCI 22319436) or RING domains are involved in protein interactions and regulation. RING domains may be related to signaling pathways or protein turnover processes. Potassium Voltage-Gated Channel Subfamily B Member 2 (KCNB2) is a voltage-gated potassium channel involved in ion transport across cell membranes. In one or more ocular surface diseases, its function could impact the excitability of cells, affecting tear production, nerve signaling, and overall ocular surface health. Ras-Related and Estrogen-Regulated Growth Inhibitor (RERG) regulates growth processes. Dysregulation of growth processes could contribute to tissue abnormalities, inflammation, and healing disruptions in one or more ocular surface diseases. Polypeptide N-Acetylgalactosaminyl Transferase 14 (GALNT14) typically adds sugar molecules to proteins. In one or more ocular surface diseases, changes in GALNT14 activity might impact the glycosylation of proteins that play a role in cell adhesion, signaling, and immune responses. Solute Carrier Family 40 Member 1 (SLC40A1 or Ferroportin-1 or Iron-Regulated Transporter 1) is involved in iron transport. Iron homeostasis is crucial for ocular health, and disruptions in iron regulation might impact various processes, including inflammation and oxidative stress. Semaphorin-3E (SEMA3E) is involved in cell signaling and guidance. In one or more ocular surface diseases, changes in SEMA3E levels could affect blood vessel growth, nerve signaling, and tissue remodeling. Nuclear Receptor- Interacting Protein 2 (NRIP2) interacts with nuclear receptors, affecting gene expression and cellular responses. A change in NRIP2, in one or more ocular surface diseases, may result in inflammation and or reduced or no cell proliferation. Aquaporin 1 (AQP1), Aquaporin 5 (AQP5) are members of aquaporin family of water channels, which are integral membrane proteins that facilitate the transport of water across cell membranes, such as kidneys, the brain, lungs, and red blood cells, exocrine glands, such as the salivary glands, lacrimal glands (tear glands), and sweat glands, and so on. Notably, Aquaporin 1 (AQP1) and Aquaporin 5 (AQP5) play critical roles in maintaining corneal function, particularly in regulating corneal hydration and transparency. Any disruption in their function can lead to various corneal disorders, including corneal edema, dry eye syndrome, and other conditions affecting corneal clarity and vision, such as keratoconus. Moreover, among these proteins, PLA2G2A emerges as a pivotal diagnostic tool and a promising therapeutic target for a spectrum of one or more ocular surface diseases.

[0023] Herein, the in-vitro method comprises acquiring the ocular sample. In this regard, typically, the in-vitro method comprises collecting biological material or ocular sample from the eye or its surrounding structures of the subject, by using techniques known to a person skilled in the art. Optionally, the subject is a human being or an animal.

[0024] Optionally, said ocular sample is selected from a group comprising at least one of: tear fluid, eyewashes, impression cytology from cornea and conjunctiva, corneal scrapings, surgically -removed corneal epithelium or corneal tissues. In this regard, tear fluid or tears can be collected non-invasively using techniques like Schirmer's test or tear strip tests. The impression cytology from cornea and conjunctiva is collected for cytological or microbiological analysis. The corneal scraping involves gently removing cells from the surface of the cornea, for diagnosing corneal infections or epithelial disorders. In cases where the interior of the eye needs to be sampled, vitreous aspiration is performed. Typically, vitreous aspiration involves inserting a fine needle through the eye wall to aspirate a small amount of vitreous fluid, the gel-like substance, or aqueous humor, inside the eye. For more invasive studies or when specific tissues need to be examined, a biopsy or excision (surgically removing) is performed to remove a small piece of tissue from the eye or surrounding structures (such as corneal epithelium or corneal tissues). It will be appreciated that while ocular samples, that are located in and around the eyes, are sampled for assessing ocular conditions, blood samples can provide information about systemic conditions that might affect the eye. Blood tests can be used to measure biomarkers, assess overall health, and identify potential risk factors for ocular diseases. Moreover, acquiring the ocular samples should be performed by specialized professionals using specialized equipment under sterile conditions, to ensure the safety and comfort of the subject.

[0025] Moreover, the in-vitro method comprises processing the ocular sample to quantify an expression level of said at least one biomarker therein. Herein, the term "processing" relates to one or more operations performed on the acquired ocular samples to analyse the ocular sample and measure the amount or levels of the at least one biomarker, under-expression or over-expression of which, with respect to a control reference sample, is associated with the one or more ocular surface disease. This process involves various laboratory techniques and methodologies to measure the quantity of the biomarker of interest.

[0026] Optionally, processing the ocular sample involves preprocessing (or preparing) the collected ocular sample for analysis. Optionally, preprocessing includes steps such as centrifugation of the acquired ocular sample to separate cellular components thereof, homogenization of tissue samples, filtration thereof to remove debris, or subjecting the ocular sample to chemical treatments to stabilize the ocular sample. For example, the acquired ocular sample is subjected to lysis to release the contents (e.g., mRNA, protein), RNA extraction or protein extraction using extraction kits or by using precipitation or affinity purification methods. Optionally, the preprocessing includes labeling of the sample with a fluorescent, radioactive, or enzymatic tag to facilitate detection of the at least one biomarker. Thus, the preprocessed ocular sample is used for determining expression level of various biomarkers, such as the aforementioned at least one biomarker. The expression level of a biomarker refers to the amount of that molecule, for example protein biomarker or RNA biomarker, present in the ocular sample. The process of quantifying biomarker expression levels involves using various laboratory techniques such as immunoassays (e.g., ELISA), Polymerase Chain Reaction (PCR) for gene expression analysis, Western blotting, mass spectrometry, and so forth.

[0027] Optionally, said processing of said ocular sample comprises employing at least one technique selected from a group comprising at least one of: quantitative polymerase chain reaction (qPCR), RNA sequencing, validation of differential expressions, immunoassays, enzyme-linked immunosorbent assays (ELISA), immunohistochemistry (IHC), and molecular analysis. Quantitative Polymerase Chain Reaction (qPCR) technique is used to amplify and quantify specific DNA sequences, to measure gene expression levels in ocular samples. RNA sequencing technique is used to identify and quantify the types and amounts of RNA molecules present in a sample, to provide insights into gene expression and help identify changes in gene expression patterns in ocular tissues. Validation of differential expressions (or expression levels compared to control group) refers to confirming the results of differential gene expression analyses to ensure the accuracy and reliability of the findings.

