AAV vectors encoding oxidoreductase enzymes and uses thereof

Abstract

Provided are recombinant adeno-associated virus (rAAV) vectors, methods for treating ocular diseases, pharmaceutical compositions, and other compositions and methods, where the rAAV vector comprises a polynucleotide encoding an oxidoreductase. Treatment methods can include administration to the lacrimal gland.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of the priority dates of U.S. Provisional Patent Application No. 63 / 375,613, filed September 14, 2022, and U.S. Provisional Patent Application No. 63 / 383,055, filed November 9, 2022, the disclosures of which are incorporated by reference in their entirety.

[0002] Reference to sequence listing The contents of the electronic sequence listing (OYST_028_03WO_SeqList_ST26.xml (56KB)), created on September 9, 2023, are hereby incorporated by reference in their entirety. [Background technology]

[0003] The thioredoxin system is a highly conserved redox system that plays a key role in maintaining a reducing environment in cells. Thioredoxins (TRXs) are major and ubiquitous disulfide It is a reductase that is reduced by thioredoxin reductase (TRXR) to form NAPDH Thiol-disulfide exchange reactions control protein function through the redox state of structural or catalytic sulfhydryl groups. Oxidation of critical sulfhydryl groups generally leads to alterations in the biological function of proteins. Thus, thiol redox regulation is a major regulatory mechanism in signal transduction, and the increased production of reactive oxygen species that oxidize protein thiols, balanced by thioredoxin- and glutathione-dependent reactions, has widespread functions in cell physiology and pathological conditions (Arner and Holmgren 2000).

[0004] The thioredoxin system in the eye lens has been shown to gradually weaken with age (Xing and Lou, Invest Ophthalmol Vis Sci. 2010 Dec;51(12):6598-6604), and both the generation of reactive oxygen species and the decrease in endogenous antioxidants contribute to cataract formation.

[0005] Related oxidoreductases include protein disulfide isomerase (PDI) The first report of PDI was in 1963, when protein folding was It was thought that PDI functions as a chaperone for the ATPase inhibitor PDI (Goldberger, RF et al., J.Biol. Chem 1963; 238; 628-635). A common response to stress is protein misfolding, and PDI PDI plays an important role in this defense. Functionally, PDI reduces disulfide bonds, This 58 kDa calcium-binding chaperone protein is localized to the endoplasmic reticulum (ER) (Maattanen, et al (2010) Semin Cell Dev Biol 21:500-11).

[0006] Presbyopia is a common eye disease that affects many people worldwide, especially those over the age of 40. It is predicted that 1.8 billion people will suffer from presbyopia by 2050 (Grzybowski et al., Asia Pac J Ophthalmol (Phila) 2020;9:226-233). Presbyopia is caused by a decrease in the flexibility of the lens. It gets rubbed.

[0007] Treatments are known for eye diseases that affect the human lens, such as cataract formation and presbyopia. Medical treatments often include prescription lenses to assist with near vision due to loss of elasticity in the natural human lens. Surgical treatments often involve removing the natural human lens and replacing it with an intraocular lens.

[0008] Additionally, pharmacological treatments for presbyopia are being investigated. These pharmacological treatments are primarily These drugs work by exerting a pinhole effect and increasing the depth of field (e.g., NSAIDs, parasympathomimetics, or COX2 inhibitors) or by softening the lens (e.g., EV06, lipoic acid choline ester), but with limited success (Grzybowski 2020).

[0009] Despite available treatments, these eye diseases remain a challenge for ophthalmologists, with treatment strategies starting with vision correction, aiming to compensate for the loss of accommodation and visual acuity, and often ending with surgical removal of the lens.

[0010] Improved medical treatments for human lens aging and disease are desirable, and there remains an unmet clinical need for long-term, effective treatments for presbyopia and other ocular diseases.

[0011] Munemasa, Y., Ahn, JH, Kwong, JMK, Caprioli, J., & Piri, N. (2009). Redox proteins thioredoxin 1 and thioredoxin 2 support retinal ganglion cell survival in experimental glaucoma. Gene Therapy, 16(1), 17-25.

