AAV vectors encoding oxidoreductases and uses thereof

Abstract

The present disclosure provides recombinant adeno-associated virus (rAAV) vectors, methods of treating ocular conditions, pharmaceutical compositions, and other compositions and methods, wherein the rAAV vectors comprise a polynucleotide encoding an oxidoreductase. Methods of treatment may include administration to the lacrimal gland.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of the priority dates of U.S. Provisional Patent Application Nos. 63 / 375,613, filed on September 14, 2022, and 63 / 383,055, filed on November 9, 2022, the disclosures of which are incorporated herein by reference in their entireties.

[0003] References to sequence listings

[0004] The contents of the electronic sequence listing (OYST_028_03WO_SeqList_ST26.xml (56KB) created on September 9, 2023 are incorporated herein by reference in their entirety. Background Art

[0005] The redox system is a highly conservative redox system that plays a key role in maintaining the reducing environment of the cell. Thioredoxin (TRX) is a major, ubiquitous disulfide reductase that is reduced by thioredoxin reductase (TRXR), which transfers electrons from NAPDH to TRX. See Arner and Holmgren, Eur. J. Biochem. 267, 6102-6109 (2000). Thiol-disulfide exchange reactions control protein function through the redox state of structural or catalytic SH groups. Oxidation of key SH groups will generally cause changes in the biological function of proteins. Therefore, thiol redox control is the main regulatory mechanism for signal transduction and increased production of reactive oxygen species oxidized protein thiols, and it has a wide range of functions in cell physiology and pathological conditions through thioredoxin and glutathione glutaredoxin dependent reaction balance (Arner and Holmgren 2000).

[0006] The thioredoxin system in the lens of the eye has been shown to become progressively weaker with age (Xing and Lou, Invest Ophthalmol Vis Sci. 2010 Dec;51(12):6598-6604), and both the generation of reactive oxygen species and a reduction in endogenous antioxidants contribute to cataract formation.

[0007] A related oxidoreductase is protein disulfide isomerase (PDI). The discovery of PDI was first reported in 1963, and it is thought to serve as a chaperone for protein folding (Goldberger, RF et al., J. Biol. Chem 1963; 238; 628-635). A common response to stress is protein misfolding, against which PDI serves as a key defense. Functionally, PDI is also able to reduce, form and rearrange disulfide bonds, which further supports the localization of this 58 kilodalton calcium-binding chaperone in the endoplasmic reticulum (ER) as a further support for maintaining protein structural integrity. The catalytic activities of PDI, including thiol oxidation / reduction, disulfide bond isomerization, and redox-regulated chaperone activity, are central to ER function (Maattanen et al. (2010) Semin Cell Dev Biol 21: 500-11).

[0008] Presbyopia is a common eye condition that affects millions of people worldwide, especially those over 40 years old. By 2050, it is predicted that 1.8 billion individuals will be affected by presbyopia (Grzybowski et al., AsiaPac J Ophthalmol (Phila) 2020; 9:226-233). Presbyopia is caused by a decrease in the flexibility of the lens.

[0009] Medical treatments for eye conditions that affect the human lens, such as cataract formation and / or presbyopia, are known. Medical treatments typically include prescription lenses to allow for nearsightedness due to loss of elasticity of the human lens. Surgical treatments typically include removal of the natural human lens and replacement with an artificial lens.

[0010] In addition, pharmacological treatments for presbyopia are being investigated, which work primarily by exerting a pinhole effect and increasing depth of field (e.g., NSAIDs, parasympathomimetics, or COX2 inhibitors) or softening the lens (e.g., EV06, lipoic acid cholinesterases), but have limited success (Gribovsky 2020).

[0011] Despite available treatments, these ocular conditions remain a challenge for ophthalmologists, and treatment strategies begin with vision correction to account for the loss of accommodation and visual acuity, and often culminate in surgical removal of the lens.

[0012] Improvements in the medical treatment of human lens aging and disorders would be desirable, and there remains an unmet clinical need for long-term, effective treatments for presbyopia and other ocular diseases.

[0013] Munemasa, Y., Ahn, J.H., Kwong, J.M.K., Caprioli, J., and Piri, N. (2009). Redox proteins thioredoxin 1 and thioredoxin 2 support retinal ganglion cell survival in experimental glaucoma. Gene Therapy, 16(1), 17-25.

[0014] Despite available treatments, these ocular conditions remain a challenge for ophthalmologists, and treatment strategies begin with vision correction to account for the loss of accommodation and vision, and often lead to surgical removal of the lens.

[0015] Improvements in the medical treatment of human lens aging and disorders would be desirable, and there remains an unmet clinical need for long-term, effective treatments for presbyopia and other ocular diseases. Summary of the invention

[0016] In some aspects, the present disclosure provides a recombinant adeno-associated virus (rAAV) vector comprising an AAV capsid and an expression cassette, wherein the expression cassette comprises a polynucleotide encoding an oxidoreductase operably linked to a promoter.

[0017] In some embodiments, the oxidoreductase is a thioredoxin (TRX). In some embodiments, the oxidoreductase is a protein disulfide isomerase (PDI). In some embodiments, the polynucleotide comprises a sequence encoding an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 26. In some embodiments, the polynucleotide comprises a sequence encoding an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 25.

[0018] In some embodiments, the polynucleotide comprises a sequence that is at least 95% identical to SEQ ID NO:2 or SEQ ID NO:28. In some embodiments, the polynucleotide comprises a sequence that is at least 95% identical to SEQ ID NO:2. In some embodiments, the polynucleotide comprises a sequence that is at least 95% identical to SEQ ID NO:28. In some embodiments, the polynucleotide comprises a sequence that is at least 95% identical to SEQ ID NO:24. In some embodiments, the polynucleotide comprises a sequence that is at least 95% identical to SEQ ID NO:30.

[0019] In some embodiments, the promoter is a CMV promoter comprising the nucleotide sequence set forth in SEQ ID NO:17.

[0020] In some embodiments, the expression cassette comprises a CMV promoter and a CMV enhancer. In some embodiments, the expression cassette comprises a polyadenylation (poly A) sequence. In some embodiments, the poly A sequence is a BGH poly A sequence.

[0021] In some embodiments, the expression cassette comprises a Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE). In some embodiments, the expression cassette comprises a Kozak sequence.

[0022] In some embodiments, the present disclosure provides a composition comprising a rAAV vector, wherein the rAAV vector comprises: (a) an AAV capsid, and (b) an expression cassette, wherein the expression cassette comprises a polynucleotide comprising a nucleotide sequence that shares at least 95% identity with a nucleotide sequence comprising SEQ ID NO: 2 or SEQ ID NO: 28, and wherein the polynucleotide is linked to a promoter.

[0023] In some embodiments, the present disclosure provides a rAAV vector composition, wherein the rAAV vector comprises: (a) an AAV shell, and (b) an expression cassette, wherein the expression cassette comprises a polynucleotide comprising a nucleotide sequence that shares at least 95% identity with a nucleotide sequence comprising SEQ ID NO: 2, and wherein the polynucleotide is linked to a promoter.

[0024] In some embodiments, the present disclosure provides a composition comprising an rAAV vector, wherein the rAAV vector comprises: (a) an AAV capsid, and (b) an expression cassette, wherein the expression cassette comprises a polynucleotide comprising a nucleotide sequence that shares at least 95% identity with a nucleotide sequence comprising SEQ ID NO: 28, and wherein the polynucleotide is linked to a promoter.

[0025] In some embodiments, the present disclosure provides a composition comprising an rAAV vector, wherein the rAAV vector comprises: (a) an AAV capsid, and (b) an expression cassette, wherein the expression cassette comprises a polynucleotide comprising a nucleotide sequence that shares at least 95% identity with a nucleotide sequence comprising SEQ ID NO: 30, and wherein the polynucleotide is linked to a promoter.

[0026] In some embodiments, the expression cassette is flanked by two inverted terminal repeats (ITRs). In some embodiments, the ITRs are AAV2 ITRs.

[0027] In some embodiments, the expression cassette comprises a nucleotide sequence that shares at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO:16.

[0028] In some embodiments, the AAV capsid comprises a VP3 that shares at least 95%, 98%, or 100% identity with AAV2 VP3 (SEQ ID NO: 8), AAV5 VP3 (SEQ ID NO: 10), AAV8 VP3 (SEQ ID NO: 12), or AAV9 VP3 (SEQ ID NO: 14). In some embodiments, the AAV capsid comprises a VP3 that shares at least 95%, 98%, or 100% identity with AAV9 (SEQ ID NO: 14).

[0029] In other embodiments, the present disclosure provides a rAAV vector composition, wherein the rAAV vector comprises: (a) an AAV2, AAV5, AAV8 or AAV9 capsid, and (b) an expression cassette, wherein the expression cassette comprises a polynucleotide comprising a nucleotide sequence that shares at least 95% identity with a nucleotide sequence comprising SEQ ID NO: 2 or SEQ ID NO: 28, and wherein the polynucleotide is linked to a promoter.

[0030] In other embodiments, the present disclosure provides a rAAV vector composition, wherein the rAAV vector comprises: (a) an AAV2, AAV5, AAV8 or AAV9 capsid, and (b) an expression cassette, wherein the expression cassette comprises a polynucleotide comprising a nucleotide sequence that shares at least 95% identity with a nucleotide sequence comprising SEQ ID NO: 2, and wherein the polynucleotide is linked to a promoter.

[0031] In other embodiments, the present disclosure provides a rAAV vector composition, wherein the rAAV vector comprises: (a) an AAV2, AAV5, AAV8 or AAV9 capsid, and (b) an expression cassette, wherein the expression cassette comprises a polynucleotide comprising a nucleotide sequence that shares at least 95% identity with a nucleotide sequence comprising SEQ ID NO: 28, and wherein the polynucleotide is linked to a promoter.

[0032] In other embodiments, the present disclosure provides a rAAV vector composition, wherein the rAAV vector comprises: (a) an AAV2, AAV5, AAV8 or AAV9 capsid, and (b) an expression cassette, wherein the expression cassette comprises a polynucleotide comprising a nucleotide sequence that shares at least 95% identity with a nucleotide sequence comprising SEQ ID NO: 30, and wherein the polynucleotide is linked to a promoter.

[0033] In other embodiments, the present disclosure provides a rAAV vector composition, wherein the rAAV vector comprises: (a) an AAV2, AAV5, AAV8 or AAV9 capsid, and (b) an expression cassette, wherein the expression cassette comprises a polynucleotide sequence that shares at least 95% identity with SEQ ID NO:2 or SEQ ID NO:28.

[0034] In other embodiments, the present disclosure provides a rAAV vector composition, wherein the rAAV vector comprises: (a) an AAV2, AAV5, AAV8 or AAV9 capsid, and (b) an expression cassette, wherein the expression cassette comprises a polynucleotide sequence that shares at least 95% identity with SEQ ID NO:2.

[0035] In other embodiments, the present disclosure provides a rAAV vector composition, wherein the rAAV vector comprises: (a) an AAV2, AAV5, AAV8 or AAV9 capsid, and (b) an expression cassette, wherein the expression cassette comprises a polynucleotide sequence that shares at least 95% identity with SEQ ID NO:28.

[0036] In other embodiments, the present disclosure provides a rAAV vector composition, wherein the rAAV vector comprises: (a) an AAV2, AAV5, AAV8 or AAV9 capsid, and (b) an expression cassette, wherein the expression cassette comprises a polynucleotide sequence that shares at least 95% identity with SEQ ID NO:30.

[0037] In other embodiments, the present disclosure provides a rAAV vector composition, wherein the rAAV vector comprises: (a) rAAV2 / 1, rAAV2 / 3, rAAV2 / 4, rAAV2 / 5, rAAV2 / 6, rAAV2 / 7, rAAV2 / 8 or rAAV2 / 9 capsid, and (b) an expression cassette, wherein the expression cassette comprises a polynucleotide comprising a nucleotide sequence that shares at least 95% identity with a nucleotide sequence comprising SEQ ID NO:2 or SEQ ID NO:28, and wherein the polynucleotide is linked to a promoter.

[0038] In other embodiments, the present disclosure provides a rAAV vector composition, wherein the rAAV vector comprises: (a) rAAV2 / 1, rAAV2 / 3, rAAV2 / 4, rAAV2 / 5, rAAV2 / 6, rAAV2 / 7, rAAV2 / 8 or rAAV2 / 9 capsid, and (b) an expression cassette, wherein the expression cassette comprises a polynucleotide comprising a nucleotide sequence that shares at least 95% identity with a nucleotide sequence comprising SEQ ID NO: 2, and wherein the polynucleotide is linked to a promoter.

[0039] In other embodiments, the present disclosure provides a rAAV vector composition, wherein the rAAV vector comprises: (a) rAAV2 / 1, rAAV2 / 3, rAAV2 / 4, rAAV2 / 5, rAAV2 / 6, rAAV2 / 7, rAAV2 / 8 or rAAV2 / 9 capsid, and (b) an expression cassette, wherein the expression cassette comprises a polynucleotide comprising a nucleotide sequence that shares at least 95% identity with a nucleotide sequence comprising SEQ ID NO: 28, and wherein the polynucleotide is linked to a promoter.

[0040] In other embodiments, the present disclosure provides a rAAV vector composition, wherein the rAAV vector comprises: (a) rAAV2 / 1, rAAV2 / 3, rAAV2 / 4, rAAV2 / 5, rAAV2 / 6, rAAV2 / 7, rAAV2 / 8 or rAAV2 / 9 capsid, and (b) an expression cassette, wherein the expression cassette comprises a polynucleotide comprising a nucleotide sequence that shares at least 95% identity with a nucleotide sequence comprising SEQ ID NO: 30, and wherein the polynucleotide is linked to a promoter.

[0041] In other embodiments, the present disclosure provides a rAAV vector composition, wherein the rAAV vector comprises: (a) rAAV2 / 1, rAAV2 / 3, rAAV2 / 4, rAAV2 / 5, rAAV2 / 6, rAAV2 / 7, rAAV2 / 8 or rAAV2 / 9 capsid, and (b) an expression cassette, wherein the expression cassette comprises a polynucleotide sequence that shares at least 95% identity with SEQ ID NO:2 or SEQ ID NO:28.

[0042] In other embodiments, the present disclosure provides a rAAV vector composition, wherein the rAAV vector comprises: (a) rAAV2 / 1, rAAV2 / 3, rAAV2 / 4, rAAV2 / 5, rAAV2 / 6, rAAV2 / 7, rAAV2 / 8 or rAAV2 / 9 capsid, and (b) an expression cassette, wherein the expression cassette comprises a polynucleotide sequence that shares at least 95% identity with SEQ ID NO:2.

[0043] In other embodiments, the present disclosure provides a rAAV vector composition, wherein the rAAV vector comprises: (a) rAAV2 / 1, rAAV2 / 3, rAAV2 / 4, rAAV2 / 5, rAAV2 / 6, rAAV2 / 7, rAAV2 / 8 or rAAV2 / 9 capsid, and (b) an expression cassette, wherein the expression cassette comprises a polynucleotide sequence that shares at least 95% identity with SEQ ID NO:28.

[0044] In other embodiments, the present disclosure provides a rAAV vector composition, wherein the rAAV vector comprises: (a) rAAV2 / 1, rAAV2 / 3, rAAV2 / 4, rAAV2 / 5, rAAV2 / 6, rAAV2 / 7, rAAV2 / 8 or rAAV2 / 9 capsid, and (b) an expression cassette, wherein the expression cassette comprises a polynucleotide sequence that shares at least 95% identity with SEQ ID NO:30.

[0045] In some embodiments, the AAV capsid is AAV2. In some embodiments, the AAV capsid is AAV5. In some embodiments, the AAV capsid is AAV9.

[0046] In some embodiments, the polynucleotide comprises a sequence encoding a signal peptide.

[0047] In some embodiments, the present disclosure provides a pharmaceutical composition comprising the rAAV vector or composition of any one of the aspects and embodiments described herein, and a pharmaceutically acceptable carrier.

[0048] In some embodiments, the composition comprises about 1×10 7About 1×10 14 In some embodiments, the composition comprises about 1×10 12 About 6.2×10 12 genome copies / mL rAAV vector.

[0049] In some embodiments, the present disclosure provides a method of treating an ocular condition in a subject in need thereof, the method comprising administering to an eye of the subject a therapeutically effective amount of the pharmaceutical composition of any one of the embodiments described herein.

[0050] In some embodiments, the pharmaceutical composition is delivered to an ocular secretory gland of an individual. In some embodiments, the pharmaceutical composition is delivered to a lacrimal gland. In some embodiments, the pharmaceutical composition is delivered to an accessory lacrimal gland. In some embodiments, the accessory lacrimal gland is a meibomian gland. In some embodiments, the pharmaceutical composition is delivered to the trabecular meshwork.

[0051] In some embodiments, about 1×10 9 About 1×10 10 , about 1×10 10 About 1×10 11 , about 1×10 11 About 1×10 12 , about 1×10 12 About 1×10 13 , or about 1×10 13 About 1×10 15 genomic copies of rAAV vectors.

[0052] In some embodiments, the eye condition is associated with increased oxidative stress. In some embodiments, the eye condition is associated with loss of expression and / or function of one or more oxidoreductases. In some embodiments, the eye condition is associated with loss of TRX expression and / or function. In some embodiments, the eye condition is associated with loss of PDI expression and / or function. In some embodiments, the eye condition is characterized by loss of near vision. In some embodiments, the eye condition is presbyopia. In some embodiments, the eye condition is cataract formation. In some embodiments, the eye condition is high intraocular pressure. In some embodiments, the eye condition is meibomian gland dysfunction (MDI). In some embodiments, the eye condition is glaucoma.

[0053] In some embodiments, the method results in the expression of an oxidoreductase in cells of the lacrimal gland and / or accessory lacrimal gland and / or in the trabecular meshwork. In some embodiments, the method results in the expression of TRX and / or PDI in cells of the lacrimal gland and / or accessory lacrimal gland and / or in the trabecular meshwork.

[0054] In some embodiments, the methods result in secretion of TRX and / or PDI into the tear film and / or onto the ocular surface of the individual.

[0055] In some embodiments, secretion of TRX and / or PDI into the tear film is stimulated by applying electrical stimulation, mechanical stimulation, ultrasonic stimulation, and / or drugs. Examples of electrical stimulation are intranasal irritants, such as Intranasal tear nerve stimulants. Examples of mechanical stimulation are e.g. 100. An example of ultrasonic stimulation is the administration of a neuromodulator developed for the treatment of dry eye disease, such as the iTear system (Olympic Ophthalmics). In some embodiments, the drug that stimulates TRX and / or PDI to be secreted into the tear film is a cholinergic agonist (e.g., pilocarpine or cevimeline). In some embodiments, the drug is a nicotinic acetylcholine receptor (nAChR) agonist (e.g., varenicline). In some embodiments, the drug is a secretagogue or mucosal protectant (e.g., diquafosol, rebamipide, or ecabet). In some embodiments, the drug that stimulates TRX and / or PDI to be secreted into the tear film is administered to the eye. In some embodiments, the drug, such as a cholinergic agonist, is administered orally. In some embodiments, the secretion of TRX and / or PDI into the tear film is stimulated by a drug (e.g., a cholinergic agonist or nAChR agonist) administered to the nasal cavity. In some embodiments, rAAV vectors or any other constructs configured to express TRX and / or PDI described herein may be administered to an individual as a synergistic therapy in combination with tear enhancement stimulation and / or drugs (e.g., the drugs described in this paragraph). In some embodiments, such synergistic therapy may follow any regimen or use any of the components or parameters described in International Patent Application Publication No. WO 2022 / 235786 (the entire contents of which are incorporated herein).

[0056] In some embodiments, the method results in an improvement in one or more symptoms of an ocular condition. In some embodiments, the method results in an improvement in vision. In some embodiments, the method results in a reduction in the need for corrective lenses. In some embodiments, the method delays progression of a condition.

[0057] In some embodiments, the methods delay progression of a condition in a subject by about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about 50%, about 50% to about 60%, about 60% to about 70%, about 70% to about 80%, about 80% to about 90%, about 90% to about 95%, or more than about 95% compared to a control subject.

[0058] In some embodiments, the method delays the onset of the condition by about 6 months to about 12 months, about 12 months to about 18 months, about 18 months to about 24 months, about 2 years to about 3 years, about 3 years to about 4 years, about 4 years to about 5 years, about 5 years to about 6 years, about 6 years to about 7 years, about 7 years to about 8 years, about 8 years to about 9 years, about 9 years to about 10 years, about 10 years to about 15 years, about 15 years to about 20 years, or more than 20 years compared to a control individual.

[0059] In some embodiments, the control individual is an age-matched individual not treated with a rAAV vector comprising an expression cassette comprising a polynucleotide encoding TRX.

[0060] In some embodiments, the individual required a corrective lens prior to administration of the rAAV, and the administration results in a constant need for the strength of a corrective lens for at least about 6 months to about 12 months, about 12 months to about 18 months, about 18 months to about 24 months, about 2 years to about 3 years, about 3 years to about 4 years, about 4 years to about 5 years, about 5 years to about 6 years, about 6 years to about 7 years, about 7 years to about 8 years, about 8 years to about 9 years, about 9 years to about 10 years, about 10 years to about 15 years, about 15 years to about 20 years, or more than 20 years after administration of the rAAV.

[0061] In some embodiments, the individual's vision remains unchanged for at least about 6 months to about 9 months, about 9 months to about 12 months, about 12 months to about 15 months, about 15 months to about 18 months, about 18 months to about 21 months, about 21 months to about 24 months, about 2 years to about 3 years, about 3 years to about 4 years, about 4 years to about 5 years, about 5 years to about 6 years, about 6 years to about 7 years, about 7 years to about 8 years, about 8 years to about 9 years, about 9 years to about 10 years, about 10 years to about 15 years, about 15 years to about 20 years, or more than 20 years after administration of the rAAV vector.

[0062] In some embodiments, the method further comprises administering one or more additional therapeutic agents. In some embodiments, the additional therapeutic agent that increases tear production is a cholinergic agonist.

[0063] In some embodiments, the subject is a human.

[0064] In some embodiments, the present disclosure provides a pharmaceutical composition of any one of the aspects and embodiments described herein for use in a method of treating an ocular condition in a subject in need thereof, the treatment comprising administering to an eye of the subject an effective amount of the pharmaceutical composition.

[0065] In some embodiments, the present disclosure provides a pharmaceutical composition of any one of the aspects and embodiments described herein for use in the manufacture of a medicament for treating an ocular condition in a subject in need thereof.

[0066] In some embodiments, the present disclosure provides a pharmaceutical composition for treating an ocular condition in a subject, wherein the pharmaceutical composition comprises a vector encoding an oxidoreductase as described herein (eg, any rAAV vector described herein) and a pharmaceutically acceptable carrier.

[0067] In some embodiments, the present disclosure provides a kit comprising the rAAV vector or composition of any one of the aspects and embodiments described herein, and a pharmaceutically acceptable carrier, and instructions for treating an ocular condition in an individual, the treatment comprising administering the pharmaceutical composition to an eye of the individual.

[0068] In some embodiments, the present disclosure provides a kit comprising the rAAV vector or composition of any one of the aspects and embodiments described herein, and a pharmaceutically acceptable carrier, and instructions for treating presbyopia in an individual, the treatment comprising administering the pharmaceutical composition to an eye of the individual.

[0069] In some embodiments, the present disclosure provides a kit comprising the rAAV vector or composition of any one of the aspects and embodiments described herein, and a pharmaceutically acceptable carrier, and instructions for treating cataract formation in an individual, wherein the treatment comprises administering the pharmaceutical composition to an eye of the individual.

[0070] In some embodiments, the present disclosure provides a kit comprising the rAAV vector or composition of any one of the aspects and embodiments described herein, and a pharmaceutically acceptable carrier, and instructions for treating loss of visual accommodation in an individual, the treatment comprising administering the pharmaceutical composition to an eye of the individual.

[0071] In some embodiments, the present disclosure provides a kit comprising the rAAV vector or composition of any one of the aspects and embodiments described herein, and a pharmaceutically acceptable carrier, and instructions for treating ocular hypertension in an individual, wherein the treatment comprises administering the pharmaceutical composition to an eye of the individual.

[0072] In some embodiments, the present disclosure provides a kit comprising the rAAV vector or composition of any one of the aspects and embodiments described herein, and a pharmaceutically acceptable carrier, and instructions for treating meibomian gland dysfunction (MGD) in an individual, the treatment comprising administering the pharmaceutical composition to an eye of the individual.

[0073] In some embodiments, the present disclosure provides a pharmaceutical composition comprising: a) a polypeptide comprising an oxidoreductase or a fragment thereof, optionally wherein the polypeptide has an amino acid sequence having at least 90% identity to an amino acid sequence selected from SEQ ID NOs: 1 and 26; and b) a pharmaceutically acceptable carrier suitable for administration to the eye of a human subject.

[0074] In some embodiments, the present disclosure provides a pharmaceutical composition comprising: a) a vector comprising a polynucleotide encoding a polypeptide comprising an oxidoreductase or a fragment thereof, optionally wherein the polypeptide has an amino acid sequence having at least 90% identity to an amino acid sequence selected from SEQ ID NOs: 1 and 26; and b) a pharmaceutically acceptable carrier suitable for administration to the eye of a human subject.

[0075] In some embodiments, the pharmaceutically acceptable carrier comprises: water; sterile water; pyrogen-free water; phosphate-buffered saline; HEPES-buffered saline; isotonic sodium chloride solution; balanced salt solution; a wetting agent; a surfactant; a tonicity agent; a pH adjuster; a viscosity adjuster; a buffer; a disaccharide, optionally sucrose or trehalose; cellulose and / or its derivatives; an amino acid, optionally histidine; or any combination thereof.

[0076] In some embodiments, the polypeptide has at least 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to SEQ ID NO:1 or SEQ ID NO:26.

[0077] In some embodiments, the polypeptide comprises at least 20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, or 500 amino acids.

[0078] In some embodiments, the formulation is a liquid formulated for administration to or into the ocular surface, or for injection into the lacrimal gland of an eye of a human subject.

[0079] In some embodiments, the pharmaceutical composition comprises a rAAV vector, wherein the vector is present in the composition in an amount effective to express 100 pg / mL to 50 μg / mL of the polypeptide (eg, TRX1 or PDI) in the tear film of the individual upon administration of the composition to the individual.

[0080] In some embodiments, the polynucleotide encoding TRX1 and / or PDI is operably linked to a promoter.

[0081] In some embodiments, the pharmaceutical composition comprises a rAAV vector, wherein the rAAV vector is engineered to constitutively express a polypeptide having an amino acid sequence having at least 90% identity to an amino acid sequence selected from SEQ ID NOs: 1 and 26.

[0082] In some embodiments, the rAAV vector as described herein comprises: a virus, optionally an adenoviral vector or a lentiviral vector; a plasmid; an episomal genome; or an artificial chromosome; and optionally comprises one or more lipids, multivalent cations, DNA-carrier proteins, histones, pseudocapsids, chimeric proteins, or endocytosis receptor proteins.

[0083] In some embodiments, the pharmaceutical composition comprises a polypeptide (e.g., any TRX1 or PDI sequence described herein), wherein the polypeptide is present in the pharmaceutical composition at a concentration of 100 pg / mL to 50 μg / mL or in an amount of 0.5 to 5 μg. In some embodiments, the polypeptide is present in the pharmaceutical composition in a unit dose.

[0084] In some embodiments, the present disclosure provides a method of treating an ocular disease, disorder or condition in an individual in need thereof, the method comprising administering an effective amount of any TRX enzyme or PDI, or any pharmaceutical composition described herein comprising the same, to at least one cell of the eye, lacrimal gland and / or nasolacrimal duct, including but not limited to acinar cells, duct cells and / or myoepithelial cells, and cells of the iris and ciliary body ("ICB"), lens epithelial cells, meibomian glands, and trabecular meshwork.

[0085] In some embodiments, the present disclosure provides a method of treating an ocular disease, disorder or condition in an individual in need thereof, the method comprising administering an effective amount of a vector encoding any TRX enzyme or PDI, or any pharmaceutical composition described herein comprising the same, to at least one cell of the eye, lacrimal gland and / or nasolacrimal duct (including but not limited to acinar cells, duct cells and / or myoepithelial cells, and cells of the iris and ciliary body ("ICB"), lens epithelial cells, meibomian glands, and trabecular meshwork.

[0086] In some embodiments, the ocular condition is a) associated with increased oxidative stress; b) associated with loss of expression and / or function of one or more oxidoreductases; c) associated with loss of expression and / or function of TRX; and / or d) associated with loss of expression and / or function of PDI. In some embodiments, the ocular condition is characterized by loss of near vision. In some embodiments, the ocular condition is: a) presbyopia; b) cataract formation; c) high intraocular pressure; d) meibomian gland dysfunction (MDI); and / or e) glaucoma.

[0087] In some embodiments, the treatment method is such that: a) the oxidoreductase is expressed in cells of the lacrimal gland and / or cells of the accessory lacrimal gland and / or in cells of the trabecular meshwork; b) TRX is expressed in cells of the lacrimal gland and / or cells of the accessory lacrimal gland and / or in cells of the trabecular meshwork; and / or c) TRX is secreted into the tear film and / or onto the ocular surface of the individual.

[0088] In some embodiments, the method of treatment results in: a) improving one or more symptoms of an ocular condition; b) improving vision; c) reducing the need for corrective lenses; and / or d) delaying the progression of an ocular condition. In some embodiments, the method results in a delay of about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about 50%, about 50% to about 60%, about 60% to about 70%, about 70% to about 80%, about 80% to about 90%, about 90% to about 95%, or more than about 95% of the progression of an ocular condition in an individual compared to a control individual. In some embodiments, the method of treatment results in the expression of a functional oxidoreductase in one or more cells of the lacrimal gland and / or accessory lacrimal gland of an individual. In some embodiments, the method of treatment results in the secretion of a functional oxidoreductase into the tear film of an individual. In some embodiments, the secretion of a functional oxidoreductase into the tear film is stimulated by a cholinergic agonist.