[0028] Immunoassays involve the use of antibodies to detect and quantify specific proteins in a sample. These assays are used to measure protein levels, identify biomarkers, and understand protein-protein interactions within ocular samples. Enzyme-Linked Immunosorbent Assays (ELISA) is a specific type of immunoassay used to detect and quantify the presence of a target protein. Immunohistochemistry (IHC) involves using antibodies to visualize the distribution and localization of specific proteins within tissue sections, to provide insights into the spatial distribution of proteins within ocular tissues.

[0029] Molecular analysis encompasses various techniques, such as DNA sequencing, RNA analysis, and protein profiling, to analyse molecular components within a sample. Optionally, the other quantitative analysis techniques include mass spectrometry, flow cytometry, and various spectroscopic methods for quantitative analysis in ocular research. Beneficially, all these techniques play crucial roles in understanding the molecular and genetic aspects of ocular health, diseases, and conditions. Additionally, beneficially, these techniques may be combined in multiple combinations to gain a comprehensive view of ocular samples and to draw meaningful conclusions from their analyses. Beneficially, the quantitative assessment of the expression levels of biomarkers in ocular samples enables a meticulous evaluation of disease severity and progression, providing clinicians with vital insights for informed decision-making or treatment response of the eye or ocular tissues. In other words, by proficiently quantifying the biomarkers and tailoring therapeutic interventions, the disclosed in-vitro method endeavors to usher in more accurate diagnosis, prognosis, and personalized treatment strategies for one or more ocular surface diseases.

[0030] Furthermore, the in-vitro method comprises comparing quantified expression level of said at least one biomarker with an expression level of a corresponding said at least one biomarker in reference samples. Herein, the term "reference samples" relates to a plurality of samples acquired from healthy control individuals (referred to as "controls" hereafter. It may be appreciated that the expression levels of said at least one biomarker is compared to a corresponding expression of said at least one biomarker in the reference samples, to determine the differential expression levels of said at least one biomarker in the acquired ocular sample as compared to the expression levels thereof in one or more reference samples. Optionally, the reference samples are ocular samples obtained from healthy control individuals which show normal or baseline expression level of a given biomarker therein.

[0031] Furthermore, the in-vitro method comprises identifying said at least one biomarker, having an altered expression level, to be associated with said one or more ocular surface diseases. Herein, the term "altered, expression level" refers to a change in the amount or activity of a given biomarker, typically a gene or protein, compared to a normal or baseline state as exhibited by said biomarker in a normal or healthy control individual. Optionally, said altered expression level of said at least one biomarker is from alteration in a gene expression level, or at a protein expression level. It may be appreciated that quantitative analysis of gene or protein is used to identify differential levels of expression of said at least one biomarker in patients and controls.

[0032] Optionally, the altered expression level of said at least one biomarker is upregulated or downregulated compared to an expression level of said corresponding at least one biomarker in the reference sample. Notably, said altered expression level can be either upregulation (increased expression) or downregulation (decreased expression) of said biomarker and is often associated with various physiological or pathological conditions, such as an ocular surface disease. Moreover, the altered expression levels of said at least one biomarker can signify disease progression, such as the transition from a stage I to a stage II (i.e., worsening of disease) of the ocular surface disease. Furthermore, monitoring changes in expression levels of said at least one biomarker can help assess how well a patient is responding to treatment. For example, the reduction of specific biomarkers might indicate the effectiveness of the treatment in treatment or alleviating symptoms of the ocular surface disease. It may be appreciated that in some cases, upregulation might be beneficial (e.g., increased expression of a biomarker 'A' in response to infection), while in others, it might be harmful (e.g., overexpression of a biomarker 'B' in keratoconus). Conversely, downregulation might indicate a loss of function (such as in the case of a biomarker 'C associated with tears production).

[0033] Optionally, the in-vitro method further comprises alleviating symptoms associated with said ocular surface disease by any one of: inhibiting activity of said at least one biomarker, modulation of an expression of said at least one biomarker, impeding progression of said ocular surface disease. It will be appreciated that inhibiting the biomarker's activity involves developing interventions or treatments that directly block the function of the identified biomarker, to interrupt the underlying molecular processes contributing to the ocular surface disease, potentially leading to symptom relief and disease management. In an example, by modulating, such as inhibiting, the activities of PLA2G2A or altering its expression, the intricate molecular mechanisms underlying one or more ocular surface diseases may be understood and highly effective therapies that are tailored to the specific needs of patients may be provided. Moreover, modulation of protein expression, using methodologies like gene therapy, RNA interference (RNAi), or pharmacological agents that regulate protein expression, to either reduce or enhance protein expression, results in mitigation or amplification, respectively, of the correlated inflammatory response, leading to the intended therapeutic effect. Furthermore, impeding progression of the ocular surface disease focuses on slowing down or halting the advancement of the ocular surface disease. This approach typically involves targeting not only the biomarker but also other relevant factors contributing to the disease's development and progression, thus, alleviating symptoms and preventing further damage to the ocular surface.

[0034] Optionally, the in-vitro method further comprises identifying a modulator corresponding to said at least one biomarker having said altered expression level, wherein said modulator when provided to said subject is adapted to mitigate progression of said one or more ocular surface diseases. Herein, the term "modulator" refers to an agent or factor that can influence the expression, activity, or levels of a biomarker in a biological system. Modulators can either upregulate (enhance) or downregulate (suppress) the biomarker's activity, directly or indirectly, leading to changes in the biological processes or pathways in which the biomarker is involved. Typically, the modulators may include chemical modulators (e.g., drugs, small molecules, or chemicals to inhibit an enzyme linked to a disease); biological modulators (such as hormones, cytokines, or other proteins that naturally regulate biomarker levels); genetic modulators (e.g., mutations, gene editing (e.g., CRISPR), or RNA interference (RNAi) for modulating a gene biomarker); environmental modulators (including external factors like diet, stress, or exposure to toxins); therapeutic modulators (including interventions such as targeted therapies, immunotherapies, or vaccines designed to modulate specific biomarkers to treat or prevent diseases).