[0012] Despite existing treatments, these eye diseases remain a challenge for ophthalmologists, with treatment strategies starting with vision correction, aiming to compensate for accommodation and visual acuity, and often culminating in surgical removal of the lens.

[0013] Improved medical treatments for aging and disorders of the human lens are desirable, and there remains an unmet clinical need for long-term, effective treatments for presbyopia and other ocular diseases. Summary of the Invention

[0014] In one aspect, the present disclosure provides a recombinant adeno-associated virus (rAAV) vector. The rAAV vector comprises an AAV capsid and an expression cassette, the expression cassette being operably linked to a promoter. The present invention includes a polynucleotide encoding an oxidoreductase enzyme tethered to the nucleotide sequence.

[0015] In some embodiments, the oxidoreductase enzyme is thioredoxin (TRX). In some embodiments, the oxidoreductase enzyme is 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.

[0016] In one embodiment, the polynucleotide has at least SEQ ID NO:2 or SEQ ID NO:28. In one embodiment, the polynucleotide comprises a sequence that is at least 95% identical to SEQ ID NO: 2. In one embodiment, the polynucleotide comprises a sequence that is at least 95% identical to SEQ ID NO: 2. In some embodiments, the polynucleotide comprises a sequence at least 95% identical to SEQ ID NO: 28. In some embodiments, the polynucleotide comprises a sequence at least 95% identical to SEQ ID NO: 24. In some embodiments, the polynucleotide comprises a sequence at least 95% identical to SEQ ID NO: 30.

[0017] In one embodiment, the promoter is a CMV promoter comprising the nucleotide sequence set forth in SEQ ID NO:17.

[0018] In one embodiment, the expression cassette comprises a CMV promoter and a CMV enhancer. In one embodiment, the expression cassette comprises a polyadenylation (polyA) sequence. In this embodiment, the polyA sequence is a BGH polyA sequence.

[0019] In some embodiments, the expression cassette comprises a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE). In some embodiments, the expression cassette comprises a Kozak sequence.

[0020] In one embodiment, the present disclosure provides a composition comprising an rAAV vector. The rAAV vector comprises: (a) an AAV capsid, and (b) an expression cassette. The expression cassette is Shares at least 95% identity with a nucleotide sequence containing SEQ ID NO: 2 or SEQ ID NO: 28 The polynucleotide comprises a nucleotide sequence encoding ...

[0021] In one embodiment, the present disclosure provides a composition comprising an rAAV vector, the rAAV vector comprising: (a) an AAV capsid, and (b) an expression cassette. The expression cassette comprises: Nucleotides sharing at least 95% identity with a nucleotide sequence comprising SEQ ID NO: 2 The polynucleotide comprises a polynucleotide comprising a sequence, which is linked to a promoter.

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

[0023] In one embodiment, the present disclosure provides a composition comprising an rAAV vector, the rAAV vector comprising: (a) an AAV capsid, and (b) an expression cassette. The expression cassette comprises: The polynucleotide comprises a nucleotide sequence that shares at least 95% identity with a nucleotide sequence comprising SEQ ID NO: 30, wherein the polynucleotide is linked to a promoter.

[0024] In one embodiment, the expression cassette is flanked by two inverted terminal repeats (ITRs). In one embodiment, the ITRs are AAV2 ITRs.

[0025] In certain embodiments, the expression cassette comprises a nucleotide sequence that shares at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:16.

[0026] In some embodiments, the AAV capsid comprises a VP3 having at least 95%, 98%, or 100% identity to 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). comprises a VP3 that is at least 95%, 98%, or 100% identical to AAV9 having SEQ ID NO: 14.

[0027] In another embodiment, the disclosure provides a composition comprising an rAAV vector. The rAAV vector comprises: (a) an AAV2, AAV5, AAV8, or AAV9 capsid, and (b) an expression cassette. The expression cassette comprises a nucleotide sequence comprising SEQ ID NO:2 or SEQ ID NO:28 and at least one nucleotide sequence comprising SEQ ID NO:2 or SEQ ID NO:28. The polynucleotide comprises a polynucleotide comprising a nucleotide sequence that shares at least 95% identity with the promoter, the polynucleotide being linked to a promoter.