[0089] In some embodiments, the method of treatment results in a reduction in one or more symptoms of an ocular disease, disorder, or condition, such as a reduction in itching, swelling, tearing, and redness. In some embodiments, the administration results in an increase in the conjunctival itch grading scale score by 0.5, 1, 1.5, 2, 2.5, 3, 3.5, or 4 points.

[0090] In some embodiments, the disclosure provides a kit comprising any of the pharmaceutical compositions described herein and instructions for use in treating a condition in a human subject, wherein the instructions comprise administering the pharmaceutical composition to an eye of the human subject.

[0091] In some embodiments, the present disclosure provides a pharmaceutical composition according to any embodiment described herein for use in the manufacture of a medicament for treating a condition in a human subject in need thereof.

[0092] In some embodiments, the present disclosure provides a pharmaceutical composition according to any embodiment described herein for use in a method of treatment according to any embodiment described herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0093] Figure 1 A vector map showing the rAAV expression cassette with inverted terminal repeats (ITRs), promoter and TRX polynucleotide elements.

[0094] Figures 2A to 2B An example showing delivery of a viral vector to the lacrimal gland of a human subject.

[0095] Figure 3 Fluorescence microscopy images of 293T cells transfected with AAV.TRX plasmid DNA (bottom row) and stained with anti-thioredoxin (anti-TRX) primary antibody and counterstained with DAPI are shown. Images show fluorescence emission of anti-TRX and DAPI (left column) at 20× and 40× magnification (middle and right columns, respectively). Control 293T cells are untransfected cells (middle row) or cells labeled with a secondary control antibody (top row).

[0096] Figures 4A to 4B Measurements are shown for untransfected and after 4 days of culture ( Figure 4A ) or 24 h after transfection with AAV.TRX plasmid ( Figure 4B ) is a bar graph of TRX in the supernatant of 293T cells.

[0097] Figure 5 Images of Western blots for detection of TRX in cell lysates obtained from untransfected (293T) cells or transfected (AAV.TRX) 293T cells are provided. Detection of GAPDH was used as a loading control. Whole cell extracts (30 μg / lane) were run on SDS-PAGE, and anti-TRX and anti-GAPDH primary antibodies were used to detect the corresponding proteins.

[0098] Figure 6 Images of Western blots for detection of TRX expressed and secreted from cell culture medium obtained from untransfected (293T) cells and transfected (AAV.TRX) 293T cells are provided. Proteins collected from conditioned cell culture medium (30 μg / lane) were run on SDS-PAGE, and anti-TRX and anti-GAPDH primary antibodies were used to detect the corresponding proteins.

[0099] Figure 7A schematic diagram depicting the elements between the ITRs of the AAV plasmid is shown. The plasmid encodes EGFP, which is linked to a secretion signal ("secEGFP") at its N-terminus under the control of a CMV promoter. A woodchuck hepatitis virus post-translational regulatory element (WPRE) is used to increase transgene expression and is in proximity to a bovine growth hormone polyadenylation (pA) signal.

[0100] Figures 8A to 8K is an image of lacrimal tissue stained with an anti-eGFP antibody. The lacrimal gland was administered an rAAV vector containing an expression cassette with an eGFP transgene by intra-lacrimal injection. The lacrimal gland tissue was stained with an anti-eGFP antibody to assess eGFP expression. The black arrow indicates staining showing eGFP expression.

[0101] Fig. 9 Images of porcine lacrimal glands that received injections of AAV-secEGFP (AAV2 or AAV9 serotypes) and were harvested on day 103 and fixed in paraffin are provided. IHC of 5 μM sections was performed using anti-GFP antibody and DAPI (nuclear) counterstaining. Images were captured using a confocal microscope at 100× magnification. Negative control animals did not receive injections.

[0102] Fig.10 Images are provided of porcine lacrimal glands injected with AAV9-secEGFP and harvested on day 103 and fixed in paraffin. IHC of 5 μM sections was performed using anti-GFP antibody and DAPI (nuclear) counterstaining. In addition to lacrimal gland acinar cells, ductile epithelial cells appear to be transduced by AAV9 (white arrows).

[0103] Fig.11 Images are provided of porcine lacrimal glands that received an injection of AAV and were collected and fixed in paraffin on day 103. H&E staining of 5 μM paraffin sections at 100× revealed no inflammatory infiltrates, macroscopic or microscopic abnormalities.

[0104] Fig.12 A schematic diagram of the treatment schedule for administration of AAV encoding a model protein by injection into the lacrimal glands of pigs in combination with OC-01 (Varenicline) by nasal spray is provided. Time points for tear collection and study termination are indicated.

[0105] Fig.13 Shown are the results of Western blot analysis performed with anti-Thioredoxin primary antibody (ThermoFisher Cat. No. 14999-1-AP) and anti-rabbit IgG HRP secondary antibody (ProMega Cat. No. A5316).

[0106] Fig.14Images of Western blots for detection of TRX in cell lysates obtained from untransfected (293T) cells or transfected (AAV.TRX) 293T cells are provided. Detection of actin was used as a loading control. Whole cell extracts (30 μg / lane) were run on SDS-PAGE, and anti-TRX and anti-actin primary antibodies were used to detect the corresponding proteins.

[0107] Fig.15 Graph showing the results of TRX activity analysis. 293T cells transfected with the thioredoxin transgenic plasmid exhibited 56.8 nM / min of thioredoxin activity, and untransfected 293T cells exhibited an endogenous thioredoxin activity level of 16.90 nM / min. This functional analysis confirmed that Figure 1 Expression, secretion and function of the transgene products of the constructs depicted in . DETAILED DESCRIPTION

[0108] The present disclosure provides a method for treating an eye condition in an individual in need. This method includes, for example, expression of an oxidoreductase (e.g., TRX and / or PDI) in the eye. As disclosed herein, a method for treating an eye condition is provided based on AAV delivery of a transgene to the eye. For transgene delivery, rAAV vectors have different advantages. Transgenes delivered by rAAV vectors are unlikely to be incorporated into the genome of transduced cells to support long-term expression and genome succession of transgenic products. The rAAV vector transports the transgene of interest across the cell membrane into the nucleus of the target cell, where the transgene cannot be integrated into the genome for transcription, but in the form of free genes. In view of the free nature of the transgene, the expression of the delivered transgene depends on cell turnover in any given tissue of the transduced cell (BioDrugs. 2017; 31(4): 317-334). The advantage is relatively controlled expression during genome succession without a transgene, especially in germ cells. In addition, rAAV vectors (e.g., rAAV virions described herein) are not as immunogenic as other viral delivery vectors (e.g., adenoviruses). AAV-based delivery vectors that use subretinal and intravitreal injections to transduce cells in the posterior segment of the eye have been described and shown to have efficacy in vivo (U.S. Pat. No. 10,308,957; Petrs-Silva et al., Mol Ther. 19:293-301 (2011); Rodriques et al., Pharm Res. 36:29 (2019)).

[0109] AAV serotypes used for AAV-based delivery of transgenes to the eye include AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, and AAV9 for delivering transgenes (Lebherz et al. J Gene Med.; 10(4):375-382 (2008)). AAV serotypes used for AAV-based delivery of transgenes to the lacrimal gland include AAV2, AAV4, AAV5, AAV5w8, AAV x5, AAV 9, AAV 12, and BAAV (Rocha et al., Ophthalmic Research and Visual Science 52:9567-9572 (2011)).

[0110] In some embodiments, the present disclosure provides a rAAV vector for expressing a polypeptide of the thioredoxin superfamily. In some embodiments, the polypeptide is TRX. In some embodiments, the polypeptide is PDI. In some embodiments, the present disclosure provides a rAAV vector for expressing TRX (e.g., human TRX). In some embodiments, the present disclosure provides a rAAV vector for expressing PDI (e.g., human PDI).

[0111] In some embodiments, cells of at least one eye and / or lacrimal gland are transduced with the rAAV vectors disclosed herein. The lacrimal gland is the main source of tear fluid responsible for promoting a healthy ocular surface and maintaining normal visual function. The main lacrimal gland contains the eyelid and orbital lobes, which are continuous with each other at the lateral edges of the aponeurosis of the levator palpebrae superioris muscle. The lobule has many alveoli and intralobular ducts, which form excretory ducts leading to the conjunctival fornix. The main lacrimal gland is composed of acinar cells, duct cells and / or myoepithelial cells (Obata Cornea.; 25(10 Suppl 1): S82-9 (2006)). The main lacrimal gland secretes the tear film aqueous layer and mucin onto the ocular surface of the individual eye (see, e.g., F. Paulsen, F. et al. (2004) Cell and tissue research, 316(2), 167-177). As used herein, the term "tear gland" refers to an individual's main tear gland, as well as the Wolfring's gland and the Krause's gland. Accessory glands called the Wolfring's gland and the Krause's gland are located in the eyelids. In the upper eyelid, there are about 2 to 5 Wolfring's glands and about 40 Krause's glands. In the lower eyelid, there are about 6 to 8 Krause's glands. The specific location and anatomy of the lacrimal gland functional unit are well known (Conrady et al., JOphthalmol. Article ID 7542929 (2016)).

[0112] Oxidative stress associated with aging is known to alter the structure and function of the tear gland and cause alveolar atrophy and fibrosis. (E. Rocha et al., Ocul Surf. 2008 Oct;6(4)). Without being bound by theory, changes in proteins in the tear gland, such as due to oxidation and the resulting sulfide bond formation, reduce the neural stimulation (efferent parasympathetic and sympathetic) and protein secretion functions of the tear gland, thereby reducing tear output in terms of quantity and quality. It is believed that this reduced tear gland function leads to dry eye disease. In addition, a similar mechanism occurs in the meibomian glands, another area of ​​the tear gland functional unit. The meibomian glands are primarily responsible for producing the lipid layer of the tear film, and loss of function leads to meibomian gland dysfunction (MGD). Meibomian gland loss or drop-out due to atrophy may be directly related to MGD (Chhadya, P. et al. Ophthalmology. 2017 Nov;124(11)), and duct occlusion / obstruction due to fibrosis may lead to atrophy and loss of function, further causing ocular surface disease.

[0113] The trabecular meshwork is an ocular drainage network located in the iridocorneal angle (where the iris and cornea meet and the sclera transitions to the cornea). The trabecular meshwork is a porous small triangle (cross section of approximately 350×50-150 μm) composed of connective tissue beams and sheets or a thin layer covered by trabecular meshwork cells (Abu-Hassan et al., Journal of Ocular Biology (JOcul Biol.) May 2014; 2(1)). Trabecular meshwork cells play an important role in maintaining intraocular pressure by regulating the outflow resistance of the eye humor. Oxidative stress changes the structure of the trabecular meshwork, thereby increasing outflow resistance and ultimately causing increased intraocular pressure (IOP) and optic nerve damage (International Journal of Medicine October 2016; Vol. 38, No. 4; 995-1002). This damage can further lead to increased and progressive visual field loss. Without being bound by theory, increased expression of TRX and / or PDI in the trabecular meshwork serves to reverse and / or prevent additional disulfide bond formation, thereby reducing outflow resistance and associated optic nerve damage.

[0114] The retina is a photoreceptor layer of neural tissue located at the back of the eye that receives light pulses and sends them to the brain as electrical signals for visual discrimination through the optic nerve. Retinal photoreceptors and retinal ganglion cells require a balance between oxygen, reactive oxygen species, and antioxidant molecules that counteract oxidative stress to survive. Oxidative stress can change the homeostasis of cells, thus causing a protective response, especially in cells such as photoreceptors and retinal ganglion cells with high metabolic rates and continuous exposure to light / oxidative stress damage ((BE Domènech, E. and G. Marfany, G.) (2020). Oxidative stress is believed to contribute to the pathogenesis and therapy of retinal dystrophy. See, for example, Antioxidants, 9 (4), 347.).

[0115] Sulfur redox proteinase

[0116] In some embodiments, the expression cassette of the present disclosure comprises a polynucleotide sequence encoding a thioredoxin (TRX) enzyme or a functional variant thereof. The TRX enzyme has a dithiol Cys-Gly-Pro-Cys (CGPC) active site motif that is highly conserved in all kingdoms of life (A. Holmgren A. Journal of Biochemistry (1968) 6: 475-484). In mammals, there are at least two enzymes capable of reducing oxidized cysteine ​​in proteins through a nicotinamide adenine dinucleotide phosphate (NADPH)-dependent reaction, including TRX1 and TRX2 (Lee et al. (2013) Antioxidant Redox Signal 18: 1165-1207). TRX1 is usually located in the cytosol and translocated to the nucleus or secreted. TRX2 is usually located in the mitochondria.

[0117] As used herein, the term "Thioredoxin", "TRX" or "TRX enzyme" refers to a TRX1 protein from any species. In some embodiments, the TRX1 protein is a human TRX1 protein. The term "functional variant" refers to a variant having a sequence substitution, insertion, deletion, and / or N-terminal or C-terminal truncation, wherein the functional variant retains one or more functions of the reference protein (e.g., native TRX enzyme). Thioredoxin is a protein that forms a homodimer and acts as an oxidoreductase.

[0118] In some embodiments, the TRX enzyme is a human TRX enzyme. In some embodiments, the human TRX enzyme is identified in a public database. Human TRX isoforms known in the art are generally identified through public databases. For example, the National Library of Medicine National Center for Biotechnology Information (NCBI) gene database (accessible through the global information network: ncbi.nlm.nih.gov / ) is a searchable gene database that provides gene nomenclature, chromosomal location, gene products, properties; associated markers; phenotypes; interactions; reference links; sequence information; information about sequence variants; gene maps; expression reports; homologs; protein domain content; and access to external databases. As understood by those skilled in the art, sequence information about human TRX isoforms known in the art can be identified by typing the appropriate access number into the NCBI gene database and selecting the sequence information in the desired computer-readable format (e.g., FASTA). Such sequence information may include, but is not limited to, a nucleotide sequence encoding a full-length gene of TRX, a nucleotide sequence encoding a pre-mRNA transcript of TRX, a nucleotide sequence encoding an mRNA of TRX, a nucleotide sequence encoding an open reading frame (ORF) of TRX, and an amino acid sequence of TRX. For example, one isoform of human TRX can be identified in the NCBI gene database by gene ID number 7295 and has an amino acid sequence set forth in SEQ ID NO: 25.

[0119] Exemplary amino acid sequences of human TRX and exemplary polynucleotide sequences (mRNA and ORF) encoding human TRX are set forth in Table 1. In some embodiments, the TRX enzyme comprises or consists of an amino acid sequence set forth in Table 1. In some embodiments, the TRX enzyme comprises or consists of an amino acid sequence encoded by an mRNA sequence set forth in Table 1. In some embodiments, the TRX enzyme comprises or consists of an amino acid sequence encoded by an ORF set forth in Table 1.

[0120] In some embodiments, the TRX enzyme comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 1. In some embodiments, the TRX enzyme comprises or consists of SEQ ID NO: 1. In some embodiments, the TRX enzyme comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 25. In some embodiments, the TRX enzyme comprises or consists of SEQ ID NO: 25.

[0121] In some embodiments, the TRX enzyme is encoded by an ORF comprising or consisting of a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 2. In some embodiments, the TRX enzyme is encoded by an ORF comprising or consisting of SEQ ID NO: 2.

[0122] In some embodiments, the TRX enzyme is encoded by an ORF comprising or consisting of a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 28. In some embodiments, the TRX enzyme is encoded by an ORF comprising or consisting of SEQ ID NO: 28.

[0123] In some embodiments, the TRX enzyme is a truncated form of any TRX enzyme sequence described herein, such as SEQ ID NO: 1 or 25. For example, in some embodiments, the TRX enzyme is a fragment comprising at least 20, 40, 60, 80, or 100 consecutive amino acids of SEQ ID NO: 1 or 25. In some embodiments, the TRX enzyme is a fragment comprising at least 20, 40, 60, 80, or 100 consecutive amino acids of SEQ ID NO: 1 or 25, wherein the fragment has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid substitutions, insertions, or deletions compared to SEQ ID NO: 1 or 25. In some embodiments, the TRX enzyme is a fragment comprising at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 consecutive amino acids of SEQ ID NO: 1 or 25, or a fragment having a length within a range where the endpoint is selected from any of the foregoing length pairs. Any truncated version of the TRX enzyme sequence described herein (e.g., SEQ ID NO: 1 or 25) may include one or more amino acid substitutions, insertions or deletions and may share at least 85, 90, 95, 96, 97, 98, 99 or 100% sequence identity with SEQ ID NO: 1 or 25.

[0124] Polynucleotide encoding TRX enzyme

[0125] In some embodiments, the expression cassette comprises a polynucleotide encoding a TRX enzyme described herein (eg, a human TRX enzyme). In some embodiments, the polynucleotide comprises an ORF encoding a TRX enzyme described herein (eg, a human TRX enzyme).

[0126] In some embodiments, the expression cassette provides increased TRX expression in at least one eye and / or lacrimal gland. In some embodiments, the expression cassette provides increased TRX expression in at least one meibomian gland. In some embodiments, the expression cassette provides increased TRX expression in the trabecular meshwork. In some embodiments, the expression cassette provides increased TRX expression in the retina. In some embodiments, the expression of TRX is increased by 5%, 10%, 15%, 20% or 25% compared to the expression of TRX in an untreated individual or in the contralateral eye of a treated individual. As used herein, "individual" means any mammal, including mice, rabbits, non-human primates (NHPs) and humans. In some embodiments, the individual is a human or NHP. In addition, "individual" or "patient" can be used interchangeably with "subject". In some embodiments, the expression of TRX is increased by at least 1.5 times, 2 times, 3 times, 4 times or 5 times compared to the expression of TRX in an untreated individual or in the contralateral eye of a treated individual. In some embodiments, TRX is expressed at any detectable level in the treated eye, whereas TRX may not be expressed or expressed at an undetectable level in an untreated individual or in the contralateral eye of a treated individual. In other words, an eye or lacrimal gland to which a rAAV vector described herein is administered may express TRX at a higher abundance than an eye or lacrimal gland that has only endogenous (i.e., native) TRX expression or an eye that has low or impaired endogenous (i.e., native) TRX secretion.

[0127] The term "functional variant" refers to a variant having sequence substitutions, insertions, deletions and / or N-terminal or C-terminal truncations, wherein the functional variant retains one or more functions of a reference protein (eg, native TRX).

[0128] In some embodiments, the expression cassette comprises a polynucleotide encoding a protein that is human TRX or a functional variant thereof. In some embodiments, the expression cassette comprises a polynucleotide comprising a nucleotide sequence encoding an amino acid sequence identified in Table 1. In some embodiments, the expression cassette comprises a polynucleotide comprising a nucleotide sequence identified in Table 1.

[0129] In some embodiments, the expression cassette comprises a polynucleotide comprising a nucleotide sequence encoding a protein having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the expression cassette comprises a polynucleotide comprising a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the expression cassette comprises a polynucleotide comprising a nucleotide sequence encoding a protein having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence set forth in SEQ ID NO: 25. In some embodiments, the expression cassette comprises a polynucleotide comprising a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 25. In some embodiments, the expression cassette comprises a polynucleotide comprising a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 2. In some embodiments, the expression cassette comprises a polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 2. In some embodiments, the expression cassette comprises a polynucleotide comprising a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 28. In some embodiments, the expression cassette comprises a polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 28.

[0130] In some embodiments, the expression cassette comprises a polynucleotide comprising a nucleotide sequence encoding a TRX enzyme, wherein the TRX enzyme comprises or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 1. In some embodiments, the TRX enzyme comprises or consists of SEQ ID NO: 1. In some embodiments, the expression cassette comprises a polynucleotide comprising a nucleotide sequence encoding a TRX enzyme, wherein the TRX enzyme comprises or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 25. In some embodiments, the TRX enzyme comprises or consists of SEQ ID NO: 25. In some embodiments, the expression cassette comprises a polynucleotide comprising a nucleotide sequence encoding a TRX enzyme, wherein the TRX enzyme comprises or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 24. In some embodiments, the TRX enzyme comprises or consists of SEQ ID NO: 24.

[0131] The percent identity can be determined using any suitable method known in the art. For example, the method for determining the percent identity of a nucleic acid sequence is genome sequencing. Methods for determining the percent identity of a nucleic acid sequence or an amino acid sequence are known to those skilled in the art.

[0132] Protein disulfide isomerase

[0133] In some embodiments, the expression cassette of the present disclosure comprises a polynucleotide sequence encoding a protein disulfide isomerase (Protein Disulfide Isomerase; PDI) or a functional variant thereof. The ability of PDI to reduce disulfide bonds is a key protection against oxidative stress and diseases associated with disulfide bond formation in an oxidative environment due to normal aging, disease, environmental or pharmacological events (e.g., cataract formation and presbyopia), and may also be involved in dry eye disease, meibomian gland dysfunction, and high intraocular pressure / glaucoma. PDI also contains a thioredoxin active site with a CXXC motif, further supporting the potential role of PDI in disulfide bond reduction to reduce disease conditions in various tissues. Protein disulfide isomerase (gene name: P4HB) is a 508 amino acid protein with a mass of approximately 57.1 kilodaltons (also known as ERBA2L, PDI, PDIA1, PO4DB).

[0134] As used herein, the term "protein disulfide isomerase" or "PDI" refers to PDI proteins of any species. The term "functional variant" refers to variants with sequence substitutions, insertions, deletions and / or N-terminal or C-terminal truncations, wherein the functional variant retains one or more functions of the reference protein (e.g., native PDI).

[0135] In some embodiments, PDI is a human PDI enzyme. In some embodiments, human PDI is identified in a public database. Human PDI isoforms known in the art are usually identified by public databases. As understood by those skilled in the art, sequence information about human PDI isoforms known in the art can be identified by typing the appropriate access number into the NCBI gene database to select the sequence information in the required computer-readable format (e.g., FASTA). Such sequence information may include, but is not limited to, the nucleotide sequence of the full-length gene encoding TRX, the nucleotide sequence of the pre-mRNA transcript encoding PDI, the nucleotide sequence of the mRNA encoding PDI, the nucleotide sequence of the open reading frame (ORF) encoding PDI, and the amino acid sequence of PDI. For example, an isoform of human PDI can be identified in the NCBI gene database with gene ID number 5034, and has the amino acid sequence set forth in SEQ ID NO:26.

[0136] Exemplary amino acid sequences of human PDI and exemplary polynucleotide sequences (mRNA and ORF) encoding human PDI are set forth in Table 2. In some embodiments, PDI comprises or consists of an amino acid sequence set forth in Table 2. In some embodiments, PDI comprises or consists of an amino acid sequence encoded by an mRNA sequence set forth in Table 2. In some embodiments, PDI comprises or consists of an amino acid sequence encoded by a nucleic acid sequence set forth in Table 2.

[0137] In some embodiments, the PDI comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 26. In some embodiments, the PDI comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a sequence spanning positions 24-474 of SEQ ID NO: 26. In some embodiments, the nucleic acid encoding the PDI comprises or consists of SEQ ID NO: 30.

[0138] In some embodiments, PDI is a truncated form of any of the PDI sequences described herein, such as SEQ ID NO: 26. For example, in some embodiments, PDI is a fragment comprising at least 20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, or 500 consecutive amino acids of SEQ ID NO: 26. In some embodiments, PDI is a fragment comprising at least 20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, or 500 consecutive amino acids of SEQ ID NO: 26, wherein the fragment has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acid substitutions, insertions, or deletions compared to SEQ ID NO: 26. In some embodiments, the PDI is a fragment comprising at least 10, 20, 30 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, or 500 consecutive amino acids of SEQ ID NO: 26, or a fragment having a length within a range where the endpoints are selected from any of the foregoing length pairs. Any truncated version of the PDI enzyme sequence described herein (e.g., SEQ ID NO: 26) may include one or more amino acid substitutions, insertions, or deletions and may share at least 85, 90, 95, 96, 97, 98, 99, or 100% sequence identity with SEQ ID NO: 26 or with a sequence spanning positions 24-474 of SEQ ID NO: 26.

[0139] Polynucleotide encoding PDI

[0140] In some embodiments, the expression cassette comprises a polynucleotide encoding a PDI enzyme described herein (eg, a human PDI enzyme). In some embodiments, the polynucleotide comprises an ORF encoding a PDI enzyme described herein (eg, a human PDI enzyme).

[0141] In some embodiments, the expression cassette provides increased PDI expression in at least one eye and / or lacrimal gland. In some embodiments, the expression cassette provides increased PDI expression in at least one meibomian gland. In some embodiments, the expression cassette provides increased PDI expression in the trabecular meshwork. In some embodiments, the expression of PDI may be increased by 5%, 10%, 15%, 20%, or 25% compared to the expression of PDI in an untreated individual or in the contralateral eye of a treated individual. In some embodiments, the expression of PDI is increased by at least 1.5 times, 2 times, 3 times, 4 times, or 5 times compared to the expression of TRX in an untreated individual or in the contralateral eye of a treated individual. In some embodiments, PDI may be expressed at any detectable level in the treated eye, while in an untreated individual or in the contralateral eye of a treated individual, PDI may not be expressed or expressed at an undetectable level. In other words, an eye or lacrimal gland to which a rAAV vector described herein is administered may express PDI at a higher abundance than an eye or lacrimal gland having only endogenous (ie, native) PDI expression or an eye having low or impaired endogenous (ie, native) PDI secretion.

[0142] The term "functional variant" refers to a variant having sequence substitutions, insertions, deletions and / or N-terminal or C-terminal truncations, wherein the functional variant retains one or more functions of a reference protein (eg, native PDI).

[0143] In some embodiments, the expression cassette comprises a polynucleotide encoding a protein that is human PDI or a functional variant thereof. In some embodiments, the expression cassette comprises a polynucleotide comprising a nucleotide sequence encoding an amino acid sequence identified in Table 2. In some embodiments, the expression cassette comprises a polynucleotide comprising a nucleotide sequence identified in Table 2.

[0144] In some embodiments, the expression cassette comprises a polynucleotide comprising a nucleotide sequence encoding a protein having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence set forth in SEQ ID NO: 26. In some embodiments, the expression cassette comprises a polynucleotide comprising a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 26. In some embodiments, the expression cassette comprises a polynucleotide comprising a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 30. In some embodiments, the expression cassette comprises a polynucleotide comprising a nucleotide sequence set forth in SEQ ID NO: 30.

[0145] In some embodiments, the expression cassette comprises a polynucleotide comprising a nucleotide sequence encoding PDI, wherein PDI comprises or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 26. In some embodiments, PDI comprises or consists of SEQ ID NO: 26. In some embodiments, the expression cassette comprises a polynucleotide comprising a nucleotide sequence encoding PDI, wherein PDI comprises or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 26. In some embodiments, PDI comprises or consists of SEQ ID NO: 26.

[0146] Codon optimization

[0147] In some embodiments, the expression cassette comprises a polynucleotide encoding a TRX enzyme or a PDI enzyme, wherein the polynucleotide comprises or consists of a nucleotide sequence that is codon-optimized for expression in a target cell. In some embodiments, the target cell is a mammalian cell. In some embodiments, the target cell is a human cell, a murine cell, or a non-human primate (NHP) cell.

[0148] A codon optimized nucleotide sequence, such as a codon optimized nucleotide sequence encoding a TRX enzyme or a PDI, is generally a sequence comprising at least one synonymous nucleobase substitution relative to a reference sequence (e.g., a wild-type ORF encoding a TRX enzyme or a wild-type ORF encoding a PDI). A codon optimized nucleotide sequence may be partially or completely different in sequence from a reference sequence. For example, a reference sequence encoding a polyserine uniformly encoded by a TCT codon may be sequence optimized by substituting 100% of its nucleobases (for each codon, T in position 1 is replaced by A, C in position 2 is replaced by G, and T in position 3 is replaced by C) to obtain a sequence encoding a polyserine uniformly encoded by an AGC codon. The percentage of sequence identity obtained from a global pairwise alignment between a reference polyserine nucleic acid sequence and a sequence optimized polyserine nucleic acid sequence will be 0%. However, the protein products from the two sequences will be 100% identical.

[0149] Codon optimization methods are known in the art and can be applied to achieve one or more desired results, such as to increase expression of the synthetic gene in a target cell. In some embodiments, the expression cassette comprises a nucleotide sequence that is sequence optimized relative to a reference sequence using a sequence optimization method. Sequence optimization methods are known in the art and include known sequence optimization tools, algorithms, and services. Non-limiting examples include those from GeneArt (Life Technologies), DNA2.0 (Menlo Park CA), and (Services of Atum, Newark, CA.

[0150] In some embodiments, the expression cassettes of the present disclosure comprise polynucleotides encoding TRX enzymes or PDI enzymes that have been sequence optimized using methods such as For example ) is sequence optimized relative to a reference sequence. In some embodiments, the reference sequence encoding TRX is set forth in SEQ ID NO: 28. In some embodiments, the expression cassette comprises a polynucleotide encoding TRX that is sequence optimized relative to a reference sequence encoding TRX. In some embodiments, the reference sequence encoding TRX is set forth in SEQ ID NO: 28, and the codon-optimized sequence encoding TRX is set forth in SEQ ID NO: 2. In some embodiments, the reference sequence encoding PDI is set forth in SEQ ID NO: 30. In some embodiments, the expression cassette comprises a polynucleotide sequence optimized relative to a reference sequence encoding PDI.

[0151] In some embodiments, the method of sequence optimization comprises the codon optimization algorithm described in US 7,561,972, US 7,561,973, US 8,126,653 and US 8,401,798, each of which is incorporated herein by reference. In some embodiments, the method of sequence optimization known in the art (e.g., For example ), the polynucleotide sequence is sequence optimized based on the codon usage preference in the host cell (e.g., mammalian cell, e.g., human cell, murine cell, non-human primate cell) relative to the reference sequence. In some embodiments, the polynucleotide sequence comprises or consists of a nucleotide sequence that is codon-optimized relative to the reference sequence using a sequence optimization method. In some embodiments, the polynucleotide sequence comprises or consists of a nucleotide sequence that is codon-optimized relative to the reference sequence for expression in a human host cell. In some embodiments, the polynucleotide sequence comprises or consists of SEQ ID NO: 2.