[0035] Notably, such modulators can be provided to patients in various ways, depending on the type of modulator, the condition being treated, and the desired therapeutic effect. Optionally, common routes of providing the modulators to the patients include, but do not limit to, oral (drugs, neutraceuticals), intravenous (IV) (monoclonal antibodies), subcutaneous, intramuscular (vaccines, hormonal therapies), topical (ointments, gels, creams, drops, serum), transdermal (hormonal replacement therapy), and localized (injections, targeted drug delivery, scleral lenses).

[0036] Optionally, said modulator is selected from any of: an agonist, an antagonist. Notably, the agonist modulator activates (i.e., increases the activity or expression of the biomarker) functions of specific receptors or biomolecules they interact with. Moreover, the agonist modulators may partially or fully activate the target receptor molecules. The antagonist modulator typically inhibits (i.e., decreases the activity or expression of the biomarker) functions of specific receptors or biomolecules they interact with, by blocking the receptor and preventing the natural ligand or an agonist from binding thereto. Typically, the antagonist modulators compete directly with the natural ligand or agonist for the same binding site on the receptor, or bind to a different site on the receptor (allosteric site), changing the receptor's shape and function, or irreversibly binding to the active site.

[0037] Optionally, the in-vitro method further comprises designing (or creating) modulators designed to target said at least one biomarker associated with said ocular surface disease, wherein said modulators are selected from a group comprising at least one of: chemical small molecules antagonists, peptides, siRNA, CRISPR-CAS gene editing, phage display antibody and peptide library, antibodybased blocking, biologies engineered to engage with said at least one biomarker and modulators their functions.

[0038] In this regard, modulators targeted for modulators of biomarker activities obstruct these proteins' activities, inflammatory processes and other disease-associated mechanisms, fostering a more favorable environment for disease management. For example, the in-vitro method comprises development of modulators specifically targeting PLA2G2A enzymatic activity. These modulators can be chemical small molecules antagonists, peptides, siRNA, CRISPR-CAS gene editing, using phage display antibody and peptide library, antibody-based blocking, biologies designed to bind to PLA2G2A and block its activity. In an embodiment, such modulators function as inhibitors of PLA2G2A. Inhibition of PLA2G2A can help reduce inflammation and related pathogenic processes in one or more ocular surface diseases.

[0039] Notably, the inhibitors are categorized into various classes based on their nature and mechanism of action. The small molecule blockers are typically low molecular weight compounds that are designed to bind to a specific site on the biomarker molecule and interfere with its activity. Small molecule blockers are often developed through medicinal chemistry approaches and are often used as drug candidates due to their ease of administration and potential for oral delivery. Peptides are short chains of amino acids that can mimic specific protein regions involved in interactions with biomarkers. They can competitively bind to the biomarker and block its function. Peptides can be synthesized to target the active sites of the biomarker or to disrupt protein-protein interactions. Small Interfering RNA (siRNA) is a molecule that can specifically target and silence the expression of a particular gene. By introducing siRNA molecules that target the mRNA of the biomarker, you can reduce its protein production and activity. CRISPR-CAS Gene Editing technology enables precise modification of specific genes, to knock out or modulate the expression of the biomarker gene, leading to reduced biomarker levels and activity. Phage display antibody and peptide library generates a diverse library of antibodies or peptides displayed on the surface of phage particles, that bind specifically to the biomarker, thus providing information on the novel inhibitors that block its function. Antibody-based blocking is achieved by using monoclonal antibodies (MAbs) to specifically target and neutralize the biomarker's activity to interact with other molecules involved in the disease pathway. Biologies, such as fusion proteins or engineered ligands, are engineered to engage with biomarkers and inhibit its activity. These molecules are often larger and more complex than traditional small molecules. A combination of different inhibitors or approaches might be adopted to achieve the desired therapeutic effect in one or more ocular surface diseases.

[0040] Optionally, the in-vitro method further comprises management of (or managing) said ocular surface disease by recommending personalized medical intervention. The inclusion of personalized medical intervention as part of the in-vitro method for managing or management of the ocular surface disease reflects a patientcentered approach to healthcare. Personalized medicine aims to tailor medical decisions and interventions to individual patients based on their unique characteristics, including genetics, biomarkers, lifestyle, and preferences. In this regard, by analysing patient-specific data, including genetic information, biomarker levels, and clinical history, healthcare providers can gain insights into the specific characteristics of the ocular surface disease in each patient. This information helps in accurate disease diagnosis and understanding disease progression. Moreover, based on the patient's diagnostic profile, personalized medical intervention involves selecting treatment options that are most likely to be effective for that individual. This could involve choosing inhibitors or therapeutic agents that target the specific biomarkers identified in the patient's ocular surface disease. Furthermore, personalized medicine considers factors such as metabolism rates, drug interactions, and individual responses to treatment. This information helps healthcare providers determine the appropriate dosage and administration schedule for medications or interventions. Notably, said one or more ocular surface diseases can vary in their severity and response to treatment. Personalized interventions involve continuous monitoring of the patient's condition and making necessary adjustments to the treatment plan based on how the disease is progressing and how the patient is responding. Personalized medicine also emphasizes patient education and engagement. Healthcare providers work closely with patients to help them understand their condition, treatment options, and the importance of following the recommended interventions. Personalized interventions may include tailored recommendations to optimize the patient's lifestyle factors, such as diet, hydration, and environmental exposures, and promote better eye health. Personalized interventions consider the long-term management of said one or more ocular surface diseases. This involves designing treatment plans that can evolve over time based on changes in the patient's condition, new research findings, and advances in medical technology. Beneficially, by incorporating personalized medical intervention into the management of said one or more ocular surface diseases, healthcare providers aim to improve treatment outcomes, minimize side effects, and enhance the overall quality of life for patients.

[0041] The present disclosure also relates to a point-of-care device as described above. Various embodiments and variants disclosed above, with respect to the aforementioned in-vitro method, apply mutatis mutandis to the point-of-care device.