[0028] In another embodiment, the disclosure provides a composition comprising an rAAV vector, the rAAV vector comprising: (a) an AAV2, AAV5, AAV8, or AAV9 capsid, and (b) an expression cassette, the expression cassette having a nucleotide sequence at least 95% identical to a nucleotide sequence comprising SEQ ID NO:2. The polynucleotide comprises a polynucleotide comprising a nucleotide sequence that shares a common identity with the promoter, the polynucleotide being linked to a promoter.

[0029] In another embodiment, the disclosure provides a composition comprising a rAAV vector, which comprises: (a) an AAV2, AAV5, AAV8, or AAV9 capsid, and (b) an expression vector. 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, wherein the polynucleotide is linked to a promoter.

[0030] In another embodiment, the disclosure provides a composition comprising an rAAV vector, the rAAV vector comprising: (a) an AAV2, AAV5, AAV8, or AAV9 capsid, and (b) an expression cassette, the expression cassette comprising a polynucleotide comprising a nucleotide sequence sharing at least 95% identity to a nucleotide sequence comprising SEQ ID NO: 30, the polynucleotide being linked to a promoter.

[0031] In another embodiment, the disclosure provides a composition comprising an rAAV vector, the rAAV vector comprising: (a) an AAV2, AAV5, AAV8, or AAV9 capsid, and (b) an expression cassette, the expression cassette having at least 95% identity to SEQ ID NO: 2 or SEQ ID NO: 28. The present invention includes a polynucleotide sequence having the following structure:

[0032] In another embodiment, the disclosure provides a composition comprising an rAAV vector, the rAAV vector comprising: (a) an AAV2, AAV5, AAV8, or AAV9 capsid, and (b) an expression cassette. The expression cassette comprises a polynucleotide sequence having at least 95% identity to SEQ ID NO:2. It contains a nucleotide sequence.

[0033] In another embodiment, the disclosure provides a composition comprising an rAAV vector, the rAAV vector comprising: (a) an AAV2, AAV5, AAV8, or AAV9 capsid, and (b) an expression cassette, the expression cassette comprising a polynucleotide sequence having at least 95% identity to SEQ ID NO:28.

[0034] In another embodiment, the disclosure also provides a composition comprising an rAAV vector, which comprises: (a) an AAV2, AAV5, AAV8, or AAV9 capsid, and (b) an expression cassette, wherein the expression cassette comprises a polynucleotide sequence having at least 95% identity to SEQ ID NO: 30.

[0035] In another embodiment, the disclosure provides a composition comprising an rAAV vector, the rAAV vector comprising: (a) an rAAV2 / 1, rAAV2 / 3, rAAV2 / 4, rAAV2 / 5, rAAV2 / 6, rAAV2 / 7, rAAV2 / 8, or rAAV2 / 9 capsid, and (b) an expression cassette, the expression cassette comprising a nucleotide sequence sharing at least 95% identity with a nucleotide sequence comprising SEQ ID NO:2 or SEQ ID NO:28. The polynucleotide comprises a nucleotide sequence, and the polynucleotide is linked to a promoter.

[0036] In another embodiment, the disclosure provides a composition comprising an rAAV vector, the rAAV vector comprising: (a) an rAAV2 / 1, rAAV2 / 3, rAAV2 / 4, rAAV2 / 5, rAAV2 / 6, rAAV2 / 7, rAAV2 / 8, or rAAV2 / 9 capsid, and (b) an expression cassette, the expression cassette comprising a polynucleotide sharing at least 95% identity to a nucleotide sequence comprising SEQ ID NO:2. The polynucleotide is linked to a promoter.

[0037] In another embodiment, the disclosure provides a composition comprising an rAAV vector comprising: (a) an 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 sharing at least 95% identity to a nucleotide sequence comprising SEQ ID NO: 28, wherein the polynucleotide is linked to a promoter.