[0152] In some embodiments, the disclosure provides an expression cassette comprising a polynucleotide comprising or consisting of a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 2. In some embodiments, the polynucleotide comprises or consists of SEQ ID NO: 2.

[0153] Table 1: Human TRX amino acid sequence and polynucleotide sequence

[0154]

[0155] Table 2: Human PDI amino acid sequence and polynucleotide sequence

[0156]

[0157] Expression Cassette

[0158] The vector of the present disclosure comprises an expression cassette. As used herein, the term "expression cassette" refers to a polynucleotide comprising at least one polynucleotide sequence encoding a protein of interest (e.g., TRX or PDI). In some embodiments, (e.g., wherein the vector is an rAAV virion), the expression cassette may further comprise inverted terminal repeats flanking at least one polynucleotide sequence encoding a protein of interest. In some embodiments, the protein of interest is a member of the thioredoxin superfamily. In some embodiments, the protein of interest is an oxidoreductase. In some embodiments, the protein of interest is TRX. In some embodiments, the protein of interest is a protein disulfide isomerase (PDI). In some embodiments, the expression cassette comprises other polynucleotide sequences, such as promoters, regulatory elements (e.g., one or more promoters), translation initiation sequences, coding sequences, and termination sequences.

[0159] In some embodiments, the expression cassette of the present disclosure comprises a polynucleotide sequence encoding an oxidoreductase (e.g., TRX or PDI) or a functional variant thereof. In some embodiments, the expression cassette provides increased TRX expression in at least one eye, lacrimal gland, and / or trabecular meshwork. In some embodiments, the expression of TRX may be increased by 5%, 10%, 15%, 20%, or 25% compared to the expression of TRX in an untreated individual or in the contralateral eye of a treated individual. In some embodiments, the expression of TRX may be increased by 1.3 times, 1.5 times, 1.7 times, 1.9 times, 2 times, 3 times, 4 times, or 5 times compared to the expression of TRX in an untreated individual or in the contralateral eye of a treated individual. In some embodiments, TRX may be expressed at any detectable level in the treated eye, while in an untreated individual or in the contralateral eye of a treated individual, TRX may not be expressed or expressed at an undetectable level. In other words, the eye, lacrimal gland, meibomian gland, and / or trabecular meshwork to which the rAAV vectors described herein are administered may express the oxidoreductase (e.g., TRX or PDI) at a higher abundance than in an eye, lacrimal gland, meibomian gland, and / or trabecular meshwork that has only endogenous (i.e., native) expression of the oxidoreductase (e.g., TRX or PDI) or in an eye that has lower or impaired secretion of the endogenous (i.e., native) form of the oxidoreductase (e.g., TRX or PDI).

[0160] The term "functional variant" refers to a variant having sequence substitutions, insertions, deletions and / or N-terminal or C-terminal truncations, wherein the functional variant retains one or more functions of a reference protein (eg, native TRX).

[0161] In some embodiments, provided herein are expression cassettes comprising a polynucleotide encoding TRX. Exemplary amino acid sequences of TRX and exemplary polynucleotide sequences encoding TRX are set forth in Table 3.

[0162] Table 3: TRX amino acid sequence and polynucleotide sequence

[0163] describe sequence Wild-type human TRX amino acid sequence SEQ ID NO:1 Mature human TRX amino acid sequence SEQ ID NO:25 Codon-optimized ORF encoding TRX (i.e. encoding SEQ ID NO: 25) SEQ ID NO:2 Signal peptide amino acid sequence SEQ ID NO:3 Polynucleotide sequence encoding signal peptide SEQ ID NO:4 Exemplary rAAV expression vectors with TRX transgene SEQ ID NO:16

[0164] In some embodiments, the polynucleotide encodes a protein that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity with SEQ ID NO: 1. In some embodiments, the polynucleotide encodes a protein that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity with SEQ ID NO: 25. In some embodiments, the polynucleotide encodes a protein that is human TRX or a functional variant thereof.

[0165] In some embodiments, the polynucleotide encoding TRX comprises a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity with SEQ ID NO: 2. In some embodiments, the polynucleotide encoding TRX comprises a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity with SEQ ID NO: 28. In some embodiments, the protein is human TRX or a functional variant thereof.

[0166] In some embodiments, the polynucleotide encoding TRX also encodes a signal peptide. The signal peptide promotes the expression and subcellular localization of the protein. In some embodiments, the signal peptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity with SEQ ID NO: 3. In some embodiments, the polynucleotide comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity with SEQ ID NO: 4.

[0167] In some embodiments, an expression cassette provided herein comprises a transgene encoding an oxidoreductase that is not TRX.

[0168] In some embodiments, the expression cassettes provided herein are bicistronic expression cassettes configured to allow simultaneous expression of two proteins using a single mRNA transcript (e.g., by incorporating an internal ribosome entry site "IRES"). For example, the bicistronic expression cassette may comprise both a TRX enzyme sequence and a PDI sequence selected from any sequence or embodiment described herein. In some embodiments, co-expression of TRX and PDI in cells of the eye may provide a synergistic therapeutic effect.

[0169] In some embodiments, the expression cassette provides increased PDI expression in at least one eye, lacrimal gland, at least one meibomian gland, and / or trabecular meshwork. In some embodiments, the expression of PDI may be increased by 5%, 10%, 15%, 20%, or 25% compared to the expression of PDI in an untreated individual or in the contralateral eye of a treated individual. In some embodiments, the expression of PDI may be increased by 1.3-fold, 1.5-fold, 1.7-fold, 1.9-fold, 2-fold, 3-fold, 4-fold, or 5-fold compared to the expression of PDI in an untreated individual or in the contralateral eye of a treated individual. In some embodiments, PDI may be expressed at any detectable level in the treated eye, while PDI may not be expressed or may be expressed at an undetectable level in an untreated individual or in the contralateral eye of a treated individual. In other words, the eye, lacrimal gland, meibomian gland, and / or trabecular meshwork to which the rAAV vectors described herein are administered may express the oxidoreductase (e.g., PDI) at a higher abundance than in an eye, lacrimal gland, meibomian gland, and / or trabecular meshwork that has only endogenous (i.e., native) expression of the oxidoreductase (e.g., PDI) or an eye that has lower or impaired secretion of the endogenous (i.e., native) form of the oxidoreductase (e.g., PDI).

[0170] The term "functional variant" refers to a variant having sequence substitutions, insertions, deletions and / or N-terminal or C-terminal truncations, wherein the functional variant retains one or more functions of a reference protein (eg, native PDI).

[0171] In some embodiments, the polynucleotide encodes a protein that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 26. In some embodiments, the polynucleotide encodes a protein that is human PDI or a functional variant thereof.

[0172] In some embodiments, the polynucleotide encoding PDI comprises a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 30. In some embodiments, the protein is human PDI or a functional variant thereof.

[0173] In some embodiments, the polynucleotide encoding PDI also encodes a signal peptide. The signal peptide promotes the expression and subcellular localization of the protein. In some embodiments, the signal peptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity with SEQ ID NO: 3. In some embodiments, the polynucleotide comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity with SEQ ID NO: 4.

[0174] In some embodiments, the expression cassette of the present disclosure comprises a promoter. As used herein, the term "promoter" refers to a DNA sequence that guides the binding of RNA polymerase and thereby promotes RNA synthesis, i.e., a minimal sequence sufficient to guide transcription. Promoters and corresponding protein or polypeptide expression may be ubiquitous, meaning that they are strongly active in a wide range of cells, tissues, and species or are cell type-specific, tissue-specific, or species-specific. A promoter may be "constitutive," meaning that it has sustained activity, or "inducible," meaning that the promoter may be activated or inactivated by the presence or absence of biological or abiotic factors. The nucleic acid construct or vector of the present disclosure also includes an enhancer sequence that may or may not be adjacent to the promoter sequence. The enhancer sequence affects promoter-dependent gene expression and may be located in the 5' or 3' region of the native gene.

[0175] Any suitable promoter region or promoter sequence therein can be used in the subject polynucleotide cassette, so long as the promoter region promotes expression of a polynucleotide sequence encoding an oxidoreductase (e.g., TRX or PDI) in at least one eye, lacrimal gland, at least one meibomian gland, and / or trabecular meshwork. In some embodiments, the promoter promotes expression of the gene in a mammalian eye, lacrimal gland, meibomian gland, and / or trabecular meshwork. In some embodiments, the expression cassette comprises a cell type-specific promoter. The promoter can specifically promote transcription in cells of the eye, cells of the lacrimal gland, cells of the meibomian gland, or cells of the trabecular meshwork. For example, in some embodiments, the promoter may include: a corneal stroma-specific promoter, such as a keratin promoter (Carlson EC et al., "In Vivo Gene Delivery and Visualization of Corneal Stromal Cells Using an Adenoviral Vector and Keratocyte-Specific Promoter". Investigative Ophthalmology & Visual Science, July 2004, Vol. 45, 2194-2200); a corneal epithelial / limbal stem cell-specific promoter, such as a keratin-12 or Pax-6 promoter (Wang, I. et al., "Cis-regulatory elements of the mouse Krt1.12 gene". Molecular Vision, 2003). Vision), 8, 94-101 (2002); M. Yoshihara, M. et al. "High-resolution promoter map of human limbal epithelial cells cultured with keratinocyte growth factor and rho kinase inhibitor".Sci Rep 7, 2845 (2017); trabecular meshwork cell-specific promoters, such as the chitinase 3-like 1 promoter (Liton PB et al., "Specific targeting of gene expression to a subset of human trabecular meshwork cells using the chitinase 3-like 1 promoter". Ophthalmic Research and Visual Science 2005 Jan; 46(1): 183-90); or iridophore cell-specific promoters, such as the phosphodiesterase 11A promoter (Janssen SF et al., "Gene expression and functional annotation of the human ciliary body epithelia". PLoS One. 2012; 7(9): e44973). .

[0176] In some embodiments, the promoter is a CAG promoter. In some embodiments, the promoter comprises a nucleotide sequence having at least 90%, 95%, 96%, 97%, 98% or 99% identity to the nucleotide sequence set forth in SEQ ID NO:5. In some embodiments, the promoter comprises SEQ ID NO:5.

[0177] In some embodiments, the promoter is a CMV promoter. In some embodiments, the promoter comprises a nucleotide sequence that is at least 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 17. In some embodiments, the promoter comprises or consists of SEQ ID NO: 17.

[0178] In some embodiments, the enhancer is a CMV enhancer. In some embodiments, the CMV enhancer comprises a nucleotide sequence that is at least 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 18. In some embodiments, the promoter comprises or consists of SEQ ID NO: 18.

[0179] In some embodiments, the expression cassette comprises a promoter and an enhancer. In some embodiments, the CMV enhancer and promoter comprise a nucleotide sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 19. In some embodiments, the enhancer and promoter comprise or consist of SEQ ID NO: 19.

[0180] The expression cassette may contain a polyadenylation (poly A) sequence. In some embodiments, the expression cassette described herein comprises a transcription termination signal. The elements that guide the effective termination and polyadenylation of heterologous nucleic acid transcripts increase heterologous gene expression. The transcription termination signal is generally found downstream of the polyadenylation signal. In some embodiments, the vector comprises a polyadenylation sequence of the polynucleotide 3' encoding the polypeptide to be expressed. As used herein, the term "poly A site" or "poly A sequence" means a DNA sequence that guides the termination and polyadenylation of nascent RNA transcripts by RNA polymerase II. The polyadenylation sequence can promote mRNA stability by adding a poly A tail at the 3' end of the coding sequence, and thus contributes to an increase in translation efficiency. Cleavage and polyadenylation are guided by the poly (A) sequence in RNA. The core poly (A) sequence of mammalian pre-mRMA has two distinguishing elements flanking the cleavage-polyadenylation site. Typically, the almost invariant AAUAAA hexamer is located 20-50 nucleotides upstream of the more variable element rich in U or GU residues. Cleavage of the nascent transcript occurs between these two elements and is coupled to the addition of up to 250 adenosines to the 5' of the cleavage product. In particular embodiments, the core poly(A) sequence is an ideal poly(A) sequence (e.g., AATAAA, ATTAAA, AGTAAA). In particular embodiments, the poly(A) sequence is an SV40 poly(A) sequence, a bovine growth hormone poly(A) sequence (BGH poly(A), a rabbit β-globulin poly(A) sequence (rβgpA), a variant thereof, or another suitable heterologous or endogenous poly(A) sequence known in the art. In some embodiments, the expression cassette described herein comprises a poly(A) sequence. In some embodiments, the poly(A) sequence is a BGH poly(A) sequence. In some embodiments, the BGH poly(A) sequence comprises or consists of a nucleotide sequence having at least 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 20. In some embodiments, the BGH poly(A) sequence comprises or consists of SEQ ID NO: 20.

[0181] In some embodiments, the rAAV vectors of the present disclosure comprise a woodchuck post-transcriptional regulatory element (WPRE). In some embodiments, the rAAV vectors comprise a WPRE comprising SEQ ID NO: 21.

[0182] Recombinant AAV vector

[0183] In some embodiments, the subject expression cassette is used to deliver an oxidoreductase (e.g., TRX or PDI) or a functional variant thereof to at least one eye and / or lacrimal gland of an individual, for example, to treat an ocular disorder. In some embodiments, the subject expression cassette is used to deliver an oxidoreductase (e.g., TRX or PDI) or a functional variant thereof to at least one meibomian gland of an individual, for example, to treat an ocular disorder. Thus, in some embodiments, the composition that provides expression of an oxidoreductase (e.g., TRX or PDI) or a functional variant thereof in at least one eye and / or lacrimal gland of an individual is a gene delivery vector, wherein the gene delivery vector comprises an expression cassette described herein.

[0184] In some embodiments, the gene delivery vector is a rAAV vector (e.g., rAAV virions). In some embodiments, the expression cassette is flanked by functional AAV inverted terminal repeat (ITR) sequences at the 5' and 3' ends. "Functional AAV ITR sequence" means that the ITR sequence functions as intended for the recovery, replication, and packaging of the AAV vector. Therefore, the AAV ITR for the gene delivery vector of the present disclosure does not have to have a wild-type nucleotide sequence, and can be changed by inserting, deleting, or replacing nucleotides, or the AAV ITR can be derived from any of several AAV serotypes, such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10. In some embodiments, AAV ITR is derived from AAV1. In some embodiments, AAVITR is derived from AAV2. In some embodiments, AAV ITR is derived from AAV3. In some embodiments, AAV ITR is derived from AAV4. In some embodiments, AAV ITR is derived from AAV5. In some embodiments, AAV ITR is derived from AAV6. In some embodiments, AAV ITR is derived from AAV7. In some embodiments, the AAV ITR is derived from AAV9. In some embodiments, the AAV ITR is derived from AAV10. In some embodiments, the rAAV vector lacks all or part of the wild-type REP and CAP genes, but retains functional flanking ITR sequences. In some embodiments, the AAV ITR is an AAV ITR identified in Table 4.

[0185] Table 4. Exemplary ITR sequences

[0186]

[0187] In some embodiments, the rAAV vector comprises an AAV shell derived from any adeno-associated virus serotype known or expected to be found in the art, including but not limited to: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, etc. For example, the AAV shell may be a wild-type (or "native") shell. In some embodiments, the rAAV vector comprises an AAV shell derived from AAV1. In some embodiments, the rAAV vector comprises an AAV shell derived from AAV2. In some embodiments, the rAAV vector comprises an AAV shell derived from AAV3. In some embodiments, the rAAV vector comprises an AAV shell derived from AAV4. In some embodiments, the rAAV vector comprises an AAV shell derived from AAV5. In some embodiments, the rAAV vector comprises an AAV shell derived from AAV6. In some embodiments, the rAAV vector comprises an AAV shell derived from AAV7. In some embodiments, the rAAV vector comprises an AAV shell derived from AAV8. In some embodiments, the rAAV vector comprises an AAV shell derived from AAV9. In some embodiments, the rAAV vector comprises an AAV shell derived from AAV10. The AAV shells that have received much attention include AAV2, AAV5, AAV8 and AAV9 (Table 5). However, like ITR, the shell does not have to have a wild-type nucleotide sequence, but can be changed by the insertion, deletion or substitution of nucleotides in the VP1, VP2 or VP3 sequence, as long as the shell can transduce the cells of the eye and / or lacrimal gland. In other words, the AAV shell can be a variant AAV shell. In some embodiments, the rAAV vector is a "pseudotyped" AAV, which is produced by using a shell (cap) gene of an AAV and rep genes and ITRs from different AAVs, such as by using rep from AAV2 and cap from AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8 or AAV9, and pseudotyped AAV2 produced by a plasmid containing an AAV2-based vector. For example, the rAAV vector can be rAAV2 / 1, rAAV2 / 3, rAAV2 / 4, rAAV2 / 5, rAAV2 / 6, rAAV2 / 7, rAAV2 / 8, rAAV2 / 9, etc. In some embodiments, the rAAV is rAAV2 / 1. In some embodiments, the rAAV is rAAV2 / 3. In some embodiments, the rAAV is rAAV2 / 4. In some embodiments, the rAAV is rAAV2 / 5. In some embodiments, the rAAV is rAAV2 / 6. In some embodiments, the rAAV is rAAV2 / 7. In some embodiments, the rAAV is rAAV2 / 8. In some embodiments, the rAAV is rAAV2 / 9.

[0188] In some embodiments, rAAV is replication-defective because the rAAV vector is unable to further replicate and package its genome independently. For example, when the eye and / or lacrimal gland is transduced with a rAAV vector, the genes are expressed in the transduced eye and / or lacrimal gland, however, because the transduced eye and / or lacrimal gland lacks the AAV rep and cap genes and the accessory function genes, the rAAV is unable to replicate.

[0189] In some embodiments, the rAAV vectors of the present disclosure encapsulating expression cassettes as described herein can be manufactured using non-assisted production. rAAV is a replication-deficient virus and generally requires components from a live helper virus (e.g., adenovirus) in host cells for encapsulating infectious rAAV vectors. The rAAV non-assisted production system allows the manufacture of infectious rAAV vectors without the use of live helper viruses. In the non-assisted system, the host encapsulation cell line is co-transfected with three plasmids. The first plasmid contains adenoviral gene products (i.e., E2A, E4, and VA RNA genes) required for encapsulating rAAV vectors. The second plasmid contains the required AAV genes (i.e., REP and CAP genes). The third plasmid contains a polynucleotide sequence encoding an oxidoreductase (e.g., TRX or PDI) or a functional variant thereof, and a promoter flanking ITR. The host encapsulation cell line may, for example, be an AAV-293 host cell. Suitable host cells contain additional components required for encapsulating infectious rAAV vectors that are not supplied by plasmids. In some embodiments, the CAP gene may encode an AAV capsid protein, such as described herein. In some embodiments, the promoter is a promoter sequence as described herein. In some embodiments, the promoter sequence is a CAG sequence. In some embodiments, the polynucleotide encodes a protein that has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 1. In some embodiments, the polynucleotide encodes a protein that has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 25. In some embodiments, the polynucleotide comprises a sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 2.

[0190] AAV serotypes shown to infect the eye and / or lacrimal gland include AAV2, AAV5, AAV 5w8, and AAV9 (Rocha et al., supra). Exemplary amino acid and nucleotide sequences of AAV capsid proteins are identified in Table 5.

[0191] In some embodiments, the AAV capsid protein shares at least 95%, 98% or 100% identity with the AAV2 VP1 protein (SEQ ID NO: 6). In some embodiments, the polynucleotide sequence encoding the AAV2 VP1 protein shares at least 95%, 98% or 100% identity with SEQ ID NO: 7. In some embodiments, the AAV capsid protein shares at least 95%, 98% or 100% identity with the AAV2 VP3 protein (SEQ ID NO: 8). In some embodiments, the polynucleotide sequence encoding the AAV2 VP3 protein shares at least 95%, 98% or 100% identity with SEQ ID NO: 9. In some embodiments, the AAV capsid protein shares at least 95%, 98% or 100% identity with the AAV5 capsid protein (SEQ ID NO: 10). In some embodiments, the polynucleotide sequence encoding the AAV5 capsid protein shares at least 95%, 98% or 100% identity with SEQ ID NO: 11. In some embodiments, the AAV capsid protein shares at least 95%, 98% or 100% identity with the AAV8 capsid protein (SEQ ID NO: 12). In some embodiments, the polynucleotide sequence encoding the AAV8 capsid protein shares at least 95%, 98% or 100% identity with SEQ ID NO: 13. In some embodiments, the AAV capsid protein shares at least 95%, 98% or 100% identity with the AAV9 capsid protein (SEQ ID NO: 14). In some embodiments, the polynucleotide sequence encoding the AAV9 capsid protein shares at least 95%, 98% or 100% identity with SEQ ID NO: 15.

[0192] Table 5: AAV capsid sequences

[0193] Shell sequence protein Nucleotide (open reading frame) AAV2 VP1 SEQ ID NO:6 SEQ ID NO:7 AAV2 VP3 SEQ ID NO:8 SEQ ID NO:9 AAV5 SEQ ID NO:10 SEQ ID NO:11 AAV8 SEQ ID NO:12 SEQ ID NO:13 AAV9 SEQ ID NO:14 SEQ ID NO:15

[0194] Exemplary rAAV Vectors

[0195] In some embodiments, the rAAV vector comprises an AAV shell. In some embodiments, the rAAV vector described herein comprises an expression cassette containing a polynucleotide comprising a nucleotide sequence encoding TRX. In some embodiments, the rAAV vector described herein comprises an expression cassette containing a polynucleotide comprising a nucleotide sequence encoding PDI. In some embodiments, the rAAV vector described herein comprises a bicistronic expression cassette containing a polynucleotide comprising a nucleotide sequence encoding both TRX and PDI. In some embodiments, the polynucleotide is operably linked to a promoter. In some embodiments, the rAAV vector comprises an AAV shell and an expression cassette, wherein the expression cassette comprises a polynucleotide operably linked to a promoter operably linked to a promoter, wherein the polynucleotide comprises a nucleotide sequence encoding TRX. In some embodiments, the rAAV vector comprises an AAV shell and an expression cassette, wherein the expression cassette comprises a polynucleotide operably linked to a promoter, wherein the polynucleotide comprises a nucleotide sequence encoding PDI. In some embodiments, the promoter is any promoter described herein or known in the art. In some embodiments, the promoter is a CAG promoter. In some embodiments, the promoter is a CMV promoter. In some embodiments, the expression cassette further comprises a 5′ITR and / or a 3′ITR. In some embodiments, the 5′ITR is an AAV2 5′ITR. In some embodiments, the 3′ITR is an AAV2 3′ITR. In some embodiments, the expression cassette comprises a nucleotide sequence comprising, from 5′ to 3′, a 5′ITR (e.g., an AAV2 5′ITR), a promoter (e.g., a CMV promoter), a 5′ untranslated region (5′UTR), a polynucleotide sequence comprising a nucleotide sequence encoding a TRX enzyme, a poly A sequence, and a 3′ITR (e.g., an AAV2 3′ITR). In some embodiments, the expression cassette comprises a nucleotide sequence comprising, from 5′ to 3′, a 5′ITR (e.g., an AAV2 5′ITR), an enhancer (e.g., a CMV enhancer), a promoter (e.g., a CMV promoter), a 5′UTR, a polynucleotide sequence comprising a nucleotide sequence encoding a TRX enzyme, a WPRE sequence, a poly A sequence, and a 3′ITR (e.g., an AAV2 3′ITR). In some embodiments, the 5′UTR comprises a Kozak sequence located immediately upstream of the nucleotide sequence encoding the TRX enzyme and adjacent to the start codon therein.

[0196] In some embodiments, the rAAV vector comprises an AAV shell and an expression cassette comprising a polynucleotide operably linked to a promoter, wherein the polynucleotide comprises a nucleotide sequence encoding a human TRX enzyme described herein. In some embodiments, the rAAV vector comprises an AAV shell and an expression cassette comprising a polynucleotide operably linked to a CAG promoter or a CMV promoter, wherein the polynucleotide comprises a nucleotide sequence encoding a TRX enzyme described herein. In some embodiments, the polynucleotide comprises from 5′ to 3′: (i) a nucleotide sequence encoding a signal peptide, and (ii) a nucleotide sequence encoding a TRX enzyme described herein.

[0197] In some embodiments, the rAAV vector comprises an AAV capsid and an expression cassette comprising a polynucleotide operably linked to a promoter (e.g., a CAG promoter or a CMV promoter), wherein the polynucleotide comprises, from 5′ to 3′: (i) a nucleotide sequence encoding a signal peptide, and (ii) a nucleotide sequence encoding a TRX enzyme, wherein the TRX enzyme comprises or consists of an amino acid sequence that shares at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 1. In some embodiments, the TRX enzyme comprises or consists of the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the rAAV vector comprises an AAV capsid and an expression cassette comprising a polynucleotide operably linked to a promoter (e.g., a CAG promoter or a CMV promoter), wherein the polynucleotide comprises, from 5′ to 3′: (i) a nucleotide sequence encoding a signal peptide, and (ii) a nucleotide sequence encoding a TRX enzyme, wherein the TRX enzyme comprises or consists of an amino acid sequence that shares at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 25. In some embodiments, the TRX enzyme comprises or consists of the amino acid sequence set forth in SEQ ID NO: 25. In some embodiments, the rAAV vector comprises an AAV capsid and an expression cassette comprising a polynucleotide operably linked to a promoter (e.g., a CAG promoter), wherein the polynucleotide comprises, from 5′ to 3′: (i) a nucleotide sequence encoding a signal peptide, and (ii) a nucleotide sequence encoding a TRX enzyme, wherein the nucleotide sequence comprises a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 2 or SEQ ID NO: 28. In some embodiments, the nucleotide sequence comprises SEQ ID NO: 2. In some embodiments, the nucleotide sequence comprises SEQ ID NO: 28.

[0198] In some embodiments, the rAAV vector comprises an AAV capsid and an expression cassette comprising a polynucleotide operably linked to a promoter (e.g., a CAG promoter or a CMV promoter), wherein the polynucleotide comprises, from 5′ to 3′: (i) a nucleotide sequence encoding a signal peptide, and (ii) a nucleotide sequence encoding a TRX enzyme, wherein the TRX enzyme comprises or consists of an amino acid sequence that shares at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 25. In some embodiments, the TRX enzyme comprises or consists of the amino acid sequence set forth in SEQ ID NO: 25.

[0199] In some embodiments, the rAAV vector comprises an AAV2 capsid and an expression cassette comprising a polynucleotide operably linked to a promoter, wherein the polynucleotide comprises a nucleotide sequence encoding a human TRX enzyme described herein. In some embodiments, the rAAV vector comprises an AAV2 capsid and an expression cassette comprising a polynucleotide operably linked to a CAG promoter or a CMV promoter, wherein the polynucleotide comprises, from 5′ to 3′: (i) a nucleotide sequence encoding a signal peptide, and (ii) a nucleotide sequence encoding a TRX enzyme described herein.

[0200] In some embodiments, the rAAV vector comprises an AAV2 capsid and an expression cassette comprising a polynucleotide operably linked to a promoter (e.g., a CAG promoter or a CMV promoter), wherein the polynucleotide comprises, from 5′ to 3′: (i) a nucleotide sequence encoding a signal peptide, and (ii) a nucleotide sequence encoding a TRX enzyme, wherein the TRX enzyme comprises or consists of an amino acid sequence that shares at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 1. In some embodiments, the TRX enzyme comprises or consists of the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the rAAV vector comprises an AAV2 capsid and an expression cassette comprising a polynucleotide operably linked to a promoter (e.g., a CAG promoter or a CMV promoter), wherein the polynucleotide comprises, from 5′ to 3′: (i) a nucleotide sequence encoding a signal peptide, and (ii) a nucleotide sequence encoding a TRX enzyme, wherein the TRX enzyme comprises or consists of an amino acid sequence that shares at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 25. In some embodiments, the TRX enzyme comprises or consists of the amino acid sequence set forth in SEQ ID NO: 25. In some embodiments, the rAAV vector comprises an AAV2 capsid and an expression cassette comprising a polynucleotide operably linked to a promoter (e.g., a CAG promoter or a CMV promoter), wherein the polynucleotide comprises, from 5′ to 3′: (i) a nucleotide sequence encoding a signal peptide, and (ii) a nucleotide encoding a TRX enzyme, wherein the nucleotide sequence comprises a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 2 or SEQ ID NO: 28. In some embodiments, the nucleotide sequence comprises SEQ ID NO: 2. In some embodiments, the nucleotide sequence comprises SEQ ID NO: 28.

[0201] In some embodiments, the rAAV vector comprises an AAV5 capsid and an expression cassette comprising a polynucleotide operably linked to a promoter, wherein the polynucleotide comprises from 5′ to 3′: (i) a nucleotide sequence encoding a signal peptide, and (ii) a nucleotide sequence encoding a human TRX enzyme described herein. In some embodiments, the rAAV vector comprises an AAV5 capsid and an expression cassette comprising a polynucleotide operably linked to a CAG promoter, wherein the polynucleotide comprises from 5′ to 3′: (i) a nucleotide sequence encoding a signal peptide, and (ii) a nucleotide sequence encoding a TRX enzyme described herein.