[0042] In a second aspect, an embodiment of the present disclosure provides a point-of- care device for identifying at least one biomarker associated with one or more ocular surface diseases, said point-of-care device comprising: a panel comprising genomic regions of a reference genome corresponding to said at least one biomarker associated with ocular surface disease; and a processor, communicably coupled with said panel, configured to: process an ocular sample to quantify an expression level of said at least one biomarker therein; compare said quantified expression level of said at least one biomarker with an expression level of a corresponding at least one biomarker in reference samples; and identify said at least one biomarker, having an altered expression level, to be associated with said ocular surface disease.

[0043] The point-of-care device achieves all the advantages and technical effects of the in-vitro method of identifying said at least one biomarker of the present disclosure. The use of the aforementioned point-of-care device contributes to identifying potential biomarkers associated with the one or more ocular surface diseases and development of novel, personalized therapeutic strategies that target the aforementioned biomarkers, using a novel, multifunctional point-of-care device. The use of the aforementioned point-of-care device also contributes to significantly enhance patient outcomes, curtail healthcare expenses, and elevate the overall quality of life for those afflicted by the one or more ocular surface diseases. Moreover, the aforementioned point-of-care device reduces complexity and long duration of wait for getting results. Additionally, the aforementioned point-of-care device enables timely alleviating the ocular surface disease by identifying even very small expression levels of biomarkers which may be associated with the presence of disease or progression thereof in the ocular sample of the subject.

[0044] The term "point-of-care device" refers to a device or unit designed to provide immediate results at or near the location of a subject (namely, patient), such as in a clinic, hospital, or even at home. The point-of-care device is designed for ease of use, requiring minimal training to deliver quick results, thus, enabling rapid decision-making and treatment. The point-of-care device is adapted to be integrated with digital health systems, enabling automatic transmission of data from and results to electronic health records (EHRs). Optionally, the point-of-care device forms a part of a comprehensive diagnostic device configured for performing detailed analyses and providing comprehensive information about a patient's health status.

[0045] The term "panel” refers to a chip comprising genomic regions of a reference genome corresponding to said at least one biomarker associated with ocular surface disease. Herein, the reference genome is for example a human genome, specific for ocular surface disease genes and proteins. The term reference genome is obtained from a healthy control individual who does not have ocular surface disease.

[0046] The term "processor” refers to refers to a computational arrangement that is operable to execute instructions, such as instructions related to processing of the ocular sample, analyses of expression level of said at least one biomarker associated with the one or more ocular surface diseases, and so on. Examples of the processor include, but are not limited to, a microprocessor, a microcontroller, a complex instruction set computing (CISC) microprocessor, a reduced instruction set (RISC) microprocessor, a very long instruction word (VLIW) microprocessor, a Field Programmable Gate Array (FPGA), or any other type of processing circuit. Furthermore, the processing arrangement may refer to one or more individual servers, processing devices and various elements associated with a processing device that may be shared by other processing devices. Additionally, one or more individual processors, processing devices and elements are arranged in various architectures for responding to and processing the instructions that execute the aforementioned steps of the in-vitro method of the present disclosure. Moreover, by being communicably coupled to the panel, the processing arrangement may be responsible for performing various operations (such as analysis, and the like) on the panel data. Optionally, the processing arrangement is communicable coupled with a database arrangement (storing patient data, scientific data about the ocular surface disease, etc.) through various means such as network connections, application programming interface (APIs), direct data access methods and the like.

[0047] Optionally, said processor is further configured to: identify a modulator corresponding to said at least one biomarker having said altered expression level, wherein said modulator when provided to said subject is adapted to mitigate progression of said one or more ocular surface diseases; and generate a recommendation for providing said modulator to said subject, to mitigate progression of said one or more ocular surface diseases.

[0048] Optionally, said panel comprises genomic regions of a reference genome corresponding to said at least one biomarker selected from a group comprising at least one of: Phospholipase A2 Group IIA (PLA2G2A), Mucin 13 (MUC13), Tissue factor pathway inhibitor 2 (TFPI2 or Placental protein 5 (PP5)), RING-type domain-containing protein (LOC 122319436), Potassium voltage-gated channel subfamily B member 2 (KCNB2), Ras-related and estrogen-regulated growth inhibitor (RERG), Polypeptide N-acetylgalactosaminyl transferase 14 (GALNT14), Solute carrier family 40 member 1 (SLC40A1 or Ferroportin-1 or Iron-regulated transporter 1), Semaphorin-3E (SEMA3E), Nuclear receptorinteracting protein 2 (NRIP2), Aquaporin 1 (AQP1), Aquaporin 5 (AQP5).

[0049] Herein, the disclosed in-vitro method strives to offer a comprehensive approach to determine a multitude of said one or more ocular surface diseases, seamlessly integrated with personalized therapeutic strategies that cater to individual patient needs. The amalgamation of advanced molecular analysis techniques and the adept design of targeted therapeutic interventions hold tremendous potential for reshaping clinical management paradigms and enhancing patient outcomes in the coming era.

[0050] Additional aspects, advantages, features and objects of the present disclosure would be made apparent from the drawings and the detailed description of the illustrative embodiments construed in conjunction with the appended claims that follow.

[0051] It will be appreciated that features of the present disclosure are susceptible to being combined in various combinations without departing from the scope of the present disclosure as defined by the appended claims.

[0052] BRIEF DESCRIPTION OF THE DRAWINGS The summary above, as well as the following detailed description of illustrative embodiments, is better understood when read in conjunction with the appended drawings. For the purpose of illustrating the present disclosure, exemplary constructions of the disclosure are shown in the drawings. However, the present disclosure is not limited to specific instrumentalities disclosed herein. Moreover, those in the art will understand that the drawings are not to scale. Wherever possible, like elements have been indicated by identical numbers.