[0038] In another embodiment, the disclosure provides a composition comprising a rAAV vector, including: (a) rAAV2 / 1, rAAV2 / 3, rAAV2 / 4, rAAV2 / 5, rAAV2 / 6, rAAV2 / 7, an rAAV2 / 8 or rAAV2 / 9 capsid, and (b) an expression cassette, the expression cassette comprising a polynucleotide that shares at least 95% identity with a nucleotide sequence comprising SEQ ID NO: 30, the polynucleotide being linked to a promoter.

[0039] In another embodiment, the disclosure provides a composition comprising an rAAV vector, the rAAV vector comprising: (a) an rAAV2 / 1, rAAV2 / 3, rAAV2 / 4, rAAV2 / 5, rAAV2 / 6, rAAV2 / 7, rAAV2 / 8, or rAAV2 / 9 capsid, and (b) an expression cassette, the expression cassette comprising a polynucleotide sequence having at least 95% identity to SEQ ID NO: 2 or SEQ ID NO: 28. .

[0040] In another embodiment, the disclosure provides a composition comprising an rAAV vector, the rAAV vector comprising: (a) an rAAV2 / 1, rAAV2 / 3, rAAV2 / 4, rAAV2 / 5, rAAV2 / 6, rAAV2 / 7, rAAV2 / 8, or rAAV2 / 9 capsid, and (b) an expression cassette, the expression cassette comprising a polynucleotide sequence having at least 95% identity to SEQ ID NO:2.

[0041] In another embodiment, the disclosure provides a composition comprising an rAAV vector, the rAAV vector comprising: (a) an rAAV2 / 1, rAAV2 / 3, rAAV2 / 4, rAAV2 / 5, rAAV2 / 6, rAAV2 / 7, rAAV2 / 8, or rAAV2 / 9 capsid, and (b) an expression cassette, the expression cassette comprising a polynucleotide sequence having at least 95% identity to SEQ ID NO:28.

[0042] In another embodiment, the disclosure provides a composition comprising an rAAV vector, the rAAV vector comprising: (a) an rAAV2 / 1, rAAV2 / 3, rAAV2 / 4, rAAV2 / 5, rAAV2 / 6, rAAV2 / 7, rAAV2 / 8, or rAAV2 / 9 capsid, and (b) an expression cassette, the expression cassette comprising a polynucleotide sequence having at least 95% identity to SEQ ID NO: 30.

[0043] In one embodiment, the AAV capsid is AAV2. In one embodiment, the AAV capsid is AAV5. In one embodiment, the AAV capsid is AAV9.

[0044] In certain embodiments, the polynucleotide comprises a sequence encoding a signal peptide.

[0045] In certain embodiments, the present disclosure provides a pharmaceutical composition comprising an rAAV vector or a composition of any aspect and embodiment described herein and a pharmaceutically acceptable carrier.

[0046] In one embodiment, the composition contains about 1×10 7 to approximately 1 x 10 14 genome copies per milliliter In one embodiment, the composition comprises about 1 x 10 rAAV vectors. 12 to approximately 6.2 x 10 12 Contains genome copies per milliliter of rAAV vector.

[0047] In certain embodiments, the present disclosure provides a method for treating an ocular disease in a subject in need thereof, comprising administering to the eye of the subject a pharmaceutical composition of any of the embodiments described herein.

[0048] In some embodiments, the pharmaceutical composition is delivered to a gland in the subject's eye. In some embodiments, the pharmaceutical composition is delivered to the 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.

[0049] In one embodiment, about 1 x 10 9 to approximately 1 x 10 10 , about 1×10 10 to approximately 1 x 10 11 , about 1×10 11 to approximately 1 x 10 12 , about 1×10 12 to approximately 1 x 10 13 , or approximately 1 × 10 13 to approximately 1 x 10 15 rAAV vector A copy of the genome is administered.