[0202] In some embodiments, the rAAV vector comprises an AAV5 capsid and an expression cassette comprising a polynucleotide operably linked to a promoter (e.g., a CAG promoter or a CMV promoter), wherein the polynucleotide comprises, from 5′ to 3′: (i) a nucleotide sequence encoding a signal peptide, and (ii) a nucleotide sequence encoding a TRX enzyme, wherein the TRX enzyme comprises or consists of an amino acid sequence that shares at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 1. In some embodiments, the TRX enzyme comprises the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the rAAV vector comprises an AAV5 capsid and an expression cassette comprising a polynucleotide operably linked to a promoter (e.g., a CAG promoter or a CMV promoter), wherein the polynucleotide comprises, from 5′ to 3′: (i) a nucleotide sequence encoding a signal peptide, and (ii) a nucleotide sequence encoding a TRX enzyme, wherein the TRX enzyme comprises or consists of an amino acid sequence that shares at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 25. In some embodiments, the TRX enzyme comprises the amino acid sequence set forth in SEQ ID NO: 25. In some embodiments, the rAAV vector comprises an AAV5 capsid and an expression cassette comprising a polynucleotide operably linked to a promoter (e.g., a CAG promoter or a CMV promoter), wherein the polynucleotide comprises, from 5′ to 3′: (i) a nucleotide sequence encoding a signal peptide, and (ii) a nucleotide encoding a TRX enzyme, wherein the nucleotide sequence comprises a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 2 or SEQ ID NO: 28. In some embodiments, the nucleotide sequence comprises SEQ ID NO: 2. In some embodiments, the nucleotide sequence comprises SEQ ID NO: 28.

[0203] In some embodiments, the rAAV vector comprises an AAV9 capsid and an expression cassette comprising a polynucleotide operably linked to a promoter, wherein the polynucleotide comprises from 5′ to 3′: (i) a nucleotide sequence encoding a signal peptide, and (ii) a nucleotide sequence encoding a human TRX enzyme described herein. In some embodiments, the rAAV vector comprises an AAV9 capsid and an expression cassette comprising a polynucleotide operably linked to a CAG promoter, wherein the polynucleotide comprises from 5′ to 3′: (i) a nucleotide sequence encoding a signal peptide, and (ii) a nucleotide sequence encoding a TRX enzyme described herein.

[0204] In some embodiments, the rAAV vector comprises an AAV9 capsid and an expression cassette comprising a polynucleotide operably linked to a promoter (e.g., a CAG promoter or a CMV promoter), wherein the polynucleotide comprises, from 5′ to 3′: (i) a nucleotide sequence encoding a signal peptide, and (ii) a nucleotide sequence encoding a TRX enzyme, wherein the TRX enzyme comprises an amino acid sequence that shares at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 1. In some embodiments, the TRX enzyme comprises the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the rAAV vector comprises an AAV9 capsid and an expression cassette comprising a polynucleotide operably linked to a promoter (e.g., a CAG promoter or a CMV promoter), wherein the polynucleotide comprises, from 5′ to 3′: (i) a nucleotide sequence encoding a signal peptide, and (ii) a nucleotide sequence encoding a TRX enzyme, wherein the TRX enzyme comprises an amino acid sequence that shares at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 25. In some embodiments, the TRX enzyme comprises the amino acid sequence set forth in SEQ ID NO: 25. In some embodiments, the rAAV vector comprises an AAV9 capsid and an expression cassette comprising a polynucleotide operably linked to a promoter (e.g., a CAG promoter or a CMV promoter), wherein the polynucleotide comprises, from 5′ to 3′: (i) a nucleotide sequence encoding a signal peptide, and (ii) a nucleotide encoding a TRX enzyme, wherein the nucleotide sequence comprises a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 2 or SEQ ID NO: 28. In some embodiments, the nucleotide sequence comprises SEQ ID NO: 2. In some embodiments, the nucleotide sequence comprises SEQ ID NO: 28.

[0205] In some embodiments, the promoter is any promoter described herein or known in the art. In some embodiments, the promoter is a CAG promoter. In some embodiments, the promoter is a CMV promoter. In some embodiments, the expression cassette further comprises a 5′ITR and / or a 3′ITR. In some embodiments, the 5′ITR is an AAV2 5′ITR. In some embodiments, the 3′ITR is an AAV2 3′ITR. In some embodiments, the expression cassette comprises the following nucleotide sequence, which comprises from 5′ to 3′: a 5′ITR (e.g., an AAV2 5′ITR), a promoter (e.g., a CMV promoter), a 5′ untranslated region (5′UTR), a polynucleotide sequence comprising a nucleotide sequence encoding PDI, a poly A sequence, and a 3′ITR (e.g., AAV2 3′ITR). In some embodiments, the expression cassette comprises a nucleotide sequence comprising, from 5′ to 3′, a 5′ ITR (e.g., AAV2 5′ ITR), an enhancer (e.g., a CMV enhancer), a promoter (e.g., a CMV promoter), a 5′ UTR, a polynucleotide sequence comprising a nucleotide sequence encoding PDI, a WPRE sequence, a poly A sequence, and a 3′ ITR (e.g., AAV2 3′ ITR). In some embodiments, the 5′ UTR comprises a Kozak sequence located immediately upstream of the nucleotide sequence encoding the PDI enzyme and adjacent to the start codon therein.

[0206] In some embodiments, the rAAV vector comprises an AAV capsid and an expression cassette comprising a polynucleotide operably linked to a promoter, wherein the polynucleotide comprises a nucleotide sequence encoding a human PDI described herein. In some embodiments, the rAAV vector comprises an AAV capsid and an expression cassette comprising a polynucleotide operably linked to a CAG promoter or a CMV promoter, wherein the polynucleotide comprises a nucleotide sequence encoding a PDI described herein.

[0207] In some embodiments, the rAAV vector comprises an AAV capsid and an expression cassette comprising a polynucleotide operably linked to a promoter (e.g., a CAG promoter or a CMV promoter), wherein the polynucleotide comprises a nucleotide sequence encoding PDI, wherein PDI comprises or consists of an amino acid sequence that shares at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 26. In some embodiments, PDI comprises or consists of the amino acid sequence set forth in SEQ ID NO: 26. In some embodiments, the rAAV vector comprises an AAV capsid and an expression cassette comprising a polynucleotide operably linked to a promoter (e.g., a CAG promoter), wherein the polynucleotide comprises a nucleotide sequence encoding PDI, wherein the nucleotide sequence comprises a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 30. In some embodiments, the nucleotide sequence comprises SEQ ID NO: 30.

[0208] In some embodiments, the rAAV vector comprises an AAV capsid and an expression cassette comprising a polynucleotide operably linked to a promoter (e.g., a CAG promoter or a CMV promoter), wherein the polynucleotide comprises a nucleotide sequence encoding PDI, wherein PDI comprises or consists of an amino acid sequence that shares at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 26. In some embodiments, PDI comprises or consists of the amino acid sequence set forth in SEQ ID NO: 26.

[0209] In some embodiments, the rAAV vector comprises an AAV2 capsid and an expression cassette comprising a polynucleotide operably linked to a promoter, wherein the polynucleotide comprises a nucleotide sequence encoding a human PDI described herein. In some embodiments, the rAAV vector comprises an AAV2 capsid and an expression cassette comprising a polynucleotide operably linked to a CAG promoter or a CMV promoter, wherein the polynucleotide comprises a nucleotide sequence encoding a PDI described herein.

[0210] In some embodiments, the rAAV vector comprises an AAV2 capsid and an expression cassette comprising a polynucleotide operably linked to a promoter (e.g., a CAG promoter or a CMV promoter), wherein the polynucleotide comprises a nucleotide sequence encoding PDI, wherein PDI comprises or consists of an amino acid sequence that shares at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 26. In some embodiments, PDI comprises or consists of the amino acid sequence set forth in SEQ ID NO: 26. In some embodiments, the rAAV vector comprises an AAV2 capsid and an expression cassette comprising a polynucleotide operably linked to a promoter (e.g., a CAG promoter or a CMV promoter), wherein the polynucleotide comprises nucleotides encoding PDI, wherein the nucleotide sequence comprises a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 30. In some embodiments, the nucleotide sequence comprises SEQ ID NO: 30.

[0211] In some embodiments, the rAAV vector comprises an AAV5 capsid and an expression cassette comprising a polynucleotide operably linked to a promoter, wherein the polynucleotide comprises a nucleotide sequence encoding a human PDI described herein. In some embodiments, the rAAV vector comprises an AAV5 capsid and an expression cassette comprising a polynucleotide operably linked to a CAG promoter, wherein the polynucleotide comprises a nucleotide sequence encoding a PDI described herein.

[0212] In some embodiments, the rAAV vector comprises an AAV5 capsid and an expression cassette comprising a polynucleotide operably linked to a promoter (e.g., a CAG promoter or a CMV promoter), wherein the polynucleotide comprises a nucleotide sequence encoding PDI, wherein PDI comprises or consists of an amino acid sequence that shares at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 26. In some embodiments, PDI comprises the amino acid sequence set forth in SEQ ID NO: 26. In some embodiments, the rAAV vector comprises an AAV5 capsid and an expression cassette comprising a polynucleotide operably linked to a promoter (e.g., a CAG promoter or a CMV promoter), wherein the polynucleotide comprises a nucleotide encoding PDI, wherein the nucleotide sequence comprises a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 30. In some embodiments, the nucleotide sequence comprises SEQ ID NO: 30.

[0213] In some embodiments, the rAAV vector comprises an AAV9 capsid and an expression cassette comprising a polynucleotide operably linked to a promoter, wherein the polynucleotide comprises a nucleotide sequence encoding a human PDI described herein. In some embodiments, the rAAV vector comprises an AAV9 capsid and an expression cassette comprising a polynucleotide operably linked to a CAG promoter, wherein the polynucleotide comprises a nucleotide sequence encoding a PDI described herein.

[0214] In some embodiments, the rAAV vector comprises an AAV9 capsid and an expression cassette comprising a polynucleotide operably linked to a promoter (e.g., a CAG promoter or a CMV promoter), wherein the polynucleotide comprises a nucleotide sequence encoding PDI, wherein PDI comprises an amino acid sequence that shares at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 26. In some embodiments, PDI comprises the amino acid sequence set forth in SEQ ID NO: 26. In some embodiments, the rAAV vector comprises an AAV9 capsid and an expression cassette comprising a polynucleotide operably linked to a promoter (e.g., a CAG promoter or a CMV promoter), wherein the polynucleotide comprises a nucleotide encoding PDI, wherein the nucleotide sequence comprises a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 30. In some embodiments, the nucleotide sequence comprises SEQ ID NO: 30.

[0215] In some embodiments, the rAAV vector comprises an AAV capsid and an expression cassette comprising a polynucleotide operably linked to a promoter (e.g., a CAG promoter or a CMV promoter), wherein the polynucleotide comprises, from 5′ to 3′: (i) a nucleotide sequence encoding a signal peptide, and (ii) a nucleotide sequence encoding PDI, wherein PDI comprises or consists of an amino acid sequence that shares at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 26. In some embodiments, PDI comprises or consists of the amino acid sequence set forth in SEQ ID NO: 26. In some embodiments, the rAAV vector comprises an AAV capsid and an expression cassette comprising a polynucleotide operably linked to a promoter (e.g., a CAG promoter), wherein the polynucleotide comprises, from 5′ to 3′: (i) a nucleotide sequence encoding a signal peptide, and (ii) a nucleotide sequence encoding PDI, wherein the nucleotide sequence comprises a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 30. In some embodiments, the nucleotide sequence comprises SEQ ID NO: 30.

[0216] In some embodiments, the rAAV vector comprises an AAV capsid and an expression cassette comprising a polynucleotide operably linked to a promoter (e.g., a CAG promoter or a CMV promoter), wherein the polynucleotide comprises, from 5′ to 3′: (i) a nucleotide sequence encoding a signal peptide, and (ii) a nucleotide sequence encoding PDI, wherein PDI comprises or consists of an amino acid sequence that shares at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 26. In some embodiments, PDI comprises or consists of the amino acid sequence set forth in SEQ ID NO: 26.

[0217] In some embodiments, the rAAV vector comprises an AAV2 capsid and an expression cassette comprising a polynucleotide operably linked to a promoter (e.g., a CAG promoter or a CMV promoter), wherein the polynucleotide comprises, from 5′ to 3′: (i) a nucleotide sequence encoding a signal peptide, and (ii) a nucleotide sequence encoding PDI, wherein PDI comprises or consists of an amino acid sequence that shares at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 26. In some embodiments, PDI comprises or consists of the amino acid sequence set forth in SEQ ID NO: 26. In some embodiments, the rAAV vector comprises an AAV2 capsid and an expression cassette comprising a polynucleotide operably linked to a promoter (e.g., a CAG promoter or a CMV promoter), wherein the polynucleotide comprises nucleotides encoding PDI, wherein the nucleotide sequence comprises a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 30. In some embodiments, the nucleotide sequence comprises SEQ ID NO: 30.

[0218] In some embodiments, the rAAV vector comprises an AAV5 capsid and an expression cassette comprising a polynucleotide operably linked to a promoter (e.g., a CAG promoter or a CMV promoter), wherein the polynucleotide comprises, from 5′ to 3′: (i) a nucleotide sequence encoding a signal peptide, and (ii) a nucleotide sequence encoding PDI, wherein PDI comprises or consists of an amino acid sequence that shares at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 26. In some embodiments, PDI comprises the amino acid sequence set forth in SEQ ID NO: 26. In some embodiments, the rAAV vector comprises an AAV5 capsid and an expression cassette comprising a polynucleotide operably linked to a promoter (e.g., a CAG promoter or a CMV promoter), wherein the polynucleotide comprises a nucleotide encoding PDI, wherein the nucleotide sequence comprises a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 30. In some embodiments, the nucleotide sequence comprises SEQ ID NO: 30.

[0219] In some embodiments, the rAAV vector comprises an AAV9 capsid and an expression cassette comprising a polynucleotide operably linked to a promoter (e.g., a CAG promoter or a CMV promoter), wherein the polynucleotide comprises, from 5′ to 3′: (i) a nucleotide sequence encoding a signal peptide, and (ii) a nucleotide sequence encoding PDI, wherein PDI comprises an amino acid sequence that shares at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 26. In some embodiments, PDI comprises the amino acid sequence set forth in SEQ ID NO: 26. In some embodiments, the rAAV vector comprises an AAV9 capsid and an expression cassette comprising a polynucleotide operably linked to a promoter (e.g., a CAG promoter or a CMV promoter), wherein the polynucleotide comprises a nucleotide encoding PDI, wherein the nucleotide sequence comprises a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 30. In some embodiments, the nucleotide sequence comprises SEQ ID NO: 30.

[0220] How to use

[0221] The methods and compositions described herein can be used to treat ocular conditions and reduce symptoms associated with ocular conditions. The terms "treatment", "treating" and similar terms are generally used herein to refer to obtaining a desired pharmacological and / or physiological effect. The effect may be preventive in terms of completely or partially preventing a disease or its symptoms, such as reducing the likelihood of an individual suffering from a disease or its symptoms, delaying the onset or progression of a disease or symptom in an individual; and / or may be therapeutic in terms of partially or completely curing a disease and / or adverse effects attributable to a disease. As used herein, "treatment" encompasses any treatment of a disease in a mammal, and includes, but is not limited to: (a) inhibiting the progression of a disease; (b) alleviating or reducing an increase in one or more symptoms of a disease; (c) alleviating or reducing an increase in one or more signs of a disease; (d) causing regression of a disease. The therapeutic agent may be administered before, during or after the onset of a disease or injury. Treatment of an ongoing disease is of particular interest, where the treatment stabilizes or reduces undesirable clinical symptoms in a patient. Such treatment is ideally performed before the function of the affected tissue is completely lost. The subject therapies are ideally administered during, and in some cases after, the symptomatic stage of disease.

[0222] As used herein, "administer," "administering," "administration," and the like refer to providing a substance (eg, a rAAV vector) to a subject in a pharmacologically suitable manner (eg, to treat a disease, disorder, or condition in the subject).

[0223] Therefore, in some embodiments, provided herein is a method for treating an eye condition of an individual, the method comprising administering to the individual a rAAV vector provided herein or a pharmaceutical composition provided herein. As in this article, the terms "individual (subject)" and "patient" are used interchangeably to refer to mammals, including but not limited to humans and non-human primates, including apes and humans; mammalian sports animals (e.g., horses); mammalian farm animals (e.g., sheep, goats, etc.); mammalian pets (dogs, cats, etc.); and rodents (e.g., mice, rats, etc.). An individual may suffer from any of the symptoms or eye conditions described herein, for example.

[0224] In some embodiments, the subject is an adult, such as an adult over the age of 40 or an adult between the ages of 45 and 55. In some embodiments, the subject is an adult over the age of 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90 years old.

[0225] In some embodiments, the ocular condition treated according to the methods described herein is a disease associated with increased oxidative stress. In some embodiments, the condition is associated with the loss of expression or function of one or more oxidoreductases. In some embodiments, the condition is associated with the loss of expression and / or function of protein disulfide isomerase (PDI). In some embodiments, the condition is associated with the loss of expression and / or function of TRX. Without wishing to be bound by theory, it is assumed that increased expression of TRX, PDI, and / or other oxidoreductases can reduce oxidative stress in tissues and thus slow the aging process and delay the onset of conditions associated with or caused by aging. In some embodiments, the condition is associated with near vision loss, such as the gradual loss of the ability of the eye to focus on nearby targets. In some embodiments, the condition is cataract formation. In some embodiments, the condition is loss of visual accommodation. In some embodiments, the condition is presbyopia. In some embodiments, the condition is meibomian gland dysfunction (MGD). In some embodiments, the condition is high intraocular pressure. In some embodiments, the condition is glaucoma. In some embodiments, neuroprotection is provided by the methods described herein.

[0226] In some embodiments, the treatment methods described herein comprise further administering to the individual one or more additional therapeutic agents. In some embodiments, the additional therapeutic agent is an agent that increases tear production. In some embodiments, the additional therapeutic agent that increases tear production is a cholinergic agonist. In some embodiments, the other therapeutic agent is another AAV-based gene therapy construct.

[0227] Drugs for tear production

[0228] In some embodiments, the treatment to increase tear production comprises administering an effective amount of a nicotinic acetylcholine receptor (nAChR) agonist or a pharmaceutically acceptable salt thereof. In some embodiments, the nAChR agonist is an nAChR agonist described herein.

[0229] nAChR is a type of pentameric ligand-gated ion channel that has high affinity and selectivity for both nicotine and acetylcholine (which is similar to the protonated form of nicotine) and comprises a combination of α and β subunits. Examples of nAChR subtypes include, but are not limited to, α3β4, α4β2, α3α5β4, and α4α6β2.

[0230] nAChR agonists can be characterized as full or partial agonists, measured by their ability to activate a given receptor to produce a response compared to the response at a given receptor for acetylcholine (ACh). Generally, if an nAChR agonist induces a response equal to or greater than the response of ACh upon binding to a given receptor, it is a full agonist. If an nAChR agonist induces a response lower than the response produced by ACh upon binding to the receptor, it is a partial agonist.

[0231] nAChR agonist responses that can be measured for receptor activation can be generated, for example, using appropriate cell-based assays. Cells engineered to express specific nAChR receptor subtypes and produce current responses when bound to and activated by nAChR agonists can be used to characterize the agonist profile of a compound and thus measure the amount of receptor activation.

[0232] In some embodiments, the nAChR agonist or a pharmaceutically acceptable salt thereof selectively binds to at least one of the following nAChR subtypes: α3β4, α3α5β4, α4β2, and α4α6β2. As used herein, "selectively binds" or "selective for..." means that the compound has a higher affinity for the nAChR subtype, and / or has a lower half-maximal effective concentration (EC50) for the nAChr subtype of at least one reference nAChR subtype. Selectivity may be associated with at least a 5-fold affinity difference in EC50 values, at least a 10-fold affinity difference in EC50 values, at least a 20-fold affinity difference in EC50 values, or at least a 50-fold affinity difference in EC50 values. In some embodiments, the nAChR agonist or a pharmaceutically acceptable salt thereof selectively binds to the nAChR subtype α3β4. In some embodiments, the nAChR agonist or a pharmaceutically acceptable salt thereof selectively binds to the nAChR subtype α3α5β4. In some embodiments, the nAChR agonist or a pharmaceutically acceptable salt thereof selectively binds to nAChR subtype α4β2. In some embodiments, the nAChR agonist or a pharmaceutically acceptable salt thereof selectively binds to nAChR subtype α4α6β2. In some embodiments, the nAChR agonist or a pharmaceutically acceptable salt thereof selectively binds to nAChR subtype α7. In some embodiments, the nAChR agonist or a pharmaceutically acceptable salt thereof does not selectively bind to nAChR subtype α7.

[0233] nAChR agonists contemplated in the present disclosure include varenicline, a pharmaceutically acceptable salt thereof, and Compound 1 or a pharmaceutically acceptable salt thereof. In some embodiments, the nAChR agonist is not varenicline.

[0234] Varenicline is characterized as a full agonist of nAChR subtype α7, and a partial agonist of subtypes α3β4, α4β2, α6β2, α3α5β4, and α4α6β2. In some embodiments, the nAChR agonist is varenicline or a pharmaceutically acceptable salt thereof. Pharmaceutically acceptable salts of varenicline include varenicline tartrate. Additional relevant information about varenicline can be found, for example, in the following: U.S. Patent 6,951,938, U.S. Patent 6,890,927, U.S. Patent 7,265,119, U.S. Patent 9,504,644, U.S. Patent 9,504,645, U.S. Patent 9,532,944, U.S. Patent 9,597,284, U.S. Patent 10,456,386, U.S. Patent 11,224,598, and U.S. Application Publication 2022 / 0233528.

[0235] As described herein, compound 1 refers to the following structure:

[0236]

[0237] An alternative structural representation of compound 1 is shown here:

[0238]

[0239] Compound 1 may also be referred to by its chemical name. For example, Compound 1 is also referred to as (R)-5-((E)-2-pyrrolidin-3-ylvinyl)pyrimidine, or variants thereof, including simpanicline 5-{(E)-2-[(3R)-pyrrolidin-3-yl]vinyl}pyrimidine and (R,E)-5-((2-pyrrolidin-3-yl)vinyl)pyrimidine.

[0240] Compound 1 is a full agonist of nAChR subtypes α4β2, α3β4, α3α5β4, and α4α6β2. Compound 1 is a full agonist of nAChR subtypes α4β2 and α3β4.

[0241] Compound 1 is a partial agonist of subtype α3β2.

[0242] Compound 1 is a weak partial agonist of subtype α 7. In one example, a 300 micromolar concentration of Compound 1 citrate induced only 25% of the maximal ACh-evoked current.

[0243] In some embodiments, the nAChR agonist may be compound 1 or a pharmaceutically acceptable salt thereof. Pharmaceutically acceptable salts of compound 1 include galactarate (e.g., hemi-galactarate dihydrate) and citrate (e.g., monocitrate). Patent-related information about compound 1 can be found, for example, in the following: U.S. Patent 7,098,331, U.S. Patent 7,714,001, U.S. Patent 8,063,068, U.S. Patent 8,067,443, U.S. Patent 8,604,191, U.S. Patent 9,145,396, U.S. Patent 9,981,949, U.S. Patent 8,633,222, U.S. Patent 8,153,821, U.S. Patent 8,633,227, U.S. Patent 10,709,707, U.S. Patent Application Publication 2020-0345734, and PCT Publication WO 2017 / 177024.

[0244] In some embodiments, the nAChR agonist is (R)-5-((E)-2-pyrrolidin-3-ylvinyl)pyrimidine or a pharmaceutically acceptable salt thereof. In some embodiments, the nAChR agonist is (R)-5-((E)-2-pyrrolidin-3-ylvinyl)pyrimidine hemi-galactarate dihydrate. In some embodiments, the nAChR agonist is (R)-5-((E)-2-pyrrolidin-3-ylvinyl)pyrimidine monocitrate.

[0245] In some embodiments, nAChR agonists increase tear production in individuals in which tear secretion is impaired.

[0246] Treatment

[0247] In some embodiments, the present disclosure provides a method of treating an individual having an ocular disease or disorder, comprising administering to the individual a rAAV comprising nucleotides encoding human TRX described herein, wherein the expression of human TRX in the individual is increased compared to the expression of human TRX in an untreated individual or in the contralateral eye of the treated individual.

[0248] In some embodiments, the present disclosure provides a rAAV vector for use in a method of treating an ocular condition in an individual in need thereof, the method comprising administering a recombinant adeno-associated virus (rAAV) vector to at least one eye of the individual, at least one lacrimal gland of the eye of the individual, or at least one meibomian gland of the individual, the rAAV vector comprising an AAV capsid and an expression cassette comprising a polynucleotide encoding human TRX operably linked to a promoter. In some embodiments, the rAAV vector for use is administered to the lacrimal gland of the individual. In some embodiments, the rAAV vector for use comprises an expression cassette encoding human TRX.

[0249] In some embodiments, the present disclosure provides rAAV vectors for use or adaptable for use in treating an individual with an ocular disease, disorder or condition. In some embodiments, the rAAV vector for use or adaptable for use comprises an AAV capsid and an expression cassette comprising a polynucleotide encoding human TRX operably linked to a promoter.

[0250] In some embodiments, the present disclosure provides a rAAV vector for use in a method of treating an ocular condition in an individual in need thereof, the method comprising administering a recombinant adeno-associated viral (rAAV) vector described herein to at least one eye of the individual or to at least one lacrimal gland of an eye of the individual.

[0251] In some embodiments, the present disclosure provides a rAAV vector for use in a method of treating an ocular condition in an individual in need thereof, the method comprising administering a recombinant adeno-associated virus (rAAV) vector to at least one eye of the individual or to at least one lacrimal gland of an eye of the individual, wherein the rAAV vector comprises an expression cassette comprising a nucleic acid sequence of SEQ ID NO: 16.

[0252] In some embodiments, the expression of human TRX is increased by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% compared to the expression of human TRX in untreated individuals or in the fellow eye of treated individuals. In some embodiments, the expression of human TRX is increased by at least 1.5 times, 2 times, 3 times, 4 times, or 5 times, 6 times, 7 times, 8 times or 9 times compared to the expression of human TRX in untreated individuals or in the fellow eye of treated individuals.

[0253] In some embodiments, expression of human TRX is increased by about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 1 year compared to expression of human TRX in an untreated individual or in the fellow eye of a treated individual.

[0254] As used herein, "about" or "approximately" as applied to one or more values ​​of interest refers to values ​​similar to the stated reference value. In certain embodiments, unless otherwise stated or otherwise apparent from the context, "about" refers to a range of values ​​that are within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less of the stated reference value in either direction (greater or less) (except where such numbers would exceed 100% of the possible values).

[0255] In some embodiments, a rAAV comprising nucleotides encoding a human TRX enzyme described herein is administered to an individual, wherein expression of the human TRX enzyme in the individual is increased compared to expression of human TRX in an untreated individual or in the fellow eye of the treated individual.

[0256] In some embodiments, the expression of human TRX enzyme is increased by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% compared to the expression of human TRX enzyme in untreated individuals or in the fellow eye of treated individuals. In some embodiments, the expression of human TRX enzyme may be increased by at least 1.5 times, 2 times, 3 times, 4 times, or 5 times, 6 times, 7 times, 8 times, or 9 times compared to the expression of human TRX enzyme in untreated individuals or in the fellow eye of treated individuals.

[0257] In some embodiments, expression of the human TRX enzyme is increased for about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 1 year compared to expression of the human TRX enzyme in an untreated individual or in the fellow eye of a treated individual.

[0258] In some embodiments, the present disclosure provides a method of treating an individual having an ocular disease or disorder, comprising administering to the individual a rAAV comprising nucleotides encoding a human TRX enzyme, wherein expression of the human TRX enzyme in the individual is increased compared to expression of the human TRX enzyme in an untreated individual or in a contralateral eye of the treated individual.

[0259] In some embodiments, the present disclosure provides a method of treating an individual having an ocular disease or disorder, comprising administering to the individual a rAAV comprising nucleotides encoding a human PDI described herein, wherein expression of human PDI in the individual is increased compared to expression of human PDI in an untreated individual or in the contralateral eye of the treated individual.

[0260] In some embodiments, the present disclosure provides a rAAV vector for use in a method of treating an ocular condition in an individual in need thereof, the method comprising administering a recombinant adeno-associated virus (rAAV) vector to at least one eye of the individual or to at least one lacrimal gland of an eye of the individual, the rAAV vector comprising an AAV capsid and an expression cassette comprising a polynucleotide encoding human PDI operably linked to a promoter. In some embodiments, the rAAV vector for use is administered to the lacrimal gland of the individual. In some embodiments, the rAAV vector for use comprises an expression cassette encoding human PDI.

[0261] In some embodiments, the present disclosure provides rAAV vectors for use or adaptable for use in treating an individual with an ocular disease, disorder or condition. In some embodiments, the rAAV vector for use or adaptable for use comprises an AAV capsid and an expression cassette comprising a polynucleotide encoding human PDI operably linked to a promoter.

[0262] In some embodiments, the present disclosure provides a rAAV vector for use in a method of treating an ocular condition in an individual in need thereof, the method comprising administering a recombinant adeno-associated viral (rAAV) vector described herein to at least one eye of the individual or to at least one lacrimal gland of an eye of the individual.

[0263] In some embodiments, the present disclosure provides a rAAV vector for use in a method of treating an ocular condition in an individual in need thereof, the method comprising administering a recombinant adeno-associated virus (rAAV) vector to at least one eye of the individual or to at least one lacrimal gland of an eye of the individual, wherein the rAAV vector comprises an expression cassette comprising a nucleic acid sequence of SEQ ID NO: 16.

[0264] In some embodiments, the expression of human PDI is increased by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% compared to the expression of human PDI in an untreated individual or in the fellow eye of a treated individual. In some embodiments, the expression of human PDI is increased by at least 1.5 times, 2 times, 3 times, 4 times, or 5 times, 6 times, 7 times, 8 times, or 9 times compared to the expression of human PDI in an untreated individual or in the fellow eye of a treated individual.

[0265] In some embodiments, expression of human PDI is increased for about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 1 year compared to expression of human PDI in an untreated individual or in the fellow eye of a treated individual.