[0053] Embodiments of the present disclosure will now be described, by way of example only, with reference to the following diagrams wherein:

[0054] FIGs. 1, 2 and 3 (Prior Arts) are graphical representations of existing databases, i.e., the GTEx (Genotype-Tissue Expression) database and the Human Protein Atlas; FIG. 4 is a flowchart of an in-vitro method of identifying a biomarker associated with ocular surface disease, in accordance with an embodiment of the present disclosure;

[0055] FIG. 5 illustrates a process of identifying and validating biomarkers in keratoconus using corneal epithelial samples from keratoconus patients undergoing corneal collagen cross-linking and individuals undergoing photorefractive keratectomy;

[0056] FIG. 6 illustrates a process of identifying and validating biomarkers in keratoconus using corneal epithelial samples, corneal tissue samples and tear fluid samples from keratoconus patients undergoing corneal collagen cross-linking and individuals undergoing photorefractive keratectomy;

[0057] FIG. 7 illustrates a gene expression analysis of PEA2G2A in corneal epithelium samples from keratoconus (KC) patients and control individuals via quantitative polymerase chain reaction (qPCR), in accordance with an embodiment of the present disclosure;

[0058] FIG. 8 illustrates a protein level analysis of PEA2G2A in the tear samples obtained from control individuals and keratoconus (KC) patients through Enzyme linked immunosorbent assay (EEISA), in accordance with an embodiment of the present disclosure; FIG. 9 illustrates an IHC analysis of PLA2G2A protein expression in corneal tissue samples obtained from control individuals and keratoconus (KC) patients, in accordance with an embodiment of the present disclosure;

[0059] FIGs. 10A and 10B illustrate a gene expression level analysis of MUC 13, sFRPl, IL8, ILlb, AQP1 and AQP5, in the corneal epithelium samples obtained from control individuals and keratoconus (KC) patients through quantitative polymerase chain reaction (qPCR), in accordance with an embodiment of the present disclosure; and

[0060] FIG. 11 illustrates a STRING interaction network of genes, in accordance with an embodiment of the present disclosure.

[0061] In the accompanying drawings, an underlined number is employed to represent an item over which the underlined number is positioned or an item to which the underlined number is adjacent. A non-underlined number relates to an item identified by a line linking the non-underlined number to the item. When a number is non-underlined and accompanied by an associated arrow, the non-underlined number is used to identify a general item at which the arrow is pointing.

[0062] DETAILED DESCRIPTION OF EMBODIMENTS

[0063] The following detailed description illustrates embodiments of the present disclosure and ways in which they can be implemented. Although some modes of carrying out the present disclosure have been disclosed, those skilled in the art would recognize that other embodiments for carrying out or practicing the present disclosure are also possible.

[0064] Referring to FIG. 4, illustrated is a flowchart 400 of steps of an in-vitro method of identifying a biomarker associated with ocular surface disease. At step 402, , an ocular sample is processed to quantify an expression level of said at least one biomarker therein. At step 404, the quantified expression level of said at least one biomarker is compared with an expression level of a corresponding at least one biomarker in reference samples. At step 406, said at least one biomarker, having an altered expression level, is identified to be associated with said one or more ocular surface diseases.

[0065] In this regard, referring to step 402, the ocular sample is acquired from the eye or its surrounding structures of the subject, by specialized professionals using specialized equipment under sterile conditions, and by using techniques known to a person skilled in the art. The ocular sample pertains to a biological material from the eye and surrounding structures of the eye, which is at least one of: tear fluid, eyewashes, impression cytology from cornea and conjunctiva, corneal scrapings, and surgically-removed corneal epithelium or corneal tissues. Before processing the acquired ocular sample, the acquired ocular sample is pre-processed which involves centrifugation to separate cellular components, filtration to remove debris, or chemical treatments to stabilize the sample, in order to ensure that proper analysis is performed. Afterwards, the pre-processed ocular sample is used for determining expression level of the said at least one biomarker therein.

[0066] Referring to step 404, the reference samples includes samples from healthy controls.

[0067] Referring to step 406, the at least one biomarker having altered expression level, are identified, after processing the acquired ocular sample and obtaining information on the expression level of the at least one biomarker. The identified the at least one biomarker is associated with an ocular surface disease, as a specific biomarker and the expression level of that biomarker corresponds to a specific physiological or pathological process, a condition, or a disease. As the biomarker expression level varies with severity and progression of at least one of a condition, a disease, a specific physiological process, or a pathological process; quantifying the expression level of the biomarker enables clear understanding of the heath of various ocular tissues and decision making regarding further treatment methodology therefor.

[0068] Referring to FIG. 5, illustrated is a process flow depicting an exemplary process of identifying and validating biomarkers in keratoconus using corneal epithelial samples from keratoconus patients undergoing corneal collagen cross-linking and individuals undergoing photorefractive keratectomy. As shown, at step 1 (left panel), RNA is isolated and sequenced, with key genes, including PLA2G2A, assessed through qPCR and ELISA in both corneal tissues and tear samples respectively. Immunohistochemistry is used to evaluate levels in tissue sections from keratoconus patients undergoing DALK (Deep Anterior Lamellar Keratoplasty) or OPK (Optical Penetrating Keratoplasty) surgeries, and DSAEK cap is collected from individuals undergoing DSAEK (Descemet's Stripping Automated Endothelial Keratoplasty) surgery. At step 2 (middle panel), data analysis is performed and biomarker levels between keratoconus patients and healthy controls are compared to establish correlations with disease pathology. At step 3 (right panel), the identified genes are used as therapeutic targets for keratoconus patients.

[0069] Referring to the above example (as depicted in FIG. 5), it may be appreciated that herein informed consent is first obtained from subjects, namely, four keratoconus (KC) patients and other individuals undergoing various procedures. Corneal epithelial tissue samples from said four keratoconus patients undergoing corneal collagen cross-linking and individuals undergoing photorefractive keratectomy are collected, followed by RNA isolation and RNA-sequencing. KC pathology- associated differentially expressed genes and enriched pathways are selected, and validated in different KC patient sets. For validation in different sets of patients, RNA is extracted from the corneal epithelial samples and converted to complementary DNA (cDNA) through reverse transcription. Quantitative PCR (qPCR) is employed to quantify the levels of PLA2G2A and other biomarkers in the corneal epithelial samples using specific primers, with gene expression levels normalized to a reference gene.