[0050] In some embodiments, the eye disease is associated with increased oxidative stress. In some embodiments, the eye disease is associated with loss of expression and / or function of one or more oxidoreductase enzymes. In some embodiments, the eye disease is associated with loss of expression and / or function of TRX. In one embodiment, the ocular disease is associated with a loss of PDI expression and / or function. In some embodiments, the eye disease is characterized by a loss of near vision. In some embodiments, the eye disease is presbyopia. In some embodiments, the eye disease is cataract formation. In some embodiments, the eye disease is ocular hypertension. In some embodiments, the eye disease is meibomian gland dysfunction (MDI). In some embodiments, the eye disease is glaucoma. It is an internal obstruction.

[0051] In some embodiments, the method causes expression of an oxidoreductase enzyme in cells of the lacrimal gland and / or accessory lacrimal gland, or trabecular meshwork. In some embodiments, the method causes expression of TRX and / or oxidoreductase enzymes in cells of the lacrimal gland and / or accessory lacrimal gland, or trabecular meshwork. or PDI expression is induced.

[0052] In one embodiment, the method causes secretion of TRX and / or PDI into the tear film and / or ocular surface of the subject.

[0053] In some embodiments, the secretion of TRX and / or PDI into the tear film is stimulated by electrical stimulation, mechanical stimulation, ultrasonic stimulation, and / or the administration of drugs. An example of electrical stimulation is the TrueTear® intranasal lacrimal nerve stimulator. An example of mechanical stimulation is vibrational energy provided by devices such as the iTEAR® 100. An example of ultrasonic stimulation is a device such as the iTear System (Olympus Opthalmics), which was developed for the treatment of dry eye syndrome. In one embodiment, the drug that stimulates the secretion of TRX and / or PDI into the tear film is a cholinergic agonist (e.g., pilocarpine or cevimeline). In another embodiment, the drug is a nicotinic acetylcholine receptor (nAChR) agonist. In some embodiments, the drug is a secretagogue or mucosal protectant (e.g., diquafosol, rebamipide, or ecabet). In some embodiments, the drug that stimulates the secretion of TRX and / or PDI into the tear film is administered ocularly. In some embodiments, the drug (e.g., a cholinergic agonist) is administered orally. In some embodiments, the drug that stimulates the secretion of TRX and / or PDI into the tear film (e.g., a cholinergic agonist or nAChR agonist) is administered intranasally. In some embodiments, a rAAV vector or other construct designed to express TRX and / or PDI is used. is administered to a subject as a co-therapy in combination with a tear-increasing stimulus and / or a drug (e.g., as described in this paragraph). In certain embodiments, such co-therapy can use any of the protocols, components, and parameters described in International Patent Application Publication No. WO 2022 / 235786, the entire contents of which are incorporated herein.

[0054] In some embodiments, the method improves one or more symptoms of the eye disease. In some embodiments, the method improves vision. In some embodiments, the method reduces the need for corrective lenses. In some embodiments, the method slows the progression of the condition.

[0055] In certain embodiments, the method slows the 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.

[0056] In one embodiment, the method reduces the onset of the condition in the subject by about 6 months compared to a control subject. From about 12 months, from about 12 months to about 18 months, from about 18 months to about 24 months, from about 2 years to about 3 years, from about 3 years from about 4 years, from about 4 to about 5 years, from about 5 to about 6 years, from about 6 to about 7 years, from about 7 to about 8 years, from about 8 to about 9 years, from about 9 to about 10 years, from about 10 to about 15 years, from about 15 to about 20 years, or more than about 20 years.

[0057] In one embodiment, the control subject is a subject that has an expression cassette comprising a polynucleotide encoding TRX. Age-matched subjects were not treated with rAAV vectors containing rAAV.

[0058] In certain embodiments, the subject required corrective lenses before administration of the rAAV and the strength of the corrective lenses remains unchanged 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 about 20 years or more after administration.

[0059] In some embodiments, visual acuity is maintained 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 20 months, or about 25 months to about 30 months after administration of the rAAV vector. Approximately 21 months, approximately 21 months to approximately 24 months, approximately 2 years to approximately 3 years, approximately 3 years to approximately 4 years, approximately 4 years to approximately 5 years, approximately 5 years to approximately 6 years, approximately 6 years to approximately 7 years, approximately 7 years to approximately 8 years, approximately 8 years to approximately 9 years, approximately 9 years to approximately 10 years , remains unchanged for about 10 to about 15 years, about 15 to about 20 years, or for more than about 20 years.