[0266] In some embodiments, a rAAV comprising nucleotides encoding a human PDI described herein is administered to an individual, wherein expression of human PDI in the individual is increased compared to expression of human PDI in an untreated individual or in the fellow eye of the treated individual.

[0267] In some embodiments, the expression of human PDI is increased by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% compared to the expression of human PDI in an untreated individual or in the fellow eye of a treated individual. In some embodiments, the expression of human PDI may be increased by at least 1.5 times, 2 times, 3 times, 4 times, or 5 times, 6 times, 7 times, 8 times, or 9 times compared to the expression of human PDI in an untreated individual or in the fellow eye of a treated individual.

[0268] In some embodiments, expression of human PDI is increased for about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 1 year compared to expression of human PDI in an untreated individual or in the fellow eye of a treated individual.

[0269] In some embodiments, the present disclosure provides a method of treating an individual having an ocular disease or disorder, comprising administering to the individual a rAAV comprising nucleotides encoding human PDI, wherein expression of human PDI in the individual is increased compared to expression of human PDI in an untreated individual or in the contralateral eye of the treated individual.

[0270] In some embodiments, the method comprises administering a dose of rAAV to an individual with a condition associated with cataract formation, wherein the condition is characterized by one or more symptoms described herein. In some embodiments, the dose is administered before the onset of one or more symptoms. In some embodiments, the administration prevents the onset of one or more symptoms. In some embodiments, the administration reduces the severity of one or more symptoms. For example, in some embodiments, the method comprises administering a dose of rAAV to an individual with cataract formation characterized by one or more symptoms (e.g., blurred vision, diplopia, color vision loss, night vision difficulties), wherein the dose of rAAV is administered and one or more symptoms are prevented or reduced in severity. In some embodiments, the dose is administered after the onset of one or more symptoms, wherein the administration reduces the severity of one or more symptoms.

[0271] In some embodiments, the method comprises administering a rAAV dosing regimen to an individual suffering from a condition associated with cataract formation, wherein the condition is characterized by one or more symptoms described herein (e.g., blurred vision, diplopia, color loss, night vision difficulties), wherein the dosing regimen comprises a first dose of rAAV and at least one additional dose, wherein the first dose of rAAV is administered to the individual before or after the onset of one or more symptoms, and wherein the at least one additional dose is administered to the individual after the first dose, thereby preventing one or more symptoms or reducing their severity. In some embodiments, the dosing regimen comprises a dosing frequency and / or dosage amount selected based on the pharmacokinetic parameters of rAAV. In some embodiments, the clinician will administer rAAV at a frequency and / or dosage to achieve or maintain one or more desired effects. In some embodiments, one or more desired effects are to prevent one or more symptoms associated with the condition of the individual. In some embodiments, one or more desired effects are to reduce the severity of one or more symptoms associated with the condition of the individual. In some embodiments, the severity of one or more symptoms is measured using methods described herein or known in the art for assessing lesions associated with cataract formation. In some embodiments, one or more desired effects are achieved immediately after the administration of the first dose of rAAV. In some embodiments, one or more desired effects occur at any time point after the administration of the first dose of rAAV. In some embodiments, one or more desired effects are achieved after the administration of at least one additional dose of rAAV. In some embodiments, one or more desired effects are achieved at any time point during the dosing regimen. In some embodiments, one or more desired effects are achieved after the administration of the first dose of rAAV, and at least one additional dose of rAAV is administered to the individual to prevent the reversal of one or more desired effects. In some embodiments, the method comprises administering a first dose of rAAV to the individual before, immediately thereafter, or during the onset of one or more symptoms, and administering additional doses after about 1 week, about 2 weeks, about 3 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 1 year, about 1.5 years, or about 2 years after the first dose.

[0272] In some embodiments, the method comprises reversing cataract formation. In some embodiments, the method comprises administering a dose of rAAV to an individual having cataract formation, wherein the administration reverses cataract formation.

[0273] In some embodiments, the method comprises administering a dose of rAAV to an individual with a condition associated with presbyopia, wherein the condition is characterized by one or more symptoms described herein. In some embodiments, the dose is administered before the onset of one or more symptoms. In some embodiments, the administration prevents the onset of one or more symptoms. In some embodiments, the administration reduces the severity of one or more symptoms. For example, in some embodiments, the method comprises administering a dose of rAAV to an individual with presbyopia characterized by one or more symptoms (e.g., blurred vision, dyslexia, headache), wherein the dose of rAAV is administered and one or more symptoms are prevented or reduced in severity. In some embodiments, the dose is administered after the onset of one or more symptoms, wherein the administration reduces the severity of one or more symptoms.

[0274] In some embodiments, the method comprises administering a rAAV dosing regimen to an individual suffering from a condition associated with presbyopia, wherein the condition is characterized by one or more symptoms described herein (e.g., blurred vision, dyslexia, headache), wherein the dosing regimen comprises a first dose of rAAV and at least one additional dose, wherein the first dose of rAAV is administered to the individual before or after the onset of one or more symptoms, and wherein at least one additional dose is administered to the individual after the first dose, thereby preventing one or more symptoms or reducing their severity. In some embodiments, the dosing regimen comprises a dosing frequency and / or dosage amount selected based on the pharmacokinetic parameters of rAAV. In some embodiments, the clinician will administer rAAV at a frequency and / or dosage to achieve or maintain one or more desired effects. In some embodiments, one or more desired effects are to prevent one or more symptoms associated with the individual's condition. In some embodiments, one or more desired effects are to reduce the severity of one or more symptoms associated with the individual's condition. In some embodiments, the severity of one or more symptoms is measured using methods described herein or known in the art for assessing lesions associated with presbyopia. In some embodiments, one or more desired effects are achieved immediately after the administration of the first dose of rAAV. In some embodiments, one or more desired effects occur at any time point after the administration of the first dose of rAAV. In some embodiments, one or more desired effects are achieved after the administration of at least one additional dose of rAAV. In some embodiments, one or more desired effects are achieved at any time point during the dosing regimen. In some embodiments, one or more desired effects are achieved after the administration of the first dose of rAAV, and at least one additional dose of rAAV is administered to the individual to prevent the reversal of one or more desired effects. In some embodiments, the method comprises administering a first dose of rAAV to the individual before, immediately thereafter, or during the onset of one or more symptoms, and administering additional doses after about 1 week, about 2 weeks, about 3 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 1 year, about 1.5 years, or about 2 years after the first dose.

[0275] In some embodiments, the method comprises administering a dose of rAAV to an individual suffering from a condition associated with loss of visual accommodation, wherein the condition is characterized by one or more symptoms described herein. In some embodiments, the dose is administered before the onset of one or more symptoms. In some embodiments, the administration prevents the onset of one or more symptoms. In some embodiments, the administration reduces the severity of one or more symptoms. For example, in some embodiments, the method comprises administering a dose of rAAV to an individual suffering from loss of visual accommodation characterized by one or more symptoms (e.g., blurred vision, dyslexia, headache), wherein the dose of rAAV is administered and one or more symptoms are prevented or reduced in severity. In some embodiments, the dose is administered after the onset of one or more symptoms, wherein the administration reduces the severity of one or more symptoms.

[0276] In some embodiments, the method comprises administering a rAAV dosing regimen to an individual suffering from a condition associated with loss of visual accommodation, wherein the condition is characterized by one or more symptoms described herein (e.g., blurred vision, dyslexia, headache), wherein the dosing regimen comprises a first dose of rAAV and at least one additional dose, wherein the first dose of rAAV is administered to the individual before or after the onset of one or more symptoms, and wherein at least one additional dose is administered to the individual after the first dose, thereby preventing one or more symptoms or reducing their severity. In some embodiments, the dosing regimen comprises a dosing frequency and / or dosage amount selected based on the pharmacokinetic parameters of rAAV. In some embodiments, the clinician will administer rAAV at a frequency and / or dosage to achieve or maintain one or more desired effects. In some embodiments, one or more desired effects are to prevent one or more symptoms associated with the individual's condition. In some embodiments, one or more desired effects are to reduce the severity of one or more symptoms associated with the individual's condition. In some embodiments, the severity of one or more symptoms is measured using methods described herein or known in the art for assessing lesions associated with loss of visual accommodation. In some embodiments, one or more desired effects are achieved immediately after the administration of the first dose of rAAV. In some embodiments, one or more desired effects occur at any time point after the administration of the first dose of rAAV. In some embodiments, one or more desired effects are achieved after the administration of at least one additional dose of rAAV. In some embodiments, one or more desired effects are achieved at any time point during the dosing regimen. In some embodiments, one or more desired effects are achieved after the administration of the first dose of rAAV, and at least one additional dose of rAAV is administered to the individual to prevent the reversal of one or more desired effects. In some embodiments, the method comprises administering a first dose of rAAV to the individual before, immediately thereafter, or during the onset of one or more symptoms, and administering additional doses after about 1 week, about 2 weeks, about 3 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 1 year, about 1.5 years, or about 2 years after the first dose.

[0277] In some embodiments, the method comprises administering a dose of rAAV to an individual with a condition associated with ocular hypertension, wherein the condition is characterized by one or more symptoms described herein. In some embodiments, the dose is administered before the onset of one or more symptoms. In some embodiments, the administration prevents the onset of one or more symptoms. In some embodiments, the administration reduces the severity of one or more symptoms. For example, in some embodiments, the method comprises administering a dose of rAAV to an individual with ocular hypertension characterized by one or more symptoms (e.g., eye pain, nausea, vomiting), wherein the dose of rAAV is administered and one or more symptoms are prevented or reduced in severity. In some embodiments, the dose is administered after the onset of one or more symptoms, wherein the administration reduces the severity of one or more symptoms.

[0278] In some embodiments, the method comprises administering a rAAV dosing regimen to an individual suffering from a condition associated with ocular hypertension, wherein the condition is characterized by one or more symptoms described herein (e.g., eye pain, nausea, vomiting), wherein the dosing regimen comprises a first dose of rAAV and at least one additional dose, wherein the first dose of rAAV is administered to the individual before or after the onset of one or more symptoms, and wherein at least one additional dose is administered to the individual after the first dose, thereby preventing one or more symptoms or reducing their severity. In some embodiments, the dosing regimen comprises a dosing frequency and / or dosage amount selected based on the pharmacokinetic parameters of rAAV. In some embodiments, the clinician will administer rAAV at a frequency and / or dosage to achieve or maintain one or more desired effects. In some embodiments, one or more desired effects are to prevent one or more symptoms associated with the individual's condition. In some embodiments, one or more desired effects are to reduce the severity of one or more symptoms associated with the individual's condition. In some embodiments, the severity of one or more symptoms is measured using methods described herein or known in the art for assessing lesions associated with ocular hypertension. In some embodiments, one or more desired effects are achieved immediately after the administration of the first dose of rAAV. In some embodiments, one or more desired effects occur at any time point after the administration of the first dose of rAAV. In some embodiments, one or more desired effects are achieved after the administration of at least one additional dose of rAAV. In some embodiments, one or more desired effects are achieved at any time point during the dosing regimen. In some embodiments, one or more desired effects are achieved after the administration of the first dose of rAAV, and at least one additional dose of rAAV is administered to the individual to prevent the reversal of one or more desired effects. In some embodiments, the method comprises administering a first dose of rAAV to the individual before, immediately thereafter, or during the onset of one or more symptoms, and administering additional doses after about 1 week, about 2 weeks, about 3 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 1 year, about 1.5 years, or about 2 years after the first dose.

[0279] In some embodiments, the method comprises administering a dose of rAAV to an individual with a condition associated with meibomian gland dysfunction (MDI), wherein the condition is characterized by one or more symptoms described herein. In some embodiments, the dose is administered before the onset of one or more symptoms. In some embodiments, the administration prevents the onset of one or more symptoms. In some embodiments, the administration reduces the severity of one or more symptoms. For example, in some embodiments, the method comprises administering a dose of rAAV to an individual with meibomian gland dysfunction (MDI) characterized by one or more symptoms (e.g., dry eyes, burning pain, itching, redness, discharge, blurred vision), wherein the dose of rAAV is administered and one or more symptoms are prevented or reduced in severity. In some embodiments, the dose is administered after the onset of one or more symptoms, wherein the administration reduces the severity of one or more symptoms.

[0280] In some embodiments, the method comprises administering a rAAV dosing regimen to an individual suffering from a condition associated with meibomian gland dysfunction (MDI), wherein the condition is characterized by one or more symptoms described herein (e.g., dry eyes, burning, itching, redness, discharge, blurred vision), wherein the dosing regimen comprises a first dose of rAAV and at least one additional dose, wherein the first dose of rAAV is administered to the individual before or after the onset of one or more symptoms, and wherein at least one additional dose is administered to the individual after the first dose, thereby preventing one or more symptoms or reducing their severity. In some embodiments, the dosing regimen comprises a dosing frequency and / or dosage amount selected based on the pharmacokinetic parameters of rAAV. In some embodiments, the clinician will administer rAAV at a frequency and / or dosage that achieves or maintains one or more desired effects. In some embodiments, the one or more desired effects are to prevent one or more symptoms associated with the individual's condition. In some embodiments, the one or more desired effects are to reduce the severity of one or more symptoms associated with the individual's condition. In some embodiments, the severity of one or more symptoms is measured using methods described herein or known in the art for assessing lesions associated with meibomian gland dysfunction (MDI). In some embodiments, one or more desired effects are achieved immediately after administration of the first dose of rAAV. In some embodiments, one or more desired effects occur at any time point after administration of the first dose of rAAV. In some embodiments, one or more desired effects are achieved after administration of at least one additional dose of rAAV. In some embodiments, one or more desired effects are achieved at any time point during the dosing regimen. In some embodiments, one or more desired effects are achieved after administration of the first dose of rAAV, and at least one additional dose of rAAV is administered to the individual to prevent reversal of one or more desired effects. In some embodiments, the method comprises administering to the individual a first dose of rAAV prior to, immediately following, or during the onset of one or more symptoms, and administering an additional dose after a period of about 1 week, about 2 weeks, about 3 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 1 year, about 1.5 years, or about 2 years after the first dose.

[0281] In some embodiments, the method comprises administering a dose of rAAV to an individual suffering from a condition associated with oxidative stress, wherein the condition is characterized by one or more symptoms described herein. In some embodiments, the dose is administered before the onset of one or more symptoms. In some embodiments, the administration prevents the onset of one or more symptoms. In some embodiments, the administration reduces the severity of one or more symptoms. For example, in some embodiments, the method comprises administering a dose of rAAV to an individual suffering from oxidative stress characterized by one or more symptoms (e.g., oxidative stress leading to retinal degeneration, cataracts, or macular degeneration), wherein the dose of rAAV is administered and one or more symptoms are prevented or reduced in severity. In some embodiments, the dose is administered after the onset of one or more symptoms, wherein the administration reduces the severity of one or more symptoms.

[0282] In some embodiments, the method comprises administering a rAAV dosing regimen to an individual suffering from a condition associated with oxidative stress, wherein the condition is characterized by one or more symptoms described herein (e.g., oxidative stress leading to retinal degeneration, cataracts, or macular degeneration), wherein the dosing regimen comprises a first dose of rAAV and at least one additional dose, wherein the first dose of rAAV is administered to the individual before or after the onset of one or more symptoms, and wherein at least one additional dose is administered to the individual after the first dose, thereby preventing one or more symptoms or reducing their severity. In some embodiments, the dosing regimen comprises a dosing frequency and / or dose amount selected based on the pharmacokinetic parameters of rAAV. In some embodiments, the clinician will administer rAAV at a frequency and / or dose to achieve or maintain one or more desired effects. In some embodiments, one or more desired effects are to prevent one or more symptoms associated with the condition of the individual. In some embodiments, one or more desired effects are to reduce the severity of one or more symptoms associated with oxidative stress. In some embodiments, the severity of one or more symptoms is measured using methods described herein or known in the art for assessing lesions associated with oxidative stress. In some embodiments, one or more desired effects are achieved immediately after the administration of the first dose of rAAV. In some embodiments, one or more desired effects occur at any time point after the administration of the first dose of rAAV. In some embodiments, one or more desired effects are achieved after the administration of at least one additional dose of rAAV. In some embodiments, one or more desired effects are achieved at any time point during the dosing regimen. In some embodiments, one or more desired effects are achieved after the administration of the first dose of rAAV, and at least one additional dose of rAAV is administered to the individual to prevent the reversal of one or more desired effects. In some embodiments, the method comprises administering a first dose of rAAV to the individual before, immediately thereafter, or during the onset of one or more symptoms, and administering additional doses after about 1 week, about 2 weeks, about 3 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 1 year, about 1.5 years, or about 2 years after the first dose.

[0283] In some embodiments, the present disclosure provides a method of treating cataract formation in an individual in need thereof, the method comprising administering to the individual one or more therapeutically effective doses of a rAAV described herein.

[0284] In some embodiments, the present disclosure provides a method of treating presbyopia in an individual in need thereof, the method comprising administering to the individual one or more therapeutically effective doses of a rAAV described herein.

[0285] In some embodiments, the present disclosure provides a method of treating oxidative stress in a subject in need thereof, the method comprising administering to the subject one or more therapeutically effective doses of a rAAV described herein.

[0286] In some embodiments, the present disclosure provides a method of treating loss of visual accommodation in an individual in need thereof, the method comprising administering to the individual one or more therapeutically effective doses of a rAAV described herein.

[0287] In some embodiments, the present disclosure provides a method of treating ocular hypertension in a subject in need thereof, the method comprising administering to the subject one or more therapeutically effective doses of a rAAV described herein.

[0288] In some embodiments, the present disclosure provides a method of treating meibomian gland dysfunction in a subject in need thereof, the method comprising administering to the subject one or more therapeutically effective doses of a rAAV described herein.

[0289] In some embodiments, the present disclosure provides a rAAV described herein for use in a method of treating cataract formation in an individual in need thereof, the method comprising administering to the individual an effective amount of a rAAV described herein.

[0290] In some embodiments, the present disclosure provides a rAAV described herein for use in the manufacture of a medicament for treating cataract formation in an individual in need thereof.

[0291] In some embodiments, the present disclosure provides a rAAV described herein for use in a method of treating presbyopia in an individual in need thereof, the method comprising administering to the individual an effective amount of a rAAV described herein.

[0292] In some embodiments, the present disclosure provides a rAAV described herein for use in the manufacture of a medicament for treating presbyopia in an individual in need thereof.

[0293] In some embodiments, the present disclosure provides a rAAV described herein for use in a method of treating loss of visual accommodation in an individual in need thereof, the method comprising administering to the individual an effective amount of a rAAV described herein.

[0294] In some embodiments, the present disclosure provides a rAAV described herein for use in the manufacture of a medicament for treating loss of visual accommodation in an individual in need thereof.

[0295] In some embodiments, the present disclosure provides a rAAV described herein for use in a method of treating oxidative stress in an individual in need thereof, the method comprising administering to the individual an effective amount of a rAAV described herein.

[0296] In some embodiments, the present disclosure provides a rAAV described herein for use in the manufacture of a medicament for treating oxidative stress in an individual in need thereof.

[0297] In some embodiments, the present disclosure provides a rAAV described herein for use in a method of treating ocular hypertension in an individual in need thereof, the method comprising administering to the individual an effective amount of a rAAV described herein.

[0298] In some embodiments, the present disclosure provides a rAAV described herein for use in the manufacture of a medicament for treating ocular hypertension in an individual in need thereof.

[0299] In some embodiments, the present disclosure provides a rAAV described herein for use in a method of treating meibomian gland dysfunction in an individual in need thereof, the method comprising administering to the individual an effective amount of a rAAV described herein.

[0300] In some embodiments, the present disclosure provides a rAAV described herein for use in the manufacture of a medicament for treating meibomian gland dysfunction in an individual in need thereof.

[0301] Mode of administration

[0302] In some embodiments, the present disclosure provides methods comprising administering to an eye of an individual, to the lacrimal gland of an eye of an individual, or to the trabecular meshwork of an eye of an individual a rAAV vector comprising an AAV capsid and an expression cassette comprising a polynucleotide encoding TRX operably linked to a promoter. In some embodiments, the present disclosure provides methods comprising administering to an eye of an individual, to the lacrimal gland of an eye of an individual, or to the trabecular meshwork of an eye of an individual a rAAV vector comprising an AAV capsid and an expression cassette comprising a polynucleotide encoding PDI operably linked to a promoter.

[0303] As noted above, the lacrimal functional unit consists of the main and accessory lacrimal glands, the ocular surface, and interconnected nerve innervations. For each eye, the main lacrimal gland is located in the frontal lacrimal fossa in the temporal orbit. Accessory glands called the Valsalva glands and Gorbachev glands are located in the eyelids. In the upper eyelid, there are about 2 to 5 Valsalva glands and about forty Gorbachev glands. In the lower eyelid, there are about 6 to 8 Gorbachev glands. The specific location and anatomical structure of the lacrimal functional unit are well known (Conrady et al. Ophthalmol; 2016:7542929 (2016)). Together, the lacrimal glands secrete the tear film onto the ocular surface through the tear ducts. The lacrimal glands also express and secrete proteins and products into the tear film that are needed for corneal regeneration and promotion of transparency, such as transforming growth factor-β and retinol (Conradi et al. Ophthalmol.; 2016:7542929 (2016); Pan et al. Optom Vis Sci.; 95:27-31 (2018)). In addition to secreting tears into the eyes, the tear ducts drain tears into the nasal cavity.

[0304] Administration of the rAAV vector to the lacrimal gland can be achieved by topical administration to the ocular surface, direct injection into the lacrimal gland, and / or topical administration to the lacrimal gland. The lacrimal gland can be accessed surgically or by manipulation of the eyelids. Manipulation of the eyelids provides access to the tissue for topical administration (e.g., by irrigating the tissue with a pharmaceutical composition comprising the rAAV vector). Direct injection into the lacrimal gland can be achieved by penetrating the skin over the lacrimal gland ( Figure 2A ) or by manipulating the eyelids to access the tear glands ( Figure 2B ). Administration of rAAV vectors to the lacrimal glands can be achieved by transconjunctival injection. In some embodiments, rAAV vectors are administered to the lacrimal glands by direct injection, such as Figure 2A In some embodiments, the rAAV vector is administered to the lacrimal gland by manipulating the eyelids, such as Figure 2B Described in.

[0305] In some embodiments, cells in the eye, lacrimal gland and / or nasolacrimal duct are transduced or transfected by rAAV vectors. Cells in the eye, lacrimal gland and / or nasolacrimal duct include but are not limited to acinar cells, ductal cells and / or myoepithelial cells, as well as cells of the iris and ciliary body ("ICB"), lens epithelial cells, meibomian glands and trabecular meshwork. In some embodiments, transduced or transfected cells in the eye, lacrimal gland and / or nasolacrimal duct express a therapeutically effective amount of TRX. In some embodiments, transduced or transfected cells in the eye, lacrimal gland and / or nasolacrimal duct secrete a therapeutically effective amount of TRX into the tear film. In some embodiments, cells in the meibomian gland are transduced or transfected by rAAV vectors. In some embodiments, cells in the trabecular meshwork are transduced or transfected by rAAV vectors. In some embodiments, a therapeutically effective amount of TRX is secreted into the nasal cavity of an individual. In some embodiments, a therapeutically effective amount of TRX is secreted onto the ocular surface of an individual.

[0306] In some embodiments, transfected or transduced cells within the eye, lacrimal gland, and / or nasolacrimal duct express a therapeutically effective amount of PDI. In some embodiments, transfected or transduced cells within the eye, lacrimal gland, and / or nasolacrimal duct secrete a therapeutically effective amount of PDI into the tear film. In some embodiments, a therapeutically effective amount of PDI is secreted into the nasal cavity of an individual. In some embodiments, a therapeutically effective amount of PDI is secreted onto the ocular surface of an individual.

[0307] Delivery of rAAV vectors to the eye and / or lacrimal glands has been demonstrated in vivo to express transgenes in the tear film. The primary lacrimal glands of mice were directly injected with rAAV vectors encoding a luciferase transgene, with serotypes of AAV2, AAV4, AAV5, AAV5w8, AAV x5, AAV 9, AAV12, and bovine AAV (BAAV). AAV9, AAV 5w8, AAV5, and AAV2 were all able to transduce both the ductal cells and the acinar cells of the lacrimal gland (Rocha et al., supra).

[0308] In some embodiments, the rAAV vector is administered to a lacrimal gland of a subject. In some embodiments, the lacrimal gland is a primary lacrimal gland. In some embodiments, the lacrimal gland is any one of the Warthwell gland or the Gorbachev gland of a subject.

[0309] The compositions and rAAV vectors of the present disclosure can be administered to the lacrimal glands of an individual by any suitable method. For example, the compositions of the present disclosure can be administered by direct injection to the primary or accessory lacrimal glands.

[0310] Access to the tear gland of a human subject can be achieved, for example, by manually lifting the upper eyelid to expose the palpebral lobe of the tear gland and delivering the therapeutic agent using a syringe (eg, with a 30G needle).

[0311] In some embodiments, the rAAV vector is administered to the trabecular meshwork of the eye of the individual.

[0312] In some embodiments, the rAAV vector is administered to endothelial cells of the cornea of ​​the individual.

[0313] The rAAV vectors of the present disclosure are generally delivered to an individual in the form of a pharmaceutical composition. The pharmaceutical composition comprises a pharmaceutically acceptable solvent (e.g., water, etc.) and one or more excipients. In some embodiments, the pharmaceutical composition comprises a buffer at about neutral pH (pH 5, 6, 7, 8, or 9). In some embodiments, the pharmaceutical composition comprises phosphate buffered saline (e.g., PBS at a pH of about 7). The pharmaceutical composition may comprise a pharmaceutically acceptable salt. The concentration of the salt may be selected to ensure that the pharmaceutical composition is isotonic or nearly isotonic with the target tissue.

[0314] In various embodiments, the pharmaceutical composition of the present disclosure comprises about 1×10 8 genome copies / mL (GC / mL), about 5×10 8 GC / mL, about 1×10 9 GC / mL, about 5×10 9 GC / mL, about 1×10 10 GC / mL, about 5×10 10 GC / mL, about 1×10 11 GC / mL, about 5×10 11 GC / mL, about 1×10 12 GC / mL, about 5×10 12 GC / mL, about 5×10 13 GC / mL, or about 1×10 14 GC / mL of rAAV vector. In various embodiments, the pharmaceutical composition of the present disclosure comprises about 1×10 8 genome copies / mL (GC / mL), about 5×10 8 GC / mL to about 1×10 9 GC / mL, about 1×10 9 GC / mL to about 5×10 9 GC / mL, about 5×10 9 GC / mL to about 1×10 10 GC / mL, about 1×10 10 GC / mL to about 5×10 10 GC / mL, about 5×10 10 GC / mL to about 1×10 11 GC / mL, about 1×10 11 GC / mL to about 5×10 11 GC / mL, about 5×10 11 GC / mL to about 1×10 12GC / mL, about 1×10 12 GC / mL to about 5×10 12 GC / mL, about 5×10 12 GC / mL to about 5×10 13 GC / mL, or about 5×10 13 GC / mL to about 1×10 14 GC / mL of rAAV vector. In various other embodiments, the pharmaceutical composition of the present disclosure comprises about 1×10 8 genome copies / mL (GC / mL), about 5×10 8 GC / mL to about 5×10 9 GC / mL, about 5×10 9 GC / mL to about 5×10 10 GC / mL, about 5×10 10 GC / mL to about 5×10 11 GC / mL, about 5×10 11 GC / mL to about 5×10 12 GC / mL, or about 5×10 12 GC / mL to about 1×10 14 GC / mL of rAAV vector. In some embodiments, the pharmaceutical composition of the present disclosure comprises about 5×10 8 GC / mL to about 5×10 10 GC / mL, about 5×10 10 GC / mL to about 5×10 12 GC / mL, or about 5×10 12 GC / mL to about 1×10 14 GC / mL of rAAV vector.

[0315] In some embodiments, the pharmaceutical compositions of the present disclosure comprise about 1×10 12 GC / mL to about 6.2×10 12 GC / mL of rAAV vector. In some embodiments, the pharmaceutical composition of the present disclosure comprises about 1×10 12 GC / mL or about 6.2×10 12 GC / mL of rAAV vector.

[0316] In some embodiments, the pharmaceutical compositions of the present disclosure are administered in a total volume of about 10 μL, about 20 μL, about 30 μL, about 40 μL, about 50 μL, about 60 μL, about 70 μL, about 80 μL, about 90 μL, about 100 μL, 110 μL, about 120 μL, about 130 μL, about 140 μL, about 150 μL, about 160 μL, about 170 μL, about 180 μL, about 190 μL, or about 200 μL. In some embodiments, the pharmaceutical compositions of the present disclosure are administered in a total volume of about 10 μL to about 20 μL, about 20 μL to about 30 μL, about 30 μL to about 40 μL, about 40 μL to about 50 μL, about 50 μL to about 60 μL, about 60 μL to about 70 μL, about 70 μL to about 80 μL, about 80 μL to about 90 μL, about 90 μL to about 100 μL, about 100 μL to 110 μL, 110 μL to about 120 μL, about 120 μL to about 130 μL, about 130 μL to about 140 μL, about 140 μL to about 150 μL, about 150 μL to about 160 μL, about 160 μL to about 170 μL, about 170 μL to about 180 μL, about 180 μL to about 190 μL, or about 190 μL to about 200 μL.

[0317] The number of genome copies per milliliter can be determined by quantitative polymerase chain reaction (qPCR) using a standard curve generated from a reference sample with a known concentration of the polynucleotide genome of the virus. For AAV, the reference sample used is usually the transfer plasmid used to generate the rAAV vector, but other reference samples can be used.