[0070] Herein, RNAseq data indicates a significant dysregulated genes in KC patients compared to control using Illumina Novaseq Platform. Differential gene expression analysis is performed at a significance level of adjusted p-adj < 0.05. Functional analysis and Gene Ontology are conducted using EnrichR. Significant differentially expressed genes are selected based on criteria including p-adj < 0.05 and log2 fold change >=+l & <=-l. For example, differential Expression of PLA2G2A in KC Patients and Control Group shows that PLA2G2A is significantly upregulated in corneal epithelium of KC patients compared to controls, with a log2 Fold Change of 21.04 and a p-value of 0.000063.

[0071] Referring to FIG. 6, illustrated is a process flow depicting an exemplary process of identifying and validating biomarkers in keratoconus using corneal epithelial samples, corneal tissue samples and tear fluid from keratoconus patients. Alternatively, besides using corneal epithelial tissue samples (left panel of FIG. 5 and as elaborated using description of FIG. 5), corneal tissue samples are used, (middle panel), to evaluate the levels of PEA2G2A in the corneal tissue of keratoconus patients compared to healthy individuals. In this regard, corneal tissue samples are collected from KC patients undergoing DAEK (Deep Anterior Lamellar Keratoplasty) or OPK (Optical Penetrating Keratoplasty) surgeries, and DSAEK cap is collected from individuals undergoing DSAEK (Descemet's Stripping Automated Endothelial Keratoplasty) surgery. The collected tissues are fixed in formalin, embedded in paraffin, and sectioned into thin slices. Immunohistochemistry (IHC) staining is performed to check the level of PLA2G2A in corneal epithelium and stroma.

[0072] Alternatively, as shown in FIG. 6 (right panel), besides using corneal epithelial tissue samples and corneal tissue samples, tear fluid sample is used, to evaluate the levels of PLA2G2A in keratoconus patients compared to healthy individuals. In this regard, Enzyme-Linked Immunosorbent Assay (ELISA) is used to measure the levels of PLA2G2A in tear fluid samples, following the manufacturer's instructions for the ELISA kit. Subsequently, data is analysed to compare the expression levels of PLA2G2A in tear fluid samples between KC patients and healthy individuals and to assess the levels of PLA2G2A in tear fluid samples for both groups. Statistical tests are applied to determine the significance of differences between groups. The collected data is used to establish correlations between the level of PLA2G2A and KC pathology. Referring to FIGs. 7-9, and 10A,10B and 11, for analysis at gene and protein expression levels, the samples are collected from control individuals group (or controls) and keratoconus (KC) patients group. Herein, the control group consists of individuals who have undergone photorefractive keratectomy surgery. The KC patient group is divided into four distinct categories:

[0073] (i) Group 1: Keratoconus (KC) patients with Vogt’s striae (with or without ocular allergic history);

[0074] (ii) Group 2: Keratoconus (KC) patients (without Vogt’s striae and ocular allergic history);

[0075] (iii) Group 3: Keratoconus (KC) patients with ocular allergic history (with or without Vogt’s striae); and

[0076] (iv) Group 4: Keratoconus (KC) patients after C3R Surgery or treatment.

[0077] Referring to FIG. 7, illustrated is a gene expression analysis of PLA2G2A in corneal epithelium samples from keratoconus (KC) patients and control individuals via quantitative polymerase chain reaction (qPCR), in accordance with an embodiment of the present disclosure. As shown, PLA2G2A is found to be upregulated in Group 1 and Group 3 KC patients, while it is downregulated in Group 2 KC patients as compared to controls. Notably, PLA2G2A levels are highest in KC patients with an allergic history (Group 3) and potentially in those with high progression of keratoconus. Therefore, PLA2G2A can be a biomarker of keratoconus disease detection and therapeutic implications. In the future, it may help identify patients who would benefit most from collagen cross-linking treatments. In the graphs of FIG. 7, error bars represent standard error of mean. A t-test was conducted (* indicates statistically significant difference, p < 0.05).

[0078] Referring to FIG. 8, illustrated is a protein level analysis of PLA2G2A in the tear samples obtained from control individuals and keratoconus (KC) patients through Enzyme linked immunosorbent assay (ELISA), in accordance with an embodiment of the present disclosure. As shown, PLA2G2A protein level is elevated in tears of Group 1 and Group 3 KC patients, but reduced in Group 4 KC patients as compared to controls. Specifically, PLA2G2A protein levels drop after treatment in Group 4 KC patients, indicating the relation of progression of keratoconus with PLA2G2A level. This suggests that PLA2G2A could be involved in disease progression and may serve as a potential therapeutic target. In the graphs of FIG. 8, error bars represent standard error of mean. A t-test was conducted (* indicates statistically significant difference, p < 0.05).

[0079] Referring to FIG. 9, illustrated is an IHC analysis of PLA2G2A protein expression in corneal tissue samples obtained from control individuals and keratoconus (KC) patients, in accordance with an embodiment of the present disclosure. As shown, the corneal tissue of control individuals shows low level or no PLA2G2A in the epithelium and stroma. In contrast, corneal tissue of KC patients shows areas of high PLA2G2A protein expression in corneal epithelium.

[0080] Referring to FIGs. 10A and 10B, illustrated is a gene expression level analysis of MUC 13, sFRPl, IL8, ILlb, AQP1 and AQP5, in the corneal epithelium samples obtained from control individuals and keratoconus (KC) patients through quantitative polymerase chain reaction (qPCR), in accordance with an embodiment of the present disclosure. As shown, similar to PLA2G2A, the levels of MUC 13, sFRPl, IL8, ILlb, AQP1 and AQP5 are upregulated across Groups 1 and 3 KC patients, while the levels of MUC 13, sFRPl, IL8, ILlb, AQP1 and AQP5 are downregulated in Group 2 KC patients as compared to controls, except for AQP5. It’s noteworthy that these marker levels are elevated in patients with an allergic history (Group 3) and possibly in those experiencing rapid progression of keratoconus. Collectively, these markers could serve as valuable indicators for monitoring keratoconus progression, with significant potential for both diagnostic and therapeutic applications. In the graphs of FIGs. 10A and 10B, error bars represent standard error of mean. A t-test was conducted (* indicates statistically significant difference, p < 0.05).