[0060] In some embodiments, the method also includes administering one or more additional therapeutic agents. In embodiments, the additional therapeutic agent that increases tear production is a cholinergic agent.

[0061] In certain embodiments, the subject is a human.

[0062] In certain embodiments, the present disclosure provides a pharmaceutical composition of any aspect and embodiment described herein for use in a method of treating an ocular disease, comprising administering to a subject in need thereof a therapeutically effective amount of the pharmaceutical composition to an eye of the subject.

[0063] In certain embodiments, the present disclosure provides a pharmaceutical composition of any aspect and embodiment described herein for use in the manufacture of a medicament for treating an ocular disease in a subject in need thereof.

[0064] In certain embodiments, the present disclosure provides a pharmaceutical composition for treating an ocular disease, the pharmaceutical composition comprising a vector encoding an oxidoreductase, the vector comprising an rAAV vector described herein and a pharmaceutically acceptable carrier.

[0065] In certain embodiments, the present disclosure provides a kit comprising an rAAV vector or a composition of any aspect or embodiment described herein, and a pharmaceutically acceptable carrier, a therapeutic procedure comprising administering the pharmaceutical composition to the eye of a subject to treat an ocular disease.

[0066] In certain embodiments, the present disclosure provides a kit comprising an rAAV vector or a composition of any aspect or embodiment described herein, and a pharmaceutically acceptable carrier, a therapeutic procedure comprising administering the pharmaceutical composition to an eye of a subject to treat presbyopia.

[0067] In certain embodiments, the present disclosure provides a kit comprising an rAAV vector or a composition of any aspect or embodiment described herein, and a pharmaceutically acceptable carrier, a therapeutic procedure, the therapeutic procedure comprising administering the pharmaceutical composition to the eye of a subject to treat cataract formation. This includes:

[0068] In certain embodiments, the present disclosure provides a kit comprising an rAAV vector or a composition of any aspect or embodiment described herein, and a pharmaceutically acceptable carrier, and a treatment procedure comprising administering the pharmaceutical composition to the eye of a subject to treat loss of accommodation.

[0069] In certain embodiments, the present disclosure provides a kit comprising an rAAV vector or a composition of any aspect or embodiment described herein, and a pharmaceutically acceptable carrier, a therapeutic procedure comprising administering the pharmaceutical composition to the eye of a subject to treat ocular hypertension.

[0070] In certain embodiments, the present disclosure provides a kit comprising an rAAV vector or a composition of any aspect or embodiment described herein, and a pharmaceutically acceptable carrier, a treatment procedure, the treatment procedure comprising administering to the eye of a subject an rAAV vector or a composition of any aspect or embodiment described herein, and a pharmaceutically acceptable carrier, the treatment procedure comprising administering to the eye of a subject an rAAV vector or a composition of any aspect or embodiment described herein, the ... the composition of any aspect or embodiment described herein, the composition of any aspect or embodiment described herein, the composition of any aspect or embodiment described herein, the composition of any aspect or embodiment described herein, the composition of any aspect or embodiment described herein, the composition of any aspect or embodiment described herein, the composition of any aspect or embodiment described herein, the composition of any aspect or embodiment described herein, the administering a pharmaceutical composition.

[0071] In certain embodiments, the present disclosure provides a pharmaceutical composition comprising: a) an oxidoreductase A polypeptide comprising an α-glucose enzyme or a fragment thereof, optionally wherein the polypeptide has 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.

[0072] In certain embodiments, the present disclosure provides a pharmaceutical composition comprising: a) an oxidoreductase a vector comprising a polynucleotide encoding a polypeptide comprising a tase enzyme or a fragment thereof, optionally wherein the polypeptide has an amino acid sequence selected from SEQ ID NOs: 1 and 26; and b) has an amino acid sequence that is at least 90% identical to that of a human subject, A pharmaceutically acceptable carrier suitable for administration.