[0318] Alternatively or additionally, the concentration of the viral vector can be determined by measuring the titer of the vector to the cell line. Viral titer is usually expressed as viral particles per unit volume (vp) (e.g., vp / mL). In various embodiments, the pharmaceutical composition of the present disclosure comprises about 1×10 8 Virus particles / ml (vp / mL), about 5×10 8 vp / mL, about 1×10 9 vp / mL, about 5×10 9 vp / mL, about 1×10 10 vp / mL, about 5×10 10 vp / mL, about 1×10 11 vp / mL, about 5×10 11 vp / mL, about 1×10 12 vp / mL, about 5×10 12 vp / mL, about 5×10 13 vp / mL, or about 1×10 14vp / mL of rAAV vector. In various other embodiments, the pharmaceutical composition of the present disclosure comprises about 1×10 8 Virus particles / ml (vp / mL) to about 5×10 8 vp / mL, about 5×10 8 vp / mL is about 1×10 9 vp / mL, about 1×10 9 vp / mL to about 5×10 9 vp / mL, about 5×10 9 vp / mL is about 1×10 10 vp / mL, about 1×10 10 vp / mL to about 5×10 10 vp / mL, about 5×10 10 vp / mL is about 1×10 11 vp / mL, about 1×10 11 vp / mL to about 5×10 11 vp / mL, about 5×10 11 vp / mL is about 1×10 12 vp / mL, about 1×10 12 vp / mL to about 5×10 12 vp / mL, about 5×10 12 vp / mL to about 5×10 13 vp / mL, or about 5×10 13 vp / mL is about 1×10 14 vp / mL of rAAV vector.

[0319] Evaluation of efficacy

[0320] A variety of tests can be used to assess the ocular condition of a subject before, during, and after treatment with any of the methods or compositions disclosed herein. In some embodiments disclosed herein, effective treatment of a subject is indicated by one or more tests, which may be, for example: a) a dry eye scoring test according to a visual analog scale, b) a Schirmer's test, c) a corneal fluorescein staining test, and d) an ocular surface disease index test. In some embodiments, effective treatment of a subject is indicated by one or more tests, which may be, for example: Symptom Assessment in Dry Eye Questionnaire (SANDE), meibomian gland atrophy / loss, glandular expression, meibomian gland imaging / meibomian gland determination, tear film lipid layer thickness, corneal fluorescein staining, tear film break up time (TBUT), distance-corrected near visual activity (DCNVA), intraocular pressure measurement (e.g., tonometry), visual acuity, slit lamp examination, or ophthalmoscopy. Tests for evaluating signs and symptoms of ocular conditions can be administered under standardized or reproducible conditions to obtain a test score for an individual. Conditions include exposing an individual to an environment artificially created to adversely stimulate the individual or one in which the environment (temperature, humidity, airflow) is monitored and carefully controlled.

[0321] The efficacy of the methods described herein can be assessed using any suitable method known in the art.

[0322] In some embodiments, the methods described herein result in a reduction in one or more symptoms of an ocular condition compared to the symptoms of the ocular condition prior to administration of the rAAV vector. As used herein, "symptoms" include any of the diagnostic criteria or symptoms associated with a given ocular condition, including those described herein. Non-limiting examples of symptoms that can be alleviated by treatment according to the methods described herein include, for example, worsening vision, and requirements for corrective lenses (e.g., glasses or contact lenses) and / or surgery.

[0323] In some embodiments, the treatment methods described herein delay the onset of an ocular condition. In some embodiments, the onset of an ocular condition is delayed compared to the onset of the ocular condition in a control individual. In some embodiments, the onset of an ocular condition is delayed compared to the onset of the ocular condition in the contralateral eye. In some embodiments, the treatment methods described herein delay the progression of an ocular condition. In some embodiments, the progression of an ocular condition is delayed compared to the progression of an ocular condition in a control individual. In some embodiments, the progression of an ocular condition is delayed compared to the progression of an ocular condition in the contralateral eye. A "control individual" may be, for example, an untreated control individual. In some embodiments, the control individual is an age-matched individual who has not been treated with an rAAV vector comprising an expression cassette comprising a polynucleotide encoding TRX or PDI. In some embodiments, the control individual is an age-matched untreated individual. "Contralateral eye" means the eye of an individual opposite to the eye that has been treated with a composition according to the present disclosure. The contralateral eye can be used as a treatment control as long as the individual suffers from bilateral disease, or, in the case of a model animal, has been subjected to an experimental protocol for treating both eyes.

[0324] The treatment methods described herein can slow the progression of an ocular condition. In some embodiments, the progression of an individual's condition is slowed by about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about 50%, about 50% to about 60%, about 60% to about 70%, about 70% to about 80%, about 80% to about 90%, about 90% to about 95%, or more than about 95% compared to a control individual.

[0325] In some embodiments, the treatment methods described herein delay the onset of a condition in a subject by about 6 months to about 9 months, about 9 months to about 12 months, about 12 months to about 15 months, about 15 months to about 18 months, about 18 months to about 21 months, about 21 months to about 24 months, about 2 years to about 3 years, about 3 years to about 4 years, about 4 years to about 5 years, about 5 years to about 6 years, about 6 years to about 7 years, about 7 years to about 8 years, about 8 years to about 9 years, about 9 years to about 10 years, about 10 years to about 15 years, about 15 years to about 20 years, or more than 20 years as compared to a control subject.

[0326] In some embodiments, the treatment methods described herein improve individual vision. In some embodiments, for example, compared to the contralateral eye or compared to a control individual, the treatment methods described herein delay individual vision reduction. Vision can be measured using a chart, on which an individual identifies letters at a distance, including, for example, a Snellen chart and an early treatment for diabetic retinopathy study (ETDRS) chart (Bailey and Kitchin, Vision Research 90 (2013) 2-9; Bennett et al., Semin Pediatr Neurol. 2019 October; 31: 30-40). Distance corrected near visual acuity (distance corrected near visual acuity; DCNVA) can also be used to assess vision.

[0327] In some embodiments, the vision of an individual treated according to the methods described herein remains unchanged for at least about 6 months to about 9 months, about 9 months to about 12 months, about 12 months to about 15 months, about 15 months to about 18 months, about 18 months to about 21 months, about 21 months to about 24 months, about 2 years to about 3 years, about 3 years to about 4 years, about 4 years to about 5 years, about 5 years to about 6 years, about 6 years to about 7 years, about 7 years to about 8 years, about 8 years to about 9 years, about 9 years to about 10 years, about 10 years to about 15 years, about 15 years to about 20 years, or more than 20 years after administration of the rAAV vector.

[0328] In some embodiments, the vision of an individual treated according to the methods described herein does not decrease by more than 5% for at least about 6 months to about 9 months, about 9 months to about 12 months, about 12 months to about 15 months, about 15 months to about 18 months, about 18 months to about 21 months, about 21 months to about 24 months, about 2 years to about 3 years, about 3 years to about 4 years, about 4 years to about 5 years, about 5 years to about 6 years, about 6 years to about 7 years, about 7 years to about 8 years, about 8 years to about 9 years, about 9 years to about 10 years, about 10 years to about 15 years, about 15 years to about 20 years, or more than 20 years after administration of the rAAV vector. In some embodiments, the vision of an individual treated according to the methods described herein does not decrease by more than 10% for at least about 6 months to about 9 months, about 9 months to about 12 months, about 12 months to about 15 months, about 15 months to about 18 months, about 18 months to about 21 months, about 21 months to about 24 months, about 2 years to about 3 years, about 3 years to about 4 years, about 4 years to about 5 years, about 5 years to about 6 years, about 6 years to about 7 years, about 7 years to about 8 years, about 8 years to about 9 years, about 9 years to about 10 years, about 10 years to about 15 years, about 15 years to about 20 years, or more than 20 years after administration of the rAAV vector.

[0329] Another indicator of vision can be the need for corrective lenses (e.g., contact lenses or glasses), which generally increase in strength as the condition progresses. In some embodiments, the individual treated according to the methods described herein required corrective lenses prior to administration of the rAAV vector, and the methods result in a constant need for corrective lens strength for at least about 6 months to about 9 months, about 9 months to about 12 months, about 12 months to about 15 months, about 15 months to about 18 months, about 18 months to about 21 months, about 21 months to about 24 months, about 2 years to about 3 years, about 3 years to about 4 years, about 4 years to about 5 years, about 5 years to about 6 years, about 6 years to about 7 years, about 7 years to about 8 years, about 8 years to about 9 years, about 9 years to about 10 years, about 10 years to about 15 years, about 15 years to about 20 years, or more than 20 years after administration of the rAAV vector.

[0330] The treatment methods described herein can slow or delay the development of cataracts in a subject compared to the fellow eye or compared to a control subject. The development of cataracts is graded using any method known in the art, including, for example, grading the severity of cataracts as defined by the Lens Opacities Classification System III.

[0331] In some embodiments, the formation of cataracts in a subject is slowed by about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about 50%, about 50% to about 60%, about 60% to about 70%, about 70% to about 80%, about 80% to about 90%, about 90% to about 95%, or more than about 95% compared to a control subject.

[0332] In some embodiments, the treatment methods described herein delay the onset of cataract formation in a subject by about 6 months to about 9 months, about 9 months to about 12 months, about 12 months to about 15 months, about 15 months to about 18 months, about 18 months to about 21 months, about 21 months to about 24 months, about 2 years to about 3 years, about 3 years to about 4 years, about 4 years to about 5 years, about 5 years to about 6 years, about 6 years to about 7 years, about 7 years to about 8 years, about 8 years to about 9 years, about 9 years to about 10 years, about 10 years to about 15 years, about 15 years to about 20 years, or more than 20 years, relative to a control subject.

[0333] In some embodiments, the individual treated according to the methods described herein had cataracts prior to administration of the rAAV vector, and the methods result in no change in the grade of cataracts for at least about 6 months to about 9 months, about 9 months to about 12 months, about 12 months to about 15 months, about 15 months to about 18 months, about 18 months to about 21 months, about 21 months to about 24 months, about 2 years to about 3 years, about 3 years to about 4 years, about 4 years to about 5 years, about 5 years to about 6 years, about 6 years to about 7 years, about 7 years to about 8 years, about 8 years to about 9 years, about 9 years to about 10 years, about 10 years to about 15 years, about 15 years to about 20 years, or more than 20 years after administration of the rAAV vector.

[0334] In some embodiments, effective treatment of an individual is indicated by the Dry Eye Questionnaire Symptom Evaluation (SANDE). In some embodiments, effective treatment of an individual is indicated by meibomian gland atrophy / loss. In some embodiments, effective treatment of an individual is indicated by glandular expression. In some embodiments, effective treatment of an individual is indicated by meibomian gland imaging / meibomian gland determination. In some embodiments, effective treatment of an individual is indicated by tear film lipid layer thickness. In some embodiments, effective treatment of an individual is indicated by corneal fluorescein staining; in some embodiments, effective treatment of an individual is indicated by tear film breakup time (TBUT). In some embodiments, effective treatment of an individual is indicated by distance-corrected near vision (DCNVA). In some embodiments, effective treatment of an individual is indicated by intraocular pressure measurement. In some embodiments, effective treatment of an individual is indicated by vision. In some embodiments, effective treatment of an individual is indicated by slit lamp examination. In some embodiments, effective treatment of an individual is indicated by ophthalmoscopy. The methods described herein for evaluating effective treatment of an individual are known to those skilled in the art.

[0335] Maintaining effective treatment over time

[0336] The efficacy of the treatment methods described herein can be assessed at any suitable time point after administration, such as about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 18 months, about 24 months, about 3 years, about 4 years, or about 5 years after administration of the rAAV vectors described herein. In some embodiments, efficacy is measured at two or more time points after administration of the rAAV vectors described herein, such as every 3 months, every 6 months, or every 12 months after administration of the rAAV vectors.

[0337] The present disclosure can provide effective treatment over a period of time during which an efficacy measure (e.g., vision) is maintained. As used in the present disclosure and as it relates to maintaining an efficacy measure (e.g., vision) in a subject's score, the term "maintain" refers to a statistically significant improvement that does not decrease below a certain threshold over time. The efficacy measure after treatment according to the disclosed methods can be maintained without additional administration or after one or more subsequent doses.

[0338] Time and method of administration

[0339] The schedule for administering doses to an individual depends on various considerations, including the duration of effect of each dose, the transduction (or transfection) efficiency of the rAAV vector, and the effect of the dose on the body. For example, in the case where the patient's condition does not improve, at the discretion of the health provider, the method for treating an eye condition as described herein may adjust the dose or repeat the administration in order to improve or otherwise control or limit the symptoms of the individual's eye condition. For example, the time period between the administration of one or more doses is extended, or the time period between the days of administering one or more doses to an individual is extended. As a non-limiting example, the administration of one or more doses is modified to administer one or more doses after measuring the symptoms of the eye condition.

[0340] As used herein, the term "dose" may refer to the dose of a pharmaceutical composition of the present disclosure, or the dose of a treatment that reduces the symptoms of an ocular condition.

[0341] In some embodiments described herein, the dosage of the rAAV vector is the dosage of the rAAV vector carrying the expression cassette. In this case, the rAAV vector of the appropriate amount / titer is applied to the target site, thereby allowing the expression of an effective amount of gene products over a period of time to achieve the delivery of an appropriate dose (e.g., an effective amount) of gene products. As used herein, "effective amount" refers to the amount or dosage of the rAAV, treatment or composition described herein that is sufficient to reduce the symptoms and / or signs of the ocular conditions described herein. As used herein, the term "amount" refers to an absolute amount (e.g., an absolute amount of a protein or rAAV particle) or a concentration (e.g., the concentration of a protein in a solution), and the amount mentioned in a given example refers to an absolute amount, a concentration, or both, which will be understood by a technician in the field based on the context provided herein.

[0342] In some embodiments, the rAAV vector is a rAAV virion. In some embodiments, the rAAV vector is administered to the lacrimal gland. In some embodiments, the rAAV vector is administered to the lacrimal gland by topical administration. In some embodiments, the rAAV vector is administered to the lacrimal gland by direct injection. In some embodiments, the rAAV vector is administered to the trabecular meshwork. In some embodiments, the dose of the rAAV vector is such that the gene product is stably produced for a period of time (e.g., about 1 day, about 2 days, about 4 days, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 9 months, about 12 months or longer). In some embodiments, the dose of the rAAV vector is such that the gene product is stably produced for about 1 week. In some embodiments, the dose of the rAAV vector is such that the gene product is stably produced for about 2 weeks. In some embodiments, the dose of the rAAV vector is such that the gene product is stably produced for about 3 weeks. In some embodiments, the dose of the rAAV vector is such that the gene product is stably produced for about 4 weeks. In some embodiments, the dose of the rAAV vector is such that the gene product is stably produced for about 1 month. In some embodiments, the dosage of the rAAV vector is such that the gene product is stably produced for about 2 months. In some embodiments, the dosage of the rAAV vector is such that the gene product is stably produced for about 3 months. In some embodiments, the dosage of the rAAV vector is such that the gene product is stably produced for about 4 months. In some embodiments, the dosage of the rAAV vector is such that the gene product is stably produced for about 5 months. In some embodiments, the dosage of the rAAV vector is such that the gene product is stably produced for about 6 months. In some embodiments, the dosage of the rAAV vector is such that the gene product is stably produced for about 9 months. In some embodiments, the dosage of the rAAV vector is such that the gene product is stably produced for about 12 months.

[0343] In some embodiments, the method described herein includes administering an effective amount of rAAV vectors described herein to an individual, wherein the rAAV vector includes a polynucleotide encoding at least one gene product (such as TRX and / or PDI). In some embodiments, the method includes delivering the first dose and the rAAV vector of one or more subsequent doses. One or more subsequent doses are administered after a period of time after the first dose. In some embodiments, the time period between the first dose and the next subsequent dose is at least 1 day, at least 3 days, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 1 month, at least 2 months, at least 4 months, at least 6 months, at least 9 months, at least 12 months or longer. In some embodiments, the time period between the first dose and the next subsequent dose is between 1-7 days, between 1-4 weeks, between 2-6 weeks, between 4-8 weeks, between 1-3 months, between 2-4 months, between 3-6 months, between 4-12 months, between 6-24 months. In some embodiments, the time period between one or more subsequent doses is at least 1 day, at least 3 days, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 1 month, at least 2 months, at least 4 months, at least 6 months, at least 9 months, at least 12 months or longer. In some embodiments, the time period between one or more subsequent doses is between 1-7 days, between 1-4 weeks, between 2-6 weeks, between 4-8 weeks, between 1-3 months, between 2-4 months, between 3-6 months, between 4-12 months, between 6-24 months.

[0344] In some embodiments, the method comprises a first dose and one or more subsequent doses of rAAV vectors. In some embodiments, one or more subsequent doses are administered after a period of time after the first dose. This time period between the first dose and the next subsequent dose is at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours or at least 8 hours. The time period between the first dose and the next subsequent dose is between 1-3 hours, 2-4 hours, 3-6 hours or 4-8 hours. The time period between one or more subsequent doses is at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours or at least 8 hours. The time period between one or more subsequent doses is between 1-3 hours, 2-4 hours, 3-6 hours or 4-8 hours.

[0345] Pharmaceutical compositions and kits

[0346] In some embodiments, the present disclosure provides a pharmaceutical composition comprising a rAAV vector described herein. In some embodiments, a pharmaceutical composition comprises a rAAV vector described herein, and a pharmaceutically acceptable carrier, delivery agent, or excipient.

[0347] In some embodiments, the present disclosure provides a use of a rAAV vector or pharmaceutical composition described herein for the manufacture of a medicament for treating an ocular disease, disorder, or condition. In some embodiments, the present disclosure provides a use of a rAAV vector or pharmaceutical composition, which is provided herein or can be adapted for use in treating an ocular disease, disorder, or condition.

[0348] In some embodiments, the pharmaceutically acceptable carrier comprises phosphate buffered saline. In some embodiments, the pharmaceutical composition is formulated to be compatible with its intended route of administration (e.g., within the lacrimal gland). In some embodiments, the pharmaceutical composition is formulated for administration into the lacrimal gland. In some embodiments, the pharmaceutical composition is formulated for administration onto the surface of the eye.

[0349] In some embodiments, the present disclosure provides a pharmaceutical composition for treating an ocular condition of a subject, wherein the pharmaceutical composition comprises a vector encoding an oxidoreductase as described herein (e.g., any rAAV vector as described herein) and a pharmaceutically acceptable carrier. Any concentration of rAAV vector suitable for effectively transducing or transfecting cells of the eye, lacrimal gland, meibomian gland, and / or trabecular meshwork cells can be prepared for contacting cells of the eye, lacrimal gland, meibomian gland, and / or trabecular meshwork in vitro or in vivo. For example, a concentration of 10 8 vector genomes / mL or higher of rAAV, e.g. 5×10 8 vector genomes / mL, 10 9 vector genomes / ml, 5×10 9 vector genomes / mL, 10 10 vector genomes / ml, 5×10 10 vector genomes / mL, 10 11 vector genomes / ml, 5×10 11 vector genomes / mL, 10 12 vector genomes / ml, 5×10 12 vector genomes / mL, 10 13 vector genomes / ml, 1.5×10 13 vector genomes / ml, 3×10 13 vector genomes / ml, 5×10 13 vector genomes / ml, 7.5×10 13 vector genomes / ml, 9×10 13 vector genomes / ml, 1×10 14 vector genomes / ml, 5×10 14 vector genomes / mL or higher, but usually no more than 1×10 15Similarly, any total number of rAAV vectors suitable for providing appropriate transduction or transfection of cells of the eye, lacrimal gland, meibomian gland, and / or trabecular meshwork to impart a desired effect or treat a disease may be administered to a mammal or to the eye of a primate. In various preferred embodiments, at least 10 vector genomes per ml are injected into each eye. 5 , 2.5×10 5 , 5×10 5 , 7.5×10 5 , 10 6 , 2.5×10 6 , 5×10 6 , 7.5×10 6 , 10 7 , 2.5×10 7 , 5×10 7 , 7.5×10 7 , 10 8 , 2.5×10 8 , 5×10 8 , 7.5×10 8 , 10 9 , 2.5×10 9 , 5×10 9 , 7.5×10 9 , 10 10 , 2.5×10 10 , 5×10 10 , 7.5×10 10 , 10 11 , 2.5×10 11 , 5×10 11 , 7.5×10 11 , 10 12 , 2.5×10 12 , 5×10 12 , 7.5×10 12 , 10 13 , 2.5×10 13 , 5×10 13 , 7.5×10 13 , 10 14 , 2.5×10 14 , 5×10 14 , 7.5×10 14 , 10 15 , 2.5×10 15 , 5×10 15 , or 7.5×10 15 , 10 16 , 2.5×10 16 , 5×10 16 or 7.5×10 16rAAV vectors or more, but usually no more than 1×10 15 For example, in some embodiments, about 1×10 9 About 1×10 10 , about 1×10 10 About 1×10 11 , about 1×10 11 About 1×10 12 , about 1×10 12 About 1×10 13 , or about 1×10 13 About 1×10 15 In some aspects, the total number of rAAV vectors administered to the eye of the treated human individual or animal may comprise a concentration within the range defined by any of the concentration pairs described in this paragraph. Any suitable number of rAAV vectors may be administered to the mammal or primate eye. In some embodiments, the method comprises a single administration; in other embodiments, multiple administrations may be performed over time as deemed appropriate by the attending clinician.

[0350] In some embodiments, a suitable amount or concentration of the rAAV vector (or any other TRX and / or PDI expression construct described herein) in the therapeutic formulation may be a concentration effective to express 100 pg / mL to 50 μg / mL TRX and / or PDI in the tear film of the individual following administration of the composition to the individual. 45, 46, 47, 48, 49, or 50 μg / mL TRX and / or PDI (or a concentration within a range defined by any of the foregoing values) is expressed in the tear film of a subject. Expression can be measured, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 hours after administration or after a longer duration, for example after 1, 2, 3, 4 or 5 days.

[0351] The rAAV vector can be formulated into any suitable unit dose, including but not limited to 1×10 8 vector genomes or more, e.g. 1×10 9 , 1×1010 , 1×10 11 , 1×10 12 or 1×10 13 vector genomes or more, in some cases 1 × 10 14 vector genomes, but usually no more than 4 × 10 15 In some embodiments, the viral vector is formulated into any suitable unit dose, including but not limited to 1×10 7 , 1×10 8 , 1×10 9 , 1×10 10 , 1×10 11 , 1×10 12 or 1×10 13 In some embodiments, the unit dose is up to about 5×10 15 vector genomes, e.g. 1×10 14 vector genomes or lower, e.g. 1×10 13 , 1×10 12 , 1×10 11 , 1×10 10 or 1×10 9 vector genomes or lower, in some cases 1 × 10 8 vector genomes or less, and usually not less than 1×10 8 In some embodiments, the unit dose is 1×10 10 Up to 1×10 11 In some cases, the unit dose is 1×10 10 Up to 3×10 12 In some embodiments, the unit dose is 1×10 9 Up to 3×10 13 In some embodiments, the unit dose is 1×10 8 Up to 3×10 14 In some embodiments, the rAAV vector comprises an AAV capsid and an expression cassette configured to express TRX and / or PDI, and the unit dose is formulated to be at least, at most, exactly, or about the following concentration: 1×10 5 , 1×10 6 , 1×10 7 , 1×10 8 , 1×10 9 , 1×10 10 , 1×10 11 , 1×10 12 , 1×10 13 , 1×10 14, 1×10 15 or 1×10 16 rAAV vector, or a concentration within the range defined by the concentration pairs described in this paragraph.

[0352] In some embodiments, the present disclosure provides a vector comprising a plasmid configured to express TRX and / or PDI, and the unit dose is formulated to be at least, at most, exactly, or about the following concentration: 1×10 7 , 1×10 8 , 1×10 9 , 1×10 10 , 1×10 11 , 1×10 12 , 1×10 13 , 1×10 14 or 1×10 15 A plasmid.

[0353] In some embodiments, the unit dose of the pharmaceutical composition can be measured using the multiplicity of infection (MOI). By MOI, it means the ratio or multiple of the viral genome of the vector or rAAV vector provided herein to the cells to which the nucleic acid can be delivered. In some embodiments, the MOI can be 1×10 6 In some embodiments, the MOI may be 1×10 5 Up to 1×10 7 In some cases, the MOI may be 1×10 4 Up to 1×10 8 In some embodiments, the recombinant virus of the present disclosure is at least about 1×10 1 , 1×10 2 , 1×10 3 , 1×10 4 , 1×10 5 , 1×10 6 , 1×10 7 , 1×10 8 , 1×10 9 , 1×10 10 , 1×10 11 , 1×10 12 , 1×10 13 , 1×10 14 , 1×10 15 , 1×10 16 , 1×10 17 and 1×10 18 In some embodiments, the MOI of the recombinant virus of the present disclosure is 1×10 8 Up to 3×10 14In some embodiments, the recombinant viruses of the present disclosure have an MOI of at most about 1×10 1 , 1×10 2 , 1×10 3 , 1×10 4 , 1×10 5 , 1×10 6 , 1×10 7 , 1×10 8 , 1×10 9 , 1×10 10 , 1×10 11 , 1×10 12 , 1×10 13 , 1×10 14 , 1×10 15 , 1×10 16 , 1×10 17 and 1×10 18 MOI.

[0354] In some embodiments, the amount of the pharmaceutical composition comprises about 1×10 8 About 1×10 15 rAAV vectors, about 1×10 9 About 1×10 14 rAAV vectors, about 1×10 10 About 1×10 13 rAAV vectors, or about 1×10 11 About 3×10 12 rAAV vectors.

[0355] In preparing the subject rAAV compositions, any host cell for producing rAAV vectors can be used, including, for example, mammalian cells (e.g., 293 cells), insect cells (e.g., SF9 cells), microorganisms, and yeast. The host cell can also be a packaging cell, in which the AAV rep and cap genes are stably maintained in the host cell or production cell, which is stably maintained and encapsulated with the rAAV vector genome. Exemplary packaging and production cells are derived from SF-9, 293, A549, or HeLa cells. The rAAV vector is purified and formulated using standard techniques known in the art.

[0356] In some embodiments, the disclosure provides uses of the rAAV vectors described herein for the manufacture of a medicament. In some embodiments, the disclosure provides uses of the rAAV vectors described herein for the manufacture of a medicament for use in the methods described herein.

[0357] In some embodiments, the present disclosure provides a kit comprising the rAAV herein and instructions for use. In some embodiments, the kit comprises the rAAV herein and a pharmaceutical package insert containing instructions for use of the kit. In some embodiments, the kit comprises the rAAV herein and a pharmaceutically acceptable carrier, or a pharmaceutical composition comprising the rAAV, and instructions for treating a disease, disorder, or condition described herein in an individual in need thereof or delaying its progression.

[0358] Exemplary Embodiments

[0359] Example I-1. A recombinant adeno-associated virus (rAAV) vector comprising an AAV capsid and an expression cassette comprising a polynucleotide encoding an oxidoreductase operably linked to a promoter.

[0360] Example I-2. The rAAV vector of Example I-1, wherein the oxidoreductase is thioredoxin (TRX).

[0361] Embodiment I-3. The rAAV vector according to Embodiment I-1, wherein the oxidoreductase is protein disulfide isomerase (PDI).

[0362] Embodiment I-4. The rAAV vector of Embodiment I-3, wherein the polynucleotide comprises a sequence encoding a protein that is at least 95% identical to SEQ ID NO: 26.

[0363] Embodiment I-5. The rAAV vector of Embodiment I-2, wherein the polynucleotide comprises a sequence encoding a protein that is at least 95% identical to SEQ ID NO: 25.

[0364] Embodiment I-6. The rAAV vector of Embodiment I-2 or I-5, wherein the polynucleotide comprises a sequence that is at least 95% identical to SEQ ID NO: 2.

[0365] Embodiment I-7. The rAAV vector of Embodiment I-3 or I-4, wherein the polynucleotide comprises a sequence that is at least 95% identical to SEQ ID NO: 30.

[0366] Embodiment I-8. The rAAV vector according to any one of Embodiments I-1 to I-7, wherein the promoter is a CMV promoter comprising the nucleotide sequence set forth in SEQ ID NO: 17.

[0367] Embodiment 1-9. The rAAV vector of any one of Embodiments 1-8, wherein the expression cassette comprises the CMV promoter and the CMV enhancer.

[0368] Embodiment I-10. The rAAV vector of any one of Embodiments I-1 to I-9, wherein the expression cassette comprises a polyadenylation (poly A) sequence.

[0369] Embodiment I-11. The rAAV vector according to Embodiment I-10, wherein the poly A sequence is a BGH poly A sequence.

[0370] Embodiment I-12. The rAAV vector of any one of Embodiments I-1 to I-11, wherein the expression cassette comprises a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE).

[0371] Embodiment I-13. The rAAV vector of any one of Embodiments I-1 to I-12, wherein the expression cassette comprises a Kozak sequence.

[0372] Example 1-14. A composition comprising a rAAV vector, wherein the rAAV vector comprises:

[0373] AAV capsid, and

[0374] An expression cassette, wherein the expression cassette comprises a polynucleotide comprising a nucleotide sequence that shares at least 95% identity with a nucleotide sequence comprising SEQ ID NO: 2, and wherein the polynucleotide is linked to a promoter.

[0375] Embodiment I-15. The rAAV vector of any one of Embodiments I-1 to I-11, wherein the expression cassette is flanked by two inverted terminal repeats (ITRs).

[0376] Embodiment I-16. The rAAV vector of Embodiment I-12, wherein the ITR is an AAV2 ITR.

[0377] Embodiment I-17. The rAAV vector of any one of Embodiments I-1 to I-2 and I-5 to I-13 or the composition of any one of Embodiments I-14 to I-16, wherein the expression cassette comprises a nucleotide sequence that shares at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO: 16.

[0378] Embodiment I-18. An rAAV vector according to any one of Embodiments I-1 to I-17, wherein the AAV shell comprises a VP3 that shares at least 95%, 98% or 100% identity with AAV2 VP3 (SEQ ID NO:8), AAV5 VP3 (SEQ ID NO:10), AAV8 VP3 (SEQ ID NO:12) or AAV9 VP3 (SEQ ID NO:14).