[0081] Referring to FIG. 11, illustrated is a STRING interaction network of genes, in accordance with an embodiment of the present disclosure. As shown, the STRING interaction network diagram illustrates the interactions among genes - PLA2G2A, sFRPl, ILlb, IL8, TNFa, MMP1, MUC13, AQP1, and AQP5. It explores how these genes interrelate and potentially collaborate in various biological pathways, offering insights into their collective role in disease mechanisms and cellular functions.

[0082] Modifications to embodiments of the invention described in the foregoing are possible without departing from the scope of the invention as defined by the accompanying claims. Expressions such as “including”, “comprising”, “incorporating”, “consisting of’, “have”, “is” used to describe and claim the present invention are intended to be construed in a non-exclusive manner, namely allowing for items, components or elements not explicitly described also to be present. Reference to the singular is also to be construed to relate to the plural. Numerals included within parentheses in the accompanying claims are intended to assist understanding of the claims and should not be construed in any way to limit subject matter claimed by these claims.

[0083] EXPERIMENTAL PART

[0084] Sample Collection: Ocular samples, such as tear fluid, removed corneal epithelium or corneal tissues during surgeries, were collected from patients suffering from an ocular surface disease i.e. keratoconus and control individuals without such conditions. Rigorous standard procedures were followed to ensure sample integrity and stability of these proteins.

[0085] It may be appreciated that besides biological samples, other details, including demographic details, time of presentation, clinical details, keratometry values (pentacam), details of treatment (medical or surgical), were also obtained from subjects (patient and other control individuals). It may be appreciated that the above-mentioned details were used for identification of the patient, establish correlation with biomarker, grading of severity and correlation with findings. Notably, the correlation between these biomarkers and clinical factors was used to enhance understanding of disease pathology. RNA Sequencing and Clue Discovery: RNA sequencing was performed on corneal epithelium samples obtained from 4 keratoconus (KC) patients and 4 control individuals. The analysis of RNA sequencing data revealed significant differential expression of these 12 proteins in the keratoconus (KC) samples compared to the control samples. The set of 12 proteins includes PLA2G2A, MUC13, TFPI2, LOC122319436, KCNB2, RERG, GALNT14, SLC40A1, SEMA3E, and NRIP2, AQP1, AQP5. This observation provided a strong indication of the potential involvement of these proteins in the pathogenesis of the ocular surface disease.

[0086] Validation of transcript level: To validate the differential levels of these 12 transcripts observed in the RNA sequencing data, the levels of these transcripts were quantified in the larger set of patient samples and control samples. This validation step confirmed the altered expression of this transcript in patients with the ocular surface disease, lending further support to their potential as biomarkers. The differential levels of the transcript were validated by qPCR. However, ELISA or other immunoassays may be performed to validate the differential levels of these 12 proteins.

[0087] Immunoassays: Immunoassays, such as enzyme-linked immunosorbent assays (ELIS As) or immunohistochemistry (IHC), will be conducted to detect and quantify the protein levels of these 12 proteins in the keratoconus patient and control samples. Specialized antibodies targeting these proteins will be employed to capture and detect them in the ocular samples. The generated optical density or fluorescence signal are expected to be proportional to the amount of these proteins present, facilitating quantitative analysis.

[0088] Data Analysis: The obtained data from qPCR were subjected to quantitative analysis. Statistical methods were employed to compare transcript levels between the ocular surface disease patient samples and control samples. The significance of the observed differential expression was determined, and potential correlations with disease severity or progression were explored. Subsequently, such 12 proteins were identified as pivotal diagnostic markers in said one or more ocular surface diseases through a comprehensive method involving sample collection, RNA sequencing, validation, and robust data analysis.

[0089] The novelty and inventiveness of the choice of the disclosed biomarker and the disclosed in-vitro method of identifying said biomarker is established by the fact that no existing databases, including The Human Protein Atlas and the GTEx (Genotype-Tissue Expression) database and references, referred to or provided information regarding expression of PLA2G2A in ocular surface tissue. Moreover, the present study revealed a novel finding through RNA sequencing data of corneal epithelium tissue samples, that the PLA2G2A expression is modulated specifically in the corneal epithelium, which is not reflected in the overall ocular surface tissue as reported in the known databases. To validate the modulation of PLA2G2A expression, a further qPCR analysis was conducted in corneal and conjunctival epithelium samples. The results of the qPCR validation confirmed the presence of PLA2G2A modulation in these specific ocular surface tissues. Therefore, while the known databases did not indicate expression of PLA2G2A in ocular surface tissue, the present invention's unique findings from RNA sequencing data and qPCR validation highlighted the novelty and inventiveness of the in-vitro method and identified biomarkers of the present disclosure. This demonstrated that the expression of PLA2G2A in ocular surface tissues, specifically the corneal and conjunctival epithelium, may differ from the overall ocular surface tissue as reported in the existing databases.

[0090] Therefore, the comprehensive search conducted in the known databases, along with the subsequent discovery of PLA2G2A modulation in corneal epithelium and conjunctival epithelium through RNA sequencing data and qPCR validation, underscores the novelty and inventiveness of the invention in identifying a previously unrecognized association between PLA2G2A and said one or more ocular surface diseases within these specific tissues. Herein, the in-vitro method relied on tear samples and impression cytology to precisely detect and quantify the expression levels of these 12 proteins within patients' ocular samples. By meticulously analysing the protein expression data, valuable insights will be gleaned for the assessment of various diseases.

[0091] Stepping into the therapeutic aspect, once a diagnosis was established, a range of targeted therapeutic strategies were strategically employed, capitalizing on the modulation of these 12 proteins. The methodology encompasses the development of inhibitors specific to these proteins' activities or approaches that modulate their expressions. The efficacy of these strategies will undergo meticulous scrutiny, encompassing preclinical investigations and subsequent clinical trials involving patients diagnosed with conditions such as keratoconus. Assessment of Therapeutic Effectiveness: The efficacy of these therapeutic approaches will be meticulously evaluated through a series of preclinical and clinical studies. Animal models and in vitro assays will be harnessed to gauge the impact of inhibiting or modulating these proteins on inflammation reduction, tissue regeneration, and clinical outcomes. Subsequently, rigorous clinical trials involving patients with said one or more ocular surface diseases will be conducted to assess the safety, efficacy, and overall therapeutic benefits of these interventions.