[0073] In certain embodiments, the pharmaceutically acceptable carrier is water, sterile water, heat-free water, phosphate buffered saline, HEPES buffered saline, isotonic sodium chloride solution, balanced salt solution, a humectant, or the like. , surfactants, tonicity agents, pH adjusters, viscosity modifiers, buffers, disaccharides (optionally sucrose or trehalose), cellulose and / or derivatives thereof, amino acids (optionally histidine) or any combination thereof.

[0074] In one embodiment, the polypeptide has at least one sequence similar to SEQ ID NO:1 or SEQ ID NO:26. At least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity It has oneness.

[0075] In certain 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.

[0076] In certain embodiments, the formulation is a liquid prepared for application or administration to the ocular surface of the eye of a human subject, or for intralacrimal injection.

[0077] In one embodiment, the pharmaceutical composition comprises an rAAV vector, wherein the vector is present in the composition in an effective amount to achieve 100 pg / mL in the tear film of a subject after administration of the composition to the subject. Express 50 µg / mL of a polypeptide (e.g., TRX1 or PDI) from

[0078] In one embodiment, the polynucleotides encoding TRX1 and / or PDI are operatively connected to the motor.

[0079] In one embodiment, the pharmaceutical composition comprising the rAAV vector comprises a vector selected from the group consisting of SEQ ID NOs: 1 and 26. It is designed to constitutively express a polypeptide having an amino acid sequence that is at least 90% identical to a selected amino acid sequence.

[0080] In certain embodiments, the rAAV vectors described herein comprise a virus (optionally an adenoviral or lentiviral vector), a plasmid, an episome, or an artificial chromosome, and optionally, one or more lipids, polycations, DNA carrier proteins, histones, pseudocapsids, chimeric proteins, or endocytic receptor proteins.

[0081] 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 μg to 5 μg. The peptide is contained in a pharmaceutical composition as a unit dosage.

[0082] In certain embodiments, the present disclosure provides a method of treating an ocular disease, disorder, or condition in a subject in need thereof, comprising administering an effective amount of any TRX enzyme or PDI, or a pharmaceutical composition comprising the same, to at least one cell of the subject's eye, lacrimal gland, and / or nasolacrimal duct. These include, but are not limited to, acinar cells, ductal cells, and / or myoepithelial cells, cells of the iris and ciliary body ("ICB"), lens epithelial cells, cells of the meibomian gland, and trabecular fibrous tissue. Contains zona cells.

[0083] In one embodiment, the present disclosure provides a method for treating an ocular disease, disorder, or condition in a subject in need thereof. The method comprises administering an effective amount of a vector encoding any TRX enzyme or PDI, or a pharmaceutical composition comprising the same, to at least one cell of the subject's eye, lacrimal gland, and / or nasolacrimal duct, including, but not limited to, acinar cells, duct cells, and / or or myoepithelial cells, cells of the iris and ciliary body ("ICB"), lens epithelial cells, meibomian gland cells of the endothelium, and cells of the trabecular meshwork.

[0084] 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 oxidoreductase enzymes, c) associated with loss of expression and / or function of TRX, 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) ocular hypertension, d) meibomian gland dysfunction (MDI), or e) glaucoma.

[0085] In one embodiment, the method of treatment results in: a) the reduction of cells in the lacrimal gland and its accessory lacrimal glands; a) expression of oxidoreductase enzymes in cells of the lacrimal gland and trabecular meshwork; expression of TRX in cells of the genu lacrimal gland or cells of the trabecular meshwork, c) secretion of TRX into the tear film and / or ocular surface of the subject.

[0086] In certain embodiments, the method of treatment results in: a) improvement in one or more symptoms of the ocular condition; b) improved visual acuity; c) reduced need for corrective lenses; d) slowed progression of the ocular condition. In some embodiments, the method slows the 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% compared to a control subject. In some embodiments, the treatment method results in the expression of a functional oxidoreductase enzyme in one or more cells of the subject's lacrimal gland and / or accessory lacrimal gland. In some embodiments, the treatment method results in the secretion of functional oxidoreductase enzymes into the tear film of the subject. In some embodiments, the secretion of functional oxidoreductase enzymes into the tear film is stimulated by a cholinergic agonist.