[0379] Embodiment I-19. The rAAV vector of any one of Embodiments I-1 to I-17, wherein the AAV capsid comprises a VP3 that shares at least 95%, 98% or 100% identity with AAV9 (SEQ ID NO: 14).

[0380] Embodiment 1-20. A composition comprising a rAAV vector, wherein the rAAV vector comprises:

[0381] AAV2, AAV5, AAV8 or AAV9 capsid, and

[0382] An expression cassette, wherein the expression cassette comprises a polynucleotide comprising a nucleotide sequence that shares at least 95% identity with a nucleotide sequence comprising SEQ ID NO: 2, and wherein the polynucleotide is linked to a promoter.

[0383] Embodiment 1-21. A composition comprising a rAAV vector, wherein the rAAV vector comprises:

[0384] AAV2, AAV5, AAV8 or AAV9 capsid, and

[0385] An expression cassette, wherein the expression cassette comprises a polynucleotide sequence that shares at least 95% identity with SEQ ID NO:2.

[0386] Embodiment I-22. A composition according to Embodiment I-20 or I-21, wherein the AAV capsid is AAV2.

[0387] Embodiment I-23. A composition according to Embodiment I-20 or I-21, wherein the AAV capsid is AAV5.

[0388] Embodiment I-24. A composition according to Embodiment I-20 or I-21, wherein the AAV capsid is AAV9.

[0389] Embodiment I-25. The rAAV vector or composition of any one of Embodiments I-1 to I-24, wherein the polynucleotide comprises a sequence encoding a signal peptide.

[0390] Embodiment I-26. A pharmaceutical composition comprising the rAAV vector or composition according to any one of Embodiments I-1 to I-25, and a pharmaceutically acceptable carrier.

[0391] Embodiment I-27. The pharmaceutical composition according to embodiment I-26, wherein the composition comprises about 1×10 7 About 1×10 14genome copies / ml of the rAAV vector.

[0392] Embodiment I-28. A pharmaceutical composition according to Embodiment I-26, wherein the composition comprises about 1×10 12 About 6.2×10 12 genome copies / ml of the rAAV vector.

[0393] Embodiment 1-29. A method of treating an ocular condition in a subject in need thereof, the method comprising administering to an eye of the subject a therapeutically effective amount of a pharmaceutical composition according to any one of Embodiments 1-26 to 1-28.

[0394] Embodiment I-30. The method of Embodiment I-29, wherein the pharmaceutical composition is delivered to an ocular gland of the subject.

[0395] Embodiment I-31. The method of Embodiment I-29 or I-30, wherein the pharmaceutical composition is delivered to the lacrimal gland.

[0396] Embodiment I-32. The method of any one of Embodiments I-29 to I-31, wherein the pharmaceutical composition is delivered to the accessory lacrimal gland.

[0397] Embodiment I-33. The method of Embodiment I-32, wherein the accessory lacrimal glands are meibomian glands.

[0398] Embodiment I-34. The method of any one of Embodiments I-29 to I-33, wherein the pharmaceutical composition is delivered to the trabecular meshwork.

[0399] Embodiment I-35. The method according to any one of embodiments I-29 to I-34, wherein about 1×10 9 About 1×10 10 , about 1×10 10 About 1×10 11 , about 1×10 11 About 1×10 12 , about 1×10 12 About 1×10 13 , or about 1×10 13 About 1×10 15 genomic copies of the rAAV vector.

[0400] Embodiment 1-36. The method of any one of embodiments 1-29 to 1-35, wherein the ocular condition is associated with increased oxidative stress.

[0401] Embodiment 1-37. The method of any one of embodiments 1-29 to 1-35, wherein the ocular condition is associated with loss of expression and / or function of one or more oxidoreductases.

[0402] Embodiment 1-38. The method of any one of Embodiments 1-29 to 1-35, wherein the ocular condition is associated with loss of TRX expression and / or function.

[0403] Embodiment 1-39. The method of any one of Embodiments 1-29 to 1-35, wherein the ocular condition is associated with loss of PDI expression and / or function.

[0404] Embodiment 1-40. The method of any one of Embodiments 1-29 to 1-35, wherein the ocular condition is characterized by loss of near vision.

[0405] Embodiment 1-41. The method of any one of embodiments 1-29 to 1-40, wherein the ocular condition is presbyopia.

[0406] Embodiment 1-42. The method of any one of embodiments 1-29 to 1-40, wherein the ocular condition is cataract formation.

[0407] Embodiment 1-43. The method of any one of Embodiments 1-29 to 1-40, wherein the ocular condition is ocular hypertension.

[0408] Embodiment 1-44. The method of any one of embodiments 1-29 to 1-40, wherein the ocular condition is meibomian gland dysfunction (MDI).

[0409] Embodiment 1-45. The method of any one of Embodiments 1-29 to 1-40, wherein the ocular condition is glaucoma.

[0410] Embodiment 1-46. The method of any one of Embodiments 1-29 to 1-45, wherein the method results in expression of the oxidoreductase in cells of the lacrimal gland and / or accessory lacrimal gland and / or in the trabecular meshwork.

[0411] Embodiment 1-47. The method of any one of Embodiments 1-29 to 1-46, wherein the method results in expression of TRX in cells of the lacrimal gland and / or accessory lacrimal gland and / or in the trabecular meshwork.

[0412] Embodiment 1-48. The method of any one of Embodiments 1-29 to 1-47, wherein the method causes secretion of TRX into the tear film and / or onto the ocular surface of the subject.

[0413] Embodiment 1-49. The method of any one of embodiments 1-29 to 1-48, wherein said method results in improvement in one or more symptoms of said ocular condition.

[0414] Embodiment 1-50. The method of any one of embodiments 1-29 to 1-49, wherein the method results in improved vision.

[0415] Embodiment 1-51. The method of any one of Embodiments 1-29 to 1-50, wherein the method results in a reduced need for corrective lenses.

[0416] Embodiment 1-52. The method of any one of Embodiments 1-29 to 1-51, wherein said method results in a delay in progression of said condition.

[0417] Embodiment I-53. A method according to Embodiment I-52, wherein the method delays the progression of the condition in the individual by about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about 50%, about 50% to about 60%, about 60% to about 70%, about 70% to about 80%, about 80% to about 90%, about 90% to about 95%, or more than about 95% compared to a control individual.

[0418] Embodiment I-54. The method of any one of Embodiments I-29 to I-53, wherein the method delays the onset of the condition by about 6 months to about 12 months, about 12 months to about 18 months, about 18 months to about 24 months, about 2 years to about 3 years, about 3 years to about 4 years, about 4 years to about 5 years, about 5 years to about 6 years, about 6 years to about 7 years, about 7 years to about 8 years, about 8 years to about 9 years, about 9 years to about 10 years, about 10 years to about 15 years, about 15 years to about 20 years, or more than 20 years, compared to a control individual.

[0419] Embodiment I-55. The method of Embodiment I-53 or I-54, wherein the control individual is an age-matched individual not treated with a rAAV vector comprising an expression cassette comprising a polynucleotide encoding TRX.

[0420] Embodiment 1-56. A method according to any one of Embodiments 1-29 to 1-55, wherein the individual required corrective lenses prior to administration of the rAAV, and the administration results in a constant requirement for the strength of corrective lenses for at least about 6 months to about 12 months, about 12 months to about 18 months, about 18 months to about 24 months, about 2 years to about 3 years, about 3 years to about 4 years, about 4 years to about 5 years, about 5 years to about 6 years, about 6 years to about 7 years, about 7 years to about 8 years, about 8 years to about 9 years, about 9 years to about 10 years, about 10 years to about 15 years, about 15 years to about 20 years, or more than 20 years after administration of the rAAV.

[0421] Embodiment 1-57. The method of any one of Embodiments 1-29 to 1-56, wherein after administration of the rAAV vector, the individual's vision remains unchanged for at least about 6 months to about 9 months, about 9 months to about 12 months, about 12 months to about 15 months, about 15 months to about 18 months, about 18 months to about 21 months, about 21 months to about 24 months, about 2 years to about 3 years, about 3 years to about 4 years, about 4 years to about 5 years, about 5 years to about 6 years, about 6 years to about 7 years, about 7 years to about 8 years, about 8 years to about 9 years, about 9 years to about 10 years, about 10 years to about 15 years, about 15 years to about 20 years, or more than 20 years.

[0422] Embodiment 1-58. The method of any one of Embodiments 1-29 to 1-57, wherein the method further comprises administering one or more additional therapeutic agents.

[0423] Embodiment 1-59. The method of any one of embodiments 1-29 to 1-58, wherein the subject is a human.

[0424] Embodiment I-60. A pharmaceutical composition according to any one of Embodiments I-26 to I-28, for use in a method of treating an ocular condition in a subject in need thereof, comprising administering an effective amount of the pharmaceutical composition to an eye of the subject.

[0425] Embodiment 1-61. A pharmaceutical composition according to any one of Embodiments 1-26 to 1-28, for use in the manufacture of a medicament for treating an ocular condition in a subject in need thereof.

[0426] Embodiment 1-62. A compound for use in treating an ocular condition in a subject, the treatment comprising administering a composition according to any one of Embodiments 1-1 to 1-25 or a pharmaceutical composition of an rAAV or an rAAV vector according to any one of Embodiments 1-26 to 1-28, and a pharmaceutically acceptable carrier.

[0427] Embodiment 1-63. A kit comprising the rAAV vector or composition of any one of Embodiments 1-1 to 1-25 or the pharmaceutical composition of any one of Embodiments 1-26 to 1-28, and a pharmaceutically acceptable carrier, and instructions for use in treating an eye condition in an individual, wherein the treatment comprises administering the pharmaceutical composition to the eye of the individual.

[0428] Embodiment 1-64. A kit comprising the rAAV vector or composition of any one of Embodiments 1-1 to 1-25 or the pharmaceutical composition of any one of Embodiments 1-26 to 1-28, and a pharmaceutically acceptable carrier, and instructions for treating presbyopia in an individual, wherein the treatment comprises administering the pharmaceutical composition to the eye of the individual.

[0429] Embodiment 1-65. A kit comprising the rAAV vector or composition of any one of Embodiments 1-1 to 1-25 or the pharmaceutical composition of any one of Embodiments 1-26 to 1-28, and a pharmaceutically acceptable carrier, and instructions for use in treating cataract formation in an individual, wherein the treatment comprises administering the pharmaceutical composition to the eye of the individual.

[0430] Embodiment 1-66. A kit comprising the rAAV vector or composition of any one of Embodiments 1-1 to 1-25 or the pharmaceutical composition of any one of Embodiments 1-26 to 1-28, and a pharmaceutically acceptable carrier, and instructions for use for treating loss of visual accommodation in an individual, wherein the treatment comprises administering the pharmaceutical composition to the eye of the individual.

[0431] Embodiment 1-67. A kit comprising the rAAV vector or composition of any one of Embodiments 1-1 to 1-25 or the pharmaceutical composition of any one of Embodiments 1-26 to 1-28, and a pharmaceutically acceptable carrier, and instructions for use in treating elevated intraocular pressure in an individual, wherein the treatment comprises administering the pharmaceutical composition to the eye of the individual.

[0432] Embodiment 1-68. A kit comprising the rAAV vector or composition of any one of Embodiments 1-1 to 1-25 or the pharmaceutical composition of any one of Embodiments 1-26 to 1-28, and a pharmaceutically acceptable carrier, and instructions for treating meibomian gland dysfunction (MGD) in an individual, wherein the treatment comprises administering the pharmaceutical composition to an eye of the individual.

[0433] Embodiment 1-69. The method of any one of Embodiments 1-29 to 1-59, further comprising administering one or more additional therapeutic agents to the individual.

[0434] Embodiment I-70. The method of Embodiment I-69, wherein the one or more additional therapeutic agents increase tear production.

[0435] Embodiment 1-71. The method of Embodiment 1-70, wherein the one or more additional therapeutic agents that increase tear production are administered by topical nasal administration.

[0436] Embodiment 1-72. The method of embodiment 1-71, wherein the topical nasal administration is by intranasal spray.

[0437] Embodiment 1-73. The method of any one of Embodiments 1-70 to 1-72, wherein the one or more additional therapeutic agents that increase tear production comprises a nicotinic acetylcholine receptor (nAChR) agonist or a pharmaceutically acceptable salt thereof.

[0438] Embodiment 1-74. The method of Embodiment 1-73, wherein the nAChR agonist is a full agonist of an nAChR subtype selected from the group consisting of α4β2, α3β4, α3α5β4, α4α6β2, and combinations thereof.

[0439] Embodiment I-75. A method according to Embodiment I-73 or I-74, wherein the nAChR agonist is varenicline or a pharmaceutically acceptable salt thereof.

[0440] Embodiment I-76. The method according to Embodiment I-73 or I-74, wherein the nAChR agonist is (R)-5-((E)-2-pyrrolidin-3-ylvinyl)pyrimidine or a pharmaceutically acceptable salt thereof.

[0441] Embodiment I-77. The method of any one of Embodiments I-70 to I-76, wherein the one or more additional therapeutic agents that increase tear production are administered before or after administering the pharmaceutical composition.

[0442] Embodiment I-78. The method of any one of Embodiments I-70 to I-776, wherein the one or more additional therapeutic agents that increase tear production are administered about 1 week after administration of the pharmaceutical composition.

[0443] Embodiment I-79. The method of any one of Embodiments I-70 to I-78, which results in expression of TRX in the tear film and / or cornea of ​​the individual following administration of the pharmaceutical composition.

[0444] Embodiment I-80. The method of Embodiment I-79, wherein expression of TRX in the tear film and / or cornea is increased for a predetermined amount of time compared to administration of the pharmaceutical composition without the one or more other therapeutic agents that increase tear production.

[0445] Embodiment I-81. The method of Embodiment I-80, wherein the predetermined amount of time is about 5 minutes.

[0446] Embodiment 1-82. The method of Embodiment 1-80, wherein the predetermined amount of time is about 1 hour.

[0447] Embodiment 1-83. The method of any one of Embodiments 1-29 to 1-82, wherein the individual is a human.

[0448] Examples

[0449] The following specific examples are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.

[0450] Example 1A: Expression of Thioredoxin

[0451] The expression of TRX was determined by ELISA using an expression cassette (eg, Figure 1 Expression of thioredoxin (TRX) in cells transfected with an rAAV vector (described in and whose sequence is SEQ ID NO: 16). The AAV transfer plasmid contains 5′ and 3′ AAV2 ITRs (SEQ ID NOs: 22 and 23, respectively). Between the 5′ and 3′ ITRs is a cDNA encoding a human TRX polypeptide and a cDNA for elements for providing optimal expression amounts of the TRX polypeptide. The encoded human TRX polypeptide has the amino acid sequence set forth in SEQ ID NO: 1. For improved expression in human cells, the cDNA encoding the human TRX polypeptide was codon-optimized and has the nucleotide sequence set forth in SEQ ID NO: 2. The nucleotide sequence from 5′ ITR to 3′ ITR is set forth in SEQ ID NO: 16. Table 6 shows the ELISA results obtained on the standard curve and on samples from two separate cell lines (triplicates).

[0452] Table 6: Expression of TRX

[0453] standard OD450 Sample ID OD450 Calculate concentration 20 0.279 HeLa-1 0.242 10.07969 8 0.225 HeLa-2 0.268 15.12382 3.2 0.128 HeLa-3 0.306 NaN 1.28 0.088 ARPE19-1 0.388 NaN 0.512 0.062 ARPE19-2 0.41 NaN 0.205 0.061 ARPE19-3 0.418 NaN 0.082 0.058 Culture medium + cells 0.193 5.920729 0 0.064 SFM 0.056 0

[0454] Next, the expression of AAV thioredoxin plasmids in 293T cells was evaluated. 5293T cells were plated in 6-well plates and transfected with 2.5 μg of AAV.TRX plasmid DNA using Lipofectamine 3000 according to the manufacturer's protocol. The cells were grown and then incubated at 5.5×10 5 Twenty-four hours after transfection, the transfected and untransfected cells were plated on 35 mm glass-bottom tissue culture plates without changing the culture medium.

[0455] MVKQIESKTAFQEALDAAGDKLVVVDFSATWCGPCKMIKPFFHSLSEKYSNVIFLEVDVDDCQDVASECEVKCMPTFQFFKKGQKVGEFSGANKEKLEATINELV; SEQ ID NO: 1) and secondary antibody tagged with AlexaFluor 488. Cells were counterstained with 4′,6-dicarboxamidino-2-phenylindole (DAPI) and then imaged on a fluorescence microscope at 20× and 40× magnification. The results shown in FIG4 confirm the expression of thioredoxin, as indicated by the bright and punctate signals present in AAV-thioredoxin transfected cells versus very little signal in untransfected control cells. 293T cells do express some level of thioredoxin, even if the amount is smaller than that of AAV.TRX plasmid transfected cells, as shown in FIG4.

[0456] Given the endogenous expression of thioredoxin in 293T cells, a study with four biological replicates was performed to compare the expression and secretion levels of thioredoxin in untransfected 293T cells and AAV.TRX plasmid DNA transfected cells. The cells were cultured and the culture medium was collected. The ELISA results of untransfected and transfected 293T cells are shown in Figure 5 A to 5B. It should be noted that the conditioned medium of non-transfected cells was collected 4 days after transfection to allow appropriate accumulation of thioredoxin for detection in ELISA; while the conditioned medium of AAV.TRX transfected cells was collected exactly 24 hours after transfection. In addition, given the amount of expression, Figure 5 The y-axes in A to 5B are clearly different. In the conditioned medium, thioredoxin was expressed and secreted by untransfected cells to an average concentration of 19.86 ng / mL, and AAV.TRX-transfected cells contained 14.3 μg / mL. The thioredoxin standard curve generated by ELISA analysis of undiluted and diluted conditioned medium (data not shown) had an R of 0.999. 2 value.

[0457] Western blot analysis was performed on 293T cells transfected with AAV.TRX. Whole cell extracts were run on Western blots 48 hours after transfection and thioredoxin and GAPDH ( Figure 6 ) to assess the thioredoxin content in whole cell extracts with GAPDH protein loading control for comparison of endogenous and AAV.TRX driven thioredoxin expression. SDS-PAGE was run with 30 μg protein / lane, and a polyclonal primary antibody for thioredoxin was used together with a secondary anti-rabbit IgG antibody with a horseradish peroxidase (HRP) tag for anti-thioredoxin protein blotting. In addition, a protein blot with a loading control using an antibody for GAPDH was run to standardize protein loading. Band density analysis was performed using Image J software developed by the National Institute of Health. Based on density analysis, whole cell extracts transfected with AAV.TRX plasmid DNA produced 3.5 times more thioredoxin than untransfected 293T cells.

[0458] Example 1B: Expression of Thioredoxin, Additional Studies

[0459] A secondary Western blot analysis was performed using an anti-Thioredoxin primary antibody (Thermo Fisher catalog number 14999-1-AP) and an anti-rabbit IgG HRP secondary antibody (Promega catalog number A5316). The results are shown in Fig.13 The results showed that the thioredoxin transgene product was present in transfected 293T cells at 2, 4, and 6 hours, but not in untransfected cells. In addition, whole cell extracts were collected 24 hours after transfection and analyzed by Western blot using an actin loading control. Transfected cells (AAV.TXN) showed a large amount of Fig.14 Protein detected with anti-thioredoxin primary antibody at relevant molecular weight as demonstrated in .

[0460] To assess the functional activity of the expressed transgenic product, a thioredoxin activity assay (Cayman Chemical Thioredoxin Fluorometric Activity Assay, catalog number 500228) was performed on the conditioned medium and whole cell lysates of the transfected cells, using untransfected cells as controls. The principle of the assay is to assess the ability of the transgenic product to reduce disulfide bonds. This is achieved by a substrate consisting of insulin to which disulfide bonds link it to eosin. The reduction of this disulfide bond releases eosin from insulin, which can be detected at a wavelength of 560 nm. The increase in thioredoxin activity is proportional to the rate of increase in fluorescence (i.e., eosin is released from the insulin-eosin substrate). The thioredoxin activity was calculated using the following equation, as recommended by the manufacturer:

[0461]

[0462] For the analysis, 293T cells were transfected with AAV-TXN and washed after 18 hours and the medium was replaced with OPTImem cell culture medium. The medium was collected for analysis 6 hours after supplementation and readings were collected every 60 seconds for 60 minutes, with recombinant thioredoxin serving as a positive control and eosin used to generate a standard curve of fluorescence at 560 nm. Fig.15 It was confirmed that the thioredoxin transgenic product expressed and secreted into the conditioned medium was active and capable of reducing disulfide bonds of eosin-labeled insulin substrate. The recombinant thioredoxin (recombinant Trx) positive control was loaded into the assay at 120 ng / well and had a thioredoxin activity of 62.45 nM / min. 293T cells transfected with the thioredoxin transgenic plasmid exhibited 56.8 nM / min of thioredoxin activity, and untransfected 293T cells showed an endogenous thioredoxin activity level of 16.90 nM / min. This functional analysis confirmed that the thioredoxin activity level was 10.1% (v / v) from the 293T cells transfected with the thioredoxin transgenic plasmid. Figure 1 The expression, secretion and function of the transgene products of the constructs depicted in the present invention are demonstrated, and the ability of the expressed thioredoxin transgene product to reduce disulfide bonds after secretion from the target cells is confirmed. This finding supports the ability of the transgene product to diffuse from the cells expressing the transgene and to reduce disulfide bonds formed by oxidative stress.

[0463] Example 2: Intra-lacrimal gene therapy using rAAV vectors

[0464] This example shows a 9-day pilot study of a single dose of rAAV vector administered as an intra-lacrimal injection to Dutch-Belted rabbits, followed by a single dose of varenicline as an intranasal dose. It evaluates the efficacy and tolerability of a group of rAAV vector embodiments administered once by injection into the lacrimal gland. Two concentrations (1×1012 GC / mL and 6.2×10 12 Each rAAV vector composition in the group was tested at 100 GC / mL. The rAAV vector group included embodiments with capsid proteins having AAV2, AAV5, AAV8, and AAV9 serotypes. The expression cassette delivered by the rAAV vector encodes an enhanced green fluorescent protein (eGFP) transgene ( Figure 7 ). On day 9 after injection of the rAAV vector, the animals were given an intranasal dose of varenicline. Varenicline induces tear production in the animals, so that eGFP delivered to the lacrimal gland by the rAAV vector and expressed in the cells of the lacrimal gland under the control of the CMV promoter will be secreted into the tear film and ocular surface of the animals. Two main objectives will be achieved using the following approach: (1) to test the feasibility of capsid protein serotypes AAV2, AAV5, AAV8 and AAV9 to deliver transgenes to cells in the lacrimal gland, resulting in measurable CMV promoter-driven expression of the transgene in the cells; and (2) to evaluate the feasibility of increasing the relative amount of the transgene encoded by the expression cassette in the tear film of the animals and on their ocular surface.

[0465] Animal studies were performed at the Charles River Laboratories (CRL) facility by CRL staff scientific staff.

[0466] Animal testing systems, husbandry and in-life monitoring

[0467] The animals used in this study were male Dutch-Belted rabbits between 4 and 5 months of age and weighing between 1.3 and 2.3 kg. The animals were acclimated for 10 days prior to the start of treatment. Each animal was housed individually and cared for using a standard care regimen, including conventional environmental conditions, feeding schedules, and veterinary care.

[0468] rAAV vector compositions and formulations

[0469] In this study, a composition set comprising a rAAV vector containing an expression cassette encoding an eGFP transgene operably linked to a CMV promoter was provided for intra-lacrimal injection under the conditions in Table 7. Each composition contained a rAAV vector having a different AAV capsid protein serotype. The compositions were labeled OC-100a-d, each corresponding to a different AAV capsid protein serotype. The dosage formulations for intra-lacrimal injection were prepared using a clean procedure by diluting with phosphate buffered saline solution to the target concentrations described in Table 7 below.

[0470] Table 7. Summary of rAAV compositions

[0471]

[0472] Intra-lacrimal injection of rAAV compositions

[0473] Animals were dosed by intra-lacrimal injection on day 1 of the study. An overview of the formulation concentrations, dose volumes, dose frequencies, and number of animals and lacrimal glands for each composition tested is found in Table 8. Prior to injection, animals were anesthetized with an intramuscular injection of dexmedetomidine (0.25 mg / kg), followed by an isoflurane / oxygen mixture provided by mask to maintain anesthesia as necessary. Topical antibiotics were applied to each eye following dosing. On day 9 of the study, animals were given intranasal administration of varenicline tartrate (50 μL per nostril, 1.2 mg / mL varenicline) to induce tear production.

[0474] Table 8. Overview of experimental design

[0475]

[0476] Biological analysis

[0477] Blood was collected from the ear vessels of all animals on day 1 before dosing, and again on days 8 and 9 (approximately 1 hour after dosing following intranasal dosing). Blood samples were placed on ice until plasma was separated by centrifugation. Plasma samples were separated into 250 μL aliquots and frozen at -80°C for subsequent analysis.

[0478] Schirmer tear test was performed on days 8 and 9 to collect intraocular moisture from the animals. The test strip was placed in the lower eyelid for approximately 1 minute. The paper was removed and placed in the corresponding tube and frozen at -80°C for subsequent analysis.

[0479] Plasma samples and Schirmer test strips were analyzed for eGFP concentration and eGFP mRNA concentration using validated procedures in the Syneos analytical laboratory.

[0480] Immunohistochemistry of lacrimal gland tissue

[0481] Animals were euthanized by intravenous injection of sodium pentobarbital on day 9 after blood and eye water collection. Five sagittal sections of the left eye and sections of the left and right lacrimal glands were prepared for immunohistochemistry (IHC) according to laboratory standard operating procedures. Lacrimal gland IHC samples were stained for eGFP and evaluated microscopically.

[0482] result

[0483] Microscopic evaluation was performed to determine the efficiency of eGFP expression in lacrimal gland tissue following in vivo administration of rAAV compositions. Isolated positive acinar cells in IHC samples had pink to red cytoplasmic staining, indicating GFP expression ( Figures 8A to 8K ; Exemplary staining is indicated by black arrows). 12 GC / mL ( Fig. 8A ) observed positive eGFP expression in rAAV compositions containing AAV2 capsid protein (OC-100a) at 1×10 12 GC / mL( Figure 8B ) and 6.2×10 12 GC / mL( Figures 8C to 8H ) were observed to contain AAV5 capsid protein, and at 6.2×10 12 GC / mL( Figures 8I to 8K ) were observed to contain AAV9 capsid protein.

[0484] in conclusion

[0485] The results of this example show that rAAV vectors can be used to deliver expression cassettes to the lacrimal gland by direct injection. The results also show that rAAV vectors containing capsid proteins having at least AAV2, AAV5, or AAV9 serotypes can be used to deliver expression cassettes to cells within the lacrimal gland. In addition, the results demonstrate that delivery of an expression cassette containing a transgene operably linked to a CAG promoter sequence results in expression of the transgene in cells of the lacrimal gland.

[0486] Example 3: Expression of EGFP transgene delivered by rAAV in porcine lacrimal glands by intra-lacrimal injection

[0487] The goal of the study was to evaluate whether the lacrimal gland could be fully exploited as a method to modify or enrich the tear film with proteins of interest in pigs. Subsequently, in vivo studies were performed to test whether EGFP could be produced in the acinar cells of the lacrimal gland and then secreted into the tear film after delivery of an adenoviral vector consisting of a plasmid encoding eGFP. In order to allow the cDNA encoding EGFP to enter the acinar cells, the approach was to inject the lacrimal gland with an adeno-associated virus (AAV) containing the cDNA encoding secreted EGFP (secEGFP). In order to produce AAVs of each of the 2 different serotypes (2 and 9) for secEGFP, an AAV transfer plasmid was generated that contained the necessary elements for secEGFP expression between the inverted terminal repeats (ITRs). The DNA sequence between the ITRs was encapsulated into the manufactured AAV ( Figure 7 )middle.

[0488] Design Analysis and Methods

[0489] Research-grade AAV for secreted EGFP (serotypes 2 and 9) was synthesized at Sirion and cultured at 5 × 10 12 AAV was provided at a stock concentration. AAV was tested in vitro by CJ Solutions using HEK 293T cells and ELISA to ensure that the manufactured AAV would transduce cells. At Texas A&M University (Texas A&M), eight domestic pigs received a single intra-tear gland injection of EGFP, with the right (OD; right eye (oculus dexter)) gland receiving a low dose and the left (OS; left eye (oculus sinister)) gland receiving a high dose. Six weeks after the first injection, a second injection with high doses of AAV2 and AAV9 was performed. The study evaluated EGFP expression at day 35. After confirmation of tear EGFP content, the study was terminated 8 weeks after the second injection to evaluate the presence of EGFP in the lacrimal gland and to evaluate any potential inflammation or glandular abnormalities. (Tables 9 and 10). In the study, nasal spray administration was applied between weeks 3 and 4 (Table 11).

[0490] Table 9: Research plan for in vivo studies of AAV2-secEGFP and AAV9-secEGFP in domestic pigs.

[0491]

[0492]

[0493] Table 10: AAV injection dose and volume.

[0494]

[0495] vg = viral genome

[0496] Table 11: OC-01 nasal spray administration was performed from day 21 to day 28.

[0497]

[0498] mcg = microgram

[0499] After the second AAV-secEGFP injection, tears were collected from each eye by Schirmer strips on day 82. Tears were collected by placing Schirmer tear test strips in the lower conjunctival sac and leaving them in place for 2 minutes. Tear proteins were extracted from the Schirmer tear test strips and subjected to mesoscale discovery (MSD) analysis to detect the presence of EGFP protein in tears.

[0500] Lacrimal glands were collected on day 103 for ocular histopathology and samples were sent to Zyagen, Inc. (San Diego, CA) for EGFP immunohistochemistry (IHC).