[0092] Table 1 below shows differentially expressed genes identified through RNA sequencing in KC patients as compared to control. Table 1 includes ensemble id, gene symbols, protein description, Log2 Fold Change and p adjusted value.

[0093] Table 1

Claims

We Claim:

1. An in-vitro method of identifying at least one biomarker associated with one or more ocular surface diseases, wherein said at least one biomarker is expressed in tear fluid, cornea, corneal epithelium, conjunctival epithelium, or ocular surface tissues, said in-vitro method comprising: processing an ocular sample to quantify an expression level of said at least one biomarker therein; comparing said quantified expression level of said at least one biomarker with an expression level of a corresponding at least one biomarker in reference samples; and identifying said at least one biomarker, having an altered expression level, to be associated with said one or more ocular surface diseases.

2. The in-vitro method as claimed in claim 1, wherein said at least one biomarker is selected by measurement of at least one of: a protein, a gene, ribonucleic acid (RNA).

3. The in-vitro method as claimed in claim 1, wherein said at least one biomarker is selected from a group comprising at least one of: Phospholipase A2 Group IIA (PLA2G2A), Mucin 13 (MUC13), Tissue factor pathway inhibitor 2 (TFPI2 or Placental protein 5 (PP5)), RING-type domain-containing protein (LOC122319436), Potassium voltage-gated channel subfamily B member 2 (KCNB2), Ras-related and estrogen-regulated growth inhibitor (RERG), Polypeptide N-acetylgalactosaminyl transferase 14 (GALNT14), Solute carrier family 40 member 1 (SLC40A1 or Ferroportin- 1 or Iron-regulated transporter 1), Semaphorin-3E (SEMA3E), Nuclear receptor-interacting protein 2 (NRIP2), Aquaporin 1 (AQP1), Aquaporin 5 (AQP5).

4. The in-vitro method as claimed in claim 1, wherein said one or more ocular surface diseases are selected from a group comprising at least one of: keratoconus (KC), vernal keratoconjunctivitis (VKC), KC with VKC, dry eye syndrome, conjunctivitis, keratitis, corneal dystrophies, and ocular surface tumors.

5. The in-vitro method as claimed in claim 1, wherein said ocular sample is selected from a group comprising at least one of: tear fluid, eyewashes, impression cytology from cornea and conjunctiva, corneal scrapings, surgically-removed corneal epithelium or corneal tissues.

6. The in-vitro method as claimed in claim 1, wherein said processing of said ocular sample comprises employing at least one technique selected from a group comprising at least one of: quantitative polymerase chain reaction (qPCR), RNA sequencing, validation of differential expressions, immunoassays, enzyme-linked immunosorbent assays (ELISA), immunohistochemistry (IHC), and molecular analysis.

7. The in-vitro method as claimed in claim 1, further comprising alleviating symptoms associated with said one or more ocular surface diseases by any one of: inhibiting activity of said at least one biomarker, modulation of an expression of said at least one biomarker, impeding progression of said one or more ocular surface diseases.

8. The in-vitro method as claimed in claim 1, further comprising identifying a modulator corresponding to said at least one biomarker having said altered expression level, wherein said modulator when provided to said subject is adapted to mitigate progression of said one or more ocular surface diseases.

9. The in-vitro method as claimed in claim 8, further comprising designing modulators designed to target said at least one biomarkers associated with said one or more ocular surface diseases, wherein said modulators are selected from a group comprising at least one of: chemical small molecules antagonists, peptides, siRNA, CRISPR-CAS gene editing, phage display antibody and peptide library, antibodybased blocking, biologies engineered to engage with said at least one biomarker and modulate their functions.

10. The in-vitro method as claimed in claim 1, further comprising management of said one or more ocular surface diseases by recommending personalized medical intervention.

11. The in-vitro method as claimed in claim 1, wherein said altered expression level of said at least one biomarker is upregulated or downregulated compared to an expression level of said corresponding at least one biomarker in said reference samples.

12. The in-vitro method as claimed in claim 1, wherein said altered expression level of said at least one biomarker is from alteration in a gene expression level, or at a protein expression level.

13. A point-of-care device for identifying at least one biomarker associated with one or more ocular surface diseases, said point-of-care device comprising: a panel comprising genomic regions of a reference genome corresponding to at least one biomarker associated with said one or more ocular surface diseases; and a processor, communicably coupled with said panel, configured to: process an ocular sample to quantify an expression level of said at least one biomarker therein; compare said quantified expression level of said at least one biomarker with an expression level of a corresponding at least one biomarker in reference samples; and identify said at least one biomarker, having an altered expression level, to be associated with said one or more ocular surface diseases.

14. The point-of-care device as claimed in claim 13, wherein said processor is further configured to: identify a modulator corresponding to said at least one biomarker having said altered expression level, wherein said modulator when provided to said subject is adapted to mitigate progression of said one or more ocular surface diseases; and generate a recommendation for providing said modulator to said subject, to mitigate progression of said one or more ocular surface diseases.

15. The point-of-care device as claimed in claim 13, wherein said panel comprises genomic regions of a reference genome corresponding to said at least one biomarker selected from a group comprising at least one of: Phospholipase A2 Group IIA (PLA2G2A), Mucin 13 (MUC13), Tissue factor pathway inhibitor 2 (TFPI2 or Placental protein 5 (PP5)), RING-type domain-containing protein(LOC122319436), Potassium voltage-gated channel subfamily B member 2 (KCNB2), Ras-related and estrogen-regulated growth inhibitor (RERG), Polypeptide N-acetylgalactosaminyl transferase 14 (GALNT14), Solute carrier family 40 member 1 (SLC40A1 or Ferroportin- 1 or Iron-regulated transporter 1), Semaphorin-3E (SEMA3E), Nuclear receptor-interacting protein 2 (NRIP2), Aquaporin 1 (AQP1), Aquaporin 5 (AQP5).

Images