[0501] ELISA results showed that the manufactured AAV serotypes could transduce HEK 293T cells and produce secreted EGFP in vitro. EGFP expression in tear samples was confirmed by MSD analysis 82 days after AAV transduction with eGFP (some levels >400 pg / mL) and by IHC ( Fig. 9 ). IHC indicated that EGFP expression was in acinar cells, and it was observed that AAV2 was more infectious to acinar cells than AAV9. In addition, transduction of ductile epithelial cells of the lacrimal gland injected with AAV9 was observed ( Fig.10 Hematoxylin and eosin staining of porcine lacrimal glands after repeated AAV injections did not reveal any inflammatory infiltrate, atrophy, or edema ( Fig.11 ).

[0502] Porcine lacrimal glands injected with AAV2-secEGFP or AAV9-secEGFP expressed the EGFP transgene product in acinar cells as well as ductile epithelial cells. EGFP expressed in the lacrimal gland was found to be secreted in the tear film. In addition, no safety signals or inflammatory infiltrates were observed in any animal following repeated injections of AAV2 or AAV9, regardless of whether they initially received a low or high dose of AAV during the first injection. The results of this study confirm that the acinar cells of the lacrimal gland are a target for gene therapy approaches to modify and / or enrich the tear film.

[0503] Example 4: Expression of rAAV transgenes in porcine lacrimal glands in combination with OC-1 nasal spray

[0504] This example describes a study in pigs to evaluate the expression of rAAV encoding a model protein after a single intra-lacrimal injection. The goal of the study was to determine the expression of mRNA encoding a model protein (referred to as "Protein_A" in the examples and figures) in the lacrimal glands and the amount of transgenic protein in the tears after a single injection of AAV encoding Protein_A (referred to as "AAV-Protein_A" in the examples and figures) to transduce the lacrimal glands of pigs. The study was further designed to confirm the expression and secretion of Protein_A after intra-lacrimal injection, and to evaluate the relative amount of protein present on the ocular surface after stimulating tear production with varenicline nasal spray. Varenicline ("OC-1") is the following compound:

[0505]

[0506] The AAV-Protein_A plasmid encodes from 5′ to 3′: AAV2 5′ ITR, CMV enhancer / promoter, intronic sequence containing Kozak, open reading frame encoding Protein_A, woodchuck hepatitis virus posttranscriptional regulatory elements, poly A sequence and AAV2 3′ ITR.

[0507] Study parameters are detailed in Table 12. Administration and dosing are shown in Table 13.

[0508] Table 12: Study plan for in vivo evaluation of intra-lacrimal injection of rAAV in domestic pigs

[0509]

[0510]

[0511] Table 13: Injected doses and volumes of AAV

[0512]

[0513] AAV-Protein A stock concentration was 5 × 10 12 vg / mL and were provided in 500 μL aliquots and stored at -20°C (short term, <2 years) or -80°C (long term). In this study, only 1 dose (1×10 11 vg).

[0514] The timeline of pig research is shown in Fig.12 For all pigs (N=14), day 0 was the injection day. On days 7, 14, 21, 28, 35, 42, 60 / 61, and 90, tears were collected from each eye by Schirmer strips (applied for approximately 2 minutes and then removed). The Schirmer strips were immediately cut above the fluid or dye line with a pair of scissors. Next, the bottom of the Schirmer strip (tear saturated) was placed in a microcentrifuge tube and kept on ice until transferred to a refrigerator (-80°C). On days 14 and 42, tear collection was performed first and then the nasal spray dose was applied. Approximately 2 minutes after the nasal spray dose was applied, tear collection for the second day was performed. For days 22 to 28, OC-01 nasal spray was applied to both nostrils twice a day (at least 6 hours between applications) on days 22 to 27, and once on day 28. Tear collection on day 28 will be performed 2 minutes after the nasal spray is applied.

[0515] At day 90, gross lesions were presented and body weights were assessed. In addition, one lacrimal gland was collected from each animal for ocular pathological histology by immunohistochemistry (IHC). After collection of the lacrimal glands, the lacrimal glands were fixed in 10% formalin for 24 to 48 hours (at room temperature), then placed in 70% EtOH and stored at 4°C. The volume in the tube was maintained at 5 times the volume of the tissue, and the tissue was completely immersed.

[0516] The second lacrimal gland was collected, rinsed with phosphate buffered saline and immediately placed in RNA-Later. The samples were snap frozen in liquid nitrogen. ~0.5-1 cm square tissue fragments were collected from the heart, kidney and liver and immediately placed in 2 mL cryovials and snap frozen in liquid nitrogen. Later, the cryovials were stored at -80°C until shipped for mRNA analysis.

[0517] mRNA analysis was performed to analyze the gene expression of AAV-derived protein_A in the lacrimal glands of domestic pigs using the developed one-step duplex RT-qPCR method. Each lacrimal gland was homogenized, and the lysate was loaded into QIASymphony for automatic RNA extraction using silica-based RNA purification, magnetic separation, and enzymatic removal of DNA. The AAV-protein_A vector contains a bovine growth hormone (bGH) poly A sequence in the 3' untranslated region of the transgene. Protein_A mRNA was analyzed using primers and probes targeting the bGH poly A sequence, wherein the sequence is shown in Table 14. Amplification was performed for the 76bp bGH sequence. Using the QuantStudio7Flex real-time PCR system and the one-step duplex RT-qPCR method, the number of bGH mRNA copies of the extracted total RNA sample and the Ct value of the pig endogenous Hprt1 mRNA on a 96-well plate were analyzed (see Table 15). Each plate includes a standard curve, a negative control, and a quality control sample, which are prepared separately to avoid cross contamination. Each standard curve is included in 10 8 , 10 7 , 10 5 , 10 4 , 10 3 , 10 2bGH standard DNA content at 1, 50, 25 and 0 copies / well. RT-qPCR of RNA samples was performed in duplicate wells up to 100 ng / well. The number of bGH mRNA copies for each RT-qPCR well was interpolated from the bGH DNA standard curve (acceptance criterion: R2 ≥ 0.980). A two-fold doubling step was used to adjust the interpolated single-stranded (ss) mRNA from the double-stranded (ds) standard curve, and the average number of copies of the two replicate wells will be reported as the number of copies of ss bGH mRNA / 100 ng RNA sample. In addition, each RNA sample was tested using qScriptXLT one-step RT-qPCR (without reverse transcriptase added) to monitor possible carrier DNA contamination in the RNA sample.

[0518] Table 14: Primer and probe sequences for bGH sequences

[0519]

[0520] Table 15: RT-qPCR analysis conditions

[0521]

[0522] ***

[0523] Finally, it should be understood that although various aspects of the present specification are highlighted by reference to specific embodiments, those skilled in the art will readily appreciate that these disclosed embodiments are merely illustrative of the principles of the subject matter disclosed herein. Therefore, it should be understood that the subject matter described herein is not limited in any way to a specific compound, composition, article, device, method, scheme and / or reagent, etc. described herein, unless explicitly stated so. In addition, those skilled in the art will recognize that certain changes, modifications, substitutions, changes, additions, deletions, and sub-combinations thereof may be made in accordance with the teachings herein without departing from the spirit of the present specification.

[0524] The use of the terms "may" or "can" in reference to an embodiment or aspect of an embodiment also has the alternative meaning of "may not" or "may not." Thus, if the specification discloses that an embodiment or an aspect of an embodiment may or may be included as part of the present subject matter, then a negative limitation or exclusion is also clear, meaning that an embodiment or an aspect of an embodiment may not or may not be included as part of the present subject matter. In a similar manner, the use of the term "optionally" in reference to an embodiment or aspect of an embodiment means that this embodiment or aspect of an embodiment may or may not be included as part of the present subject matter. Whether such a negative limitation or exclusion applies will be based on whether the negative limitation or exclusion is recited in the claimed subject matter.

[0525] Although the numerical ranges and values ​​describing the broad scope of the present invention are approximate, the numerical ranges and values ​​described in the specific examples are reported as accurately as possible. However, any numerical range or value inherently contains certain errors, which are necessarily caused by the standard deviation found in its corresponding test measurement. The description of the numerical range of values ​​herein is intended only to serve as a simplified method of referring to each individual numerical value falling within the described range individually. Unless otherwise specified herein, each individual value of the numerical range is incorporated into this specification as if it were described individually in this article (e.g., any disclosure of a range with integer endpoints should be interpreted as also describing a sub-range limited by any integer pair within a wider range).

[0526] Unless otherwise specified herein or obviously contradictory to the context, the term "a / an", "said" and similar indicators used in the case of describing the present invention (especially in the case of the appended claims) should be interpreted as covering both the singular and the plural. Further, unless otherwise specifically specified, the ordinal indicator of the element used to identify, such as "first", "second", "third", etc., is used to distinguish these elements, without indicating or implying the number of requirements or restrictions on these elements, and does not indicate the specific position or order of these elements. Unless otherwise specified herein or obviously contradictory to the context, all methods described herein can be carried out in any suitable order. The use of any and all examples or exemplary language (such as "such as") provided herein is only intended to better illustrate the present invention, and is not intended to limit the scope of the present invention to be protected in addition. Any language in this specification sheet should not be interpreted as indicating any unprotected element as necessary for practicing the present invention.

[0527] When used in a claim, whether as filed or added by amendment, the open transition term "comprising" (and its equivalent open transition phrases, such as includes, contains, and has) encompasses all explicitly recited elements, limitations, steps, and / or features, either alone or in combination with unrecited subject matter; the recited elements, limitations, and / or features are essential, but other unrecited elements, limitations, and / or features may be added and still form a construction within the scope of the claim. Specific embodiments disclosed herein may be further limited in the claims using the closed transition phrases "consisting of" or "consisting essentially of" instead of or as a modification to "comprising". When used in a claim, whether as filed or added by amendment, the closed transition phrase "consisting of" excludes any element, limitation, step, or feature not explicitly recited in the claim. The closed transition phrase "consisting essentially of" limits the scope of the claim to the explicitly recited elements, limitations, steps, and / or features and any other elements, limitations, steps, and / or features that do not materially affect the basic and novel characteristics of the claimed subject matter. Thus, the meaning of the open transition phrase "comprising" is defined as encompassing all specifically recited elements, limitations, steps and / or features and any optional otherwise unspecified elements, limitations, steps and / or features. The meaning of the closed transition phrase "consisting of" is defined as including only those elements, limitations, steps and / or features specifically recited in the claims, while the meaning of the closed transition phrase "consisting essentially of" is defined as including only those elements, limitations, steps and / or features specifically recited in the claims and those elements, limitations, steps and / or features that do not substantially affect the basic and novel features of the claimed subject matter. Thus, as a limiting case, the open transition phrase "comprising" (and its equivalent open transition phrases) includes within its meaning the claimed subject matter specified by the closed transition phrase "consisting of" or "consisting essentially of". Thus, for the phrases "consisting essentially of" and "consisting of", such embodiments as described herein or claimed for protection are explicitly or essentially explicitly described, enabled and supported herein with the phrase "comprising".

[0528] All patents, patent publications and other publications cited and identified in this specification are individually and explicitly incorporated herein by reference in their entirety for the purpose of describing and publishing compositions and methods that may be used in conjunction with the present invention, such as those described in these publications. These publications are provided solely for their disclosures prior to the filing date of this application. Nothing in this regard should be construed as an admission that the inventor of this case is not entitled to advance such a public date due to prior invention or for any other reason. All statements about dates or statements about the contents of these documents are based on information available to the applicant and do not constitute any admission of the correctness of the dates or contents of these documents.

[0529] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention, which is defined solely by the claims. Accordingly, the present invention is not to be limited as precisely shown and described.

[0530] Sequence Listing

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Claims

1. A recombinant adeno-associated virus (rAAV) vector comprising an AAV capsid and an expression cassette, wherein the expression cassette comprises a polynucleotide encoding an oxidoreductase operably linked to a promoter.

2. The rAAV vector of claim 1, wherein the oxidoreductase is thioredoxin (TRX).

3. The rAAV vector of claim 1, wherein the oxidoreductase is protein disulfide isomerase (PDI).

4. The rAAV vector of claim 3, wherein the polynucleotide comprises a sequence encoding an amino acid sequence having at least 95% sequence identity to SEQ ID NO:

26.

5. The rAAV vector of claim 2, wherein the polynucleotide comprises a sequence encoding an amino acid sequence having at least 95% sequence identity to SEQ ID NO:

25.

6. The rAAV vector of claim 2 or 5, wherein the polynucleotide comprises a sequence that is at least 95% identical to SEQ ID NO: 2 or SEQ ID NO:

28.

7. The rAAV vector of claim 3 or 4, wherein the polynucleotide comprises a sequence that is at least 95% identical to SEQ ID NO:

30.

8. The rAAV vector according to any one of claims 1 to 7, wherein the promoter is a CMV promoter comprising the nucleotide sequence set forth in SEQ ID NO:

17.

9. The rAAV vector of any one of claim 8, wherein the expression cassette comprises the CMV promoter and the CMV enhancer.

10. The rAAV vector of any one of claims 1 to 9, wherein the expression cassette comprises a polyadenylation (poly A) sequence.

11. The rAAV vector of claim 10, wherein the poly A sequence is a BGH poly A sequence.

12. The rAAV vector of any one of claims 1 to 11, wherein the expression cassette comprises a Woodchuck Hepatitis Virus Post-transcriptional Regulatory Element (WPRE).

13. The rAAV vector of any one of claims 1 to 12, wherein the expression cassette comprises a Kozak sequence.

14. A composition comprising a rAAV vector, wherein the rAAV vector comprises: (a) AAV capsid, and (b) an expression cassette, wherein the expression cassette comprises a polynucleotide comprising a nucleotide sequence that shares at least 95% identity with a nucleotide sequence comprising SEQ ID NO: 2 or SEQ ID NO: 28, and wherein the polynucleotide is linked to a promoter.

15. The rAAV vector of any one of claims 1 to 13, wherein the expression cassette is flanked by two inverted terminal repeats (ITRs).

16. The rAAV vector of claim 15, wherein the ITR is an AAV2 ITR.

17. The rAAV vector of any one of claims 1 to 2 and 5 to 13 or the composition of any one of claims 14 to 16, wherein the expression cassette comprises a nucleotide sequence that shares at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO:

16.

18. The rAAV vector or composition of any one of claims 1 to 17, wherein the AAV capsid comprises a VP3 that shares at least 95%, 98% or 100% identity with AAV2 VP3 (SEQ ID NO: 8), AAV5 VP3 (SEQ ID NO: 10), AAV8 VP3 (SEQ ID NO: 12) or AAV9 VP3 (SEQ ID NO: 14).

19. The rAAV vector or composition of any one of claims 1 to 17, wherein the AAV capsid comprises a VP3 that shares at least 95%, 98% or 100% identity with AAV9 (SEQ ID NO: 14).

20. A composition comprising a rAAV vector, wherein the rAAV vector comprises: (a) AAV2, AAV5, AAV8 or AAV9 capsid, and (b) an expression cassette, wherein the expression cassette comprises a polynucleotide comprising a nucleotide sequence that shares at least 95% identity with a nucleotide sequence comprising SEQ ID NO: 2 or SEQ ID NO: 28, and wherein the polynucleotide is linked to a promoter.

21. A composition comprising a rAAV vector, wherein the rAAV vector comprises: (a) AAV2, AAV5, AAV8 or AAV9 capsid, and (b) an expression cassette, wherein the expression cassette comprises a polynucleotide sequence that shares at least 95% identity with SEQ ID NO: 2 or SEQ ID NO:

28.

22. The composition of claim 20 or 21, wherein the AAV capsid is AAV2.

23. The composition of claim 20 or 21, wherein the AAV capsid is AAV5.

24. The composition of claim 20 or 21, wherein the AAV capsid is AAV9.

25. The rAAV vector or composition of any one of claims 1 to 24, wherein the polynucleotide comprises a sequence encoding a signal peptide.

26. A pharmaceutical composition comprising the rAAV vector or composition according to any one of claims 1 to 25 and a pharmaceutically acceptable carrier.

27. The pharmaceutical composition of claim 26, wherein the composition comprises about 1×10 7 About 1×10 14 genome copies / ml of the rAAV vector.

28. The pharmaceutical composition of claim 26, wherein the composition comprises about 1×10 12 About 6.2×10 12 genome copies / ml of the rAAV vector.

29. A method of treating an ocular condition in a subject in need thereof, the method comprising administering to an eye of the subject a therapeutically effective amount of a pharmaceutical composition according to any one of claims 26 to 28.

30. The method of claim 29, wherein the pharmaceutical composition is delivered to an ocular secretory gland of the subject.

31. The method of claim 29 or 30, wherein the pharmaceutical composition is delivered to the lacrimal gland.

32. The method of any one of claims 29 to 31, wherein the pharmaceutical composition is delivered to the accessory lacrimal gland.

33. The method of claim 32, wherein the accessory lacrimal glands are meibomian glands.

34. The method of any one of claims 29 to 33, wherein the pharmaceutical composition is delivered to the trabecular meshwork.

35. The method of any one of claims 29 to 34, wherein about 1×10 9 About 1×10 10 , about 1×10 10 About 1×10 11 , about 1×10 11 About 1×10 12 , about 1×10 12 About 1×10 13 , or about 1×10 13 About 1×10 15 genomic copies of the rAAV vector.

36. The method of any one of claims 29 to 35, wherein the ocular condition is associated with increased oxidative stress.

37. The method of any one of claims 29 to 35, wherein the ocular condition is associated with loss of expression and / or function of one or more oxidoreductases.

38. The method of any one of claims 28 to 35, wherein the ocular condition is associated with loss of TRX expression and / or function.

39. The method of any one of claims 28 to 35, wherein the ocular condition is associated with loss of PDI expression and / or function.

40. The method of any one of claims 29-35, wherein the ocular condition is characterized by loss of near vision.

41. The method of any one of claims 29-40, wherein the ocular condition is presbyopia.

42. The method of any one of claims 29-40, wherein the ocular condition is cataract formation.

43. The method of any one of claims 29-40, wherein the ocular condition is ocular hypertension.

44. The method of any one of claims 29-40, wherein the ocular condition is Meibomian gland dysfunction (MDI).

45. The method of any one of claims 29-40, wherein the ocular condition is glaucoma.

46. ​​The method of any one of claims 29 to 45, wherein the method results in expression of the oxidoreductase in cells of the lacrimal gland and / or accessory lacrimal gland and / or in the trabecular meshwork.

47. The method of any one of claims 29 to 46, wherein the method results in expression of TRX in cells of the lacrimal gland and / or accessory lacrimal gland and / or in the trabecular meshwork.

48. The method of any one of claims 29 to 47, wherein the method results in secretion of TRX into the tear film and / or onto the ocular surface of the subject.

49. The method of any one of claims 29-48, wherein the method results in improvement of one or more symptoms of the ocular condition.

50. The method of any one of claims 29 to 49, wherein the method results in improved vision.

51. The method of any one of claims 29 to 50, wherein the method results in a reduced need for corrective lenses.

52. The method of any one of claims 29-51, wherein the method results in a delay in progression of the condition.

53. The method of claim 52, wherein the method delays progression of the condition in the subject by about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about 50%, about 50% to about 60%, about 60% to about 70%, about 70% to about 80%, about 80% to about 90%, about 90% to about 95%, or more than about 95% compared to a control subject.

54. The method of any one of claims 29-53, wherein the method delays the onset of the condition by about 6 months to about 12 months, about 12 months to about 18 months, about 18 months to about 24 months, about 2 years to about 3 years, about 3 years to about 4 years, about 4 years to about 5 years, about 5 years to about 6 years, about 6 years to about 7 years, about 7 years to about 8 years, about 8 years to about 9 years, about 9 years to about 10 years, about 10 years to about 15 years, about 15 years to about 20 years, or more than 20 years compared to a control subject.

55. The method of claim 53 or 54, wherein the control individual is an age-matched individual not treated with a rAAV vector comprising an expression cassette comprising a polynucleotide encoding TRX.

56. The method of any one of claims 29 to 55, wherein the individual required corrective lenses prior to administration of the rAAV, and the administration results in a constant need for corrective lens strength for at least about 6 months to about 12 months, about 12 months to about 18 months, about 18 months to about 24 months, about 2 years to about 3 years, about 3 years to about 4 years, about 4 years to about 5 years, about 5 years to about 6 years, about 6 years to about 7 years, about 7 years to about 8 years, about 8 years to about 9 years, about 9 years to about 10 years, about 10 years to about 15 years, about 15 years to about 20 years, or more than 20 years after administration of the rAAV.

57. The method of any one of claims 29 to 55, wherein the subject's vision remains unchanged for at least about 6 months to about 9 months, about 9 months to about 12 months, about 12 months to about 15 months, about 15 months to about 18 months, about 18 months to about 21 months, about 21 months to about 24 months, about 2 years to about 3 years, about 3 years to about 4 years, about 4 years to about 5 years, about 5 years to about 6 years, about 6 years to about 7 years, about 7 years to about 8 years, about 8 years to about 9 years, about 9 years to about 10 years, about 10 years to about 15 years, about 15 years to about 20 years, or more than 20 years after administration of the rAAV vector.

58. The method of any one of claims 29-57, wherein the method further comprises administering one or more additional therapeutic agents.

59. The method of any one of claims 29-58, wherein the subject is a human.

60. The pharmaceutical composition of any one of claims 26 to 28 for use in a method of treating an ocular condition in a subject in need thereof, the method comprising administering an effective amount of the pharmaceutical composition to an eye of the subject.

61. A pharmaceutical composition according to any one of claims 26 to 28 for use in the manufacture of a medicament for treating an ocular condition in a subject in need thereof.

62. A compound for use in treating an ocular condition in a subject, the treatment comprising administering a pharmaceutical composition of any one of claims 1 to 25 or rAAV or a rAAV vector of any one of claims 26 to 28, and a pharmaceutically acceptable carrier.

63. A kit comprising the rAAV vector or composition of any one of claims 1 to 25 or the pharmaceutical composition of any one of claims 26 to 28, and a pharmaceutically acceptable carrier, and instructions for treating an ocular condition in a subject, wherein the treatment comprises administering the pharmaceutical composition to the eye of the subject.

64. A kit comprising the rAAV vector or composition of any one of claims 1 to 25 or the pharmaceutical composition of any one of claims 26 to 28, and a pharmaceutically acceptable carrier, and instructions for treating presbyopia in a subject, wherein the treatment comprises administering the pharmaceutical composition to an eye of the subject.

65. A kit comprising the rAAV vector or composition of any one of claims 1 to 25 or the pharmaceutical composition of any one of claims 26 to 28, and a pharmaceutically acceptable carrier, and instructions for treating cataract formation in a subject, wherein the treatment comprises administering the pharmaceutical composition to the eye of the subject.

66. A kit comprising the rAAV vector or composition of any one of claims 1 to 25 or the pharmaceutical composition of any one of claims 26 to 28, and a pharmaceutically acceptable carrier, and instructions for treating loss of visual accommodation in a subject, wherein the treatment comprises administering the pharmaceutical composition to an eye of the subject.

67. A kit comprising the rAAV vector or composition of any one of claims 1 to 25 or the pharmaceutical composition of any one of claims 26 to 28, and a pharmaceutically acceptable carrier, and instructions for treating ocular hypertension in a subject, wherein the treatment comprises administering the pharmaceutical composition to the eye of the subject.

68. A kit comprising the rAAV vector or composition of any one of claims 1 to 25 or the pharmaceutical composition of any one of claims 26 to 28, and a pharmaceutically acceptable carrier, and instructions for treating meibomian gland dysfunction (MGD) in a subject, wherein the treatment comprises administering the pharmaceutical composition to an eye of the subject.

69. A pharmaceutical composition comprising: a) a polypeptide comprising an oxidoreductase or a fragment thereof, optionally wherein the polypeptide has an amino acid sequence having at least 90% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 and 26; and b) a pharmaceutically acceptable carrier suitable for administration to the eye of a human subject.

70. A pharmaceutical composition comprising: a) a vector comprising a polynucleotide encoding a polypeptide comprising an oxidoreductase or a fragment thereof, optionally wherein the polypeptide has an amino acid sequence having at least 90% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 and 26; and b) a pharmaceutically acceptable carrier suitable for administration to the eye of a human subject.

71. A pharmaceutical composition according to claim 69 or 70, wherein the pharmaceutically acceptable carrier comprises: water; sterile water; pyrogen-free water; phosphate-buffered saline; HEPES-buffered saline; isotonic sodium chloride solution; balanced salt solution; a wetting agent; a surfactant; a tonicity agent; a pH adjuster; a viscosity adjuster; a buffer; a disaccharide, optionally sucrose or trehalose; cellulose and / or its derivatives; an amino acid, optionally histidine; or any combination thereof.

72. The pharmaceutical composition of any one of claims 69 to 71, wherein the polypeptide has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1 or SEQ ID NO:

26.

73. The pharmaceutical composition of any one of claims 69 to 72, wherein the polypeptide comprises at least 20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, or 500 amino acids.

74. The pharmaceutical composition of any one of claims 69 to 73, wherein the formulation is a liquid formulated for administration to or into the ocular surface of the eye of the human subject, or for injection into the lacrimal gland.

75. The pharmaceutical composition of any one of claims 70 to 74, comprising the vector, wherein the vector is present in the composition in an amount effective to express 100 pg / mL to 50 μg / mL of the polypeptide in the tear film of the individual after administration of the composition to the individual.

76. A pharmaceutical composition according to any one of claims 70 to 75, comprising the vector, wherein the polynucleotide is operably linked to a promoter.

77. The pharmaceutical composition of any one of claims 70 to 76, comprising the vector, wherein the vector is engineered to constitutively express a polypeptide having an amino acid sequence having at least 90% identity to an amino acid sequence selected from SEQ ID NOs: 1 and 26.

78. The pharmaceutical composition of any one of claims 70 to 77, comprising the vector, wherein the vector comprises a virus, optionally an adenoviral vector or a lentiviral vector; a plasmid; an episomal genome; or an artificial chromosome; and optionally comprises one or more lipids, multivalent cations, DNA carrier proteins, histones, pseudocapsids, chimeric proteins, or endocytosis receptor proteins.

79. The pharmaceutical composition of any one of claims 69 or 71 to 74, comprising the polypeptide, wherein the polypeptide is present in the pharmaceutical composition at a concentration of 100 pg / mL to 50 μg / mL.

80. The pharmaceutical composition of any one of claims 69, 71 to 74 or 79, comprising the polypeptide, wherein the polypeptide is present in the pharmaceutical composition in an amount of 500 ng to 5 μg.

81. The pharmaceutical composition of any one of claims 69, 71 to 74, 79 or 80, comprising the polypeptide, wherein the polypeptide is present in the pharmaceutical composition in a unit dose.

82. A method of treating an ocular disease, disorder or condition in a subject in need thereof, the method comprising administering to an eye of the subject an effective amount of a pharmaceutical composition according to any one of claims 69 to 81.

83. The method of claim 82, wherein the pharmaceutical composition is delivered to the accessory lacrimal gland.

84. The method of claim 83, wherein the accessory lacrimal glands are meibomian glands.

85. The method of any one of claims 82-84, wherein the pharmaceutical composition is delivered to the trabecular meshwork.

86. The method of any one of claims 82-85, wherein the ocular condition: a) associated with increased oxidative stress; b) is associated with loss of expression and / or function of one or more oxidoreductases; c) is associated with loss of TRX expression and / or function; and / or d) associated with loss of PDI expression and / or function.

87. The method of any one of claims 82-86, wherein the ocular condition is characterized by loss of near vision.

88. The method of any one of claims 82 to 87, wherein the ocular condition is: a) Presbyopia; b) Cataract formation; c) high intraocular pressure; d) Meibomian gland dysfunction (MDI); and / or e) Glaucoma.

89. The method of any one of claims 82 to 88, wherein the method results in: a) the oxidoreductase is expressed in cells of the lacrimal gland and / or accessory lacrimal gland and / or in the trabecular meshwork; b) TRX is expressed in cells of the lacrimal gland and / or accessory lacrimal gland and / or in the trabecular meshwork; and / or c) TRX is secreted into the tear film and / or ocular surface of the individual.

90. The method of any one of claims 82 to 88, wherein the method results in: a) improvement in one or more symptoms of the ocular condition; b) Improved vision; c) a reduced need for corrective lenses; and / or d) Delay in progression of said ocular condition.

91. The method of any one of claims 82-88, wherein the method delays progression of the ocular condition in the subject by about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about 50%, about 50% to about 60%, about 60% to about 70%, about 70% to about 80%, about 80% to about 90%, about 90% to about 95%, or more than about 95% as compared to a control subject.

92. The method of any one of claims 82-91, wherein the administering results in expression of a functional oxidoreductase in one or more cells of the lacrimal gland and / or accessory lacrimal gland of the individual.

93. The method of any one of claims 82-92, wherein the administering results in secretion of a functional oxidoreductase into the tear film of the subject.

94. The method of claim 93, wherein secretion of the functional oxidoreductase into the tear film is stimulated by a cholinergic agonist.

95. The method of any one of claims 82-94, wherein the administering is to or into the ocular surface of the subject, and / or to the lacrimal gland of the subject.

96. The method of any one of claims 82-95, wherein the symptom is selected from the group consisting of itching, swelling, tearing, and redness.

97. The method of any one of claims 82 to 96, wherein the administering increases the conjunctival itch grading scale score by 0.5, 1, 1.5, 2, 2.5, 3, 3.5, or 4 points.

98. The method of any one of claims 82-97, wherein the subject is a human subject.

99. A kit comprising a pharmaceutical composition according to any one of claims 69 to 81 and instructions for use in treating a condition in a human subject, wherein the instructions comprise administering the pharmaceutical composition to an eye of the human subject.

100. A pharmaceutical composition according to any one of claims 69 to 81 for use in the manufacture of a medicament for treating a condition in a human subject in need thereof.

101. A pharmaceutical composition according to any one of claims 26 to 28 for use in a method of treatment according to any one of claims 29 to 59.

102. A pharmaceutical composition according to any one of claims 69 to 81 for use in a method of treatment according to any one of claims 82 to 98.

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