[0087] In some embodiments, the method of treatment involves treating one or more symptoms of an ocular disease, disorder, or condition, e.g., In certain embodiments, administration results in a 0.5-point, 1-point, 1.5-point, 2-point, 2.5-point, 3-point, 3.5-point, or 4-point improvement on the Conjunctival Itch Rating Scale.

[0088] In some embodiments, the present disclosure provides a kit comprising any of the pharmaceutical compositions described herein and instructions for treating a condition in a human subject, wherein the instructions include administering the pharmaceutical composition to the eye of the human subject.

[0089] In certain embodiments, the present disclosure provides a pharmaceutical composition of any of the embodiments described herein for use in the manufacture of a medicament for treating a condition in a human subject in need thereof.

[0090] In certain embodiments, the present disclosure provides a pharmaceutical composition of any of the embodiments described herein for use in a method of treatment according to any of the embodiments described herein. [Brief explanation of the drawings]

[0091] [Figure 1] FIG. 1 shows a vector map of the rAAV expression cassette, including the inverted terminal repeats (ITRs), promoter, and TRX polynucleotide elements. [Figure 2A] 2A-2B show an example of viral vector delivery to the lacrimal gland of a human subject. [Figure 2B] 2A-2B show an example of viral vector delivery to the lacrimal gland of a human subject. [Figure 3] Figure 3 shows 293T cells transfected with AAV.TRX plasmid DNA (bottom row), stained with anti-thioredoxin (anti-TRX) primary antibody, and counterstained with DAPI. Images show the fluorescence of anti-TRX (middle and right rows) and DAPI (left row) at 20x and 40x magnification. Control 293T cells were either untransfected (middle row) or labeled with a secondary control antibody (top row). [Figure 4A] Figures 4A-4B show bar graphs measuring TRX in the supernatant of untransfected 293T cells after 4 days of culture (Figure 4A) or 24 hours after transfection with the AAV.TRX plasmid (Figure 4B). [Figure 4B] Figures 4A-4B show bar graphs measuring TRX in the supernatant of untransfected 293T cells after 4 days of culture (Figure 4A) or 24 hours after transfection with the AAV.TRX plasmid (Figure 4B). [Figure 5] Figure 5 shows images of Western blots for detecting TRX in cell lysates obtained from untransfected (293T) and transfected (AAV.TRX 293T) cells. Detection of GAPDH was used as a loading control. Whole cell extracts (30 μg / lane) were subjected to SDS-PAGE, and anti-TRX and anti-GAPDH primary antibodies were used to detect the respective proteins. [Figure 6] Figure 6 shows Western blot images for detecting expressed and secreted TRX in cell culture media obtained from non-transfected (293T) and transfected (AAV.TRX 293T) cells. Proteins (30 µg / lane) collected from conditioned cell culture media were subjected to SDS-PAGE, and anti-TRX and anti-GAPDH primary antibodies were used to detect the respective proteins. [Figure 7] Figure 7 is a schematic diagram showing the elements between the ITRs of the AAV plasmid. The plasmid encodes EGFP ("secEGFP") with an N-terminally linked secretion signal under the control of a CMV promoter. A woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) increases gene expression and is adjacent to the bovine growth hormone polyadenylation (pA) signal. [Figure 8A]Figures 8A-8K show images of lacrimal gland tissue stained with anti-eGFP antibody. The lacrimal gland was administered with an rAAV vector containing an expression cassette with an eGFP transgene via intralacrimal injection. The lacrimal gland tissue was stained with anti-eGFP antibody to assess eGFP expression. The black arrows indicate staining indicating eGFP expression. [Figure 8B] Figures 8A-8K show images of lacrimal gland tissue stained with anti-eGFP antibody. The lacrimal gland was administered with an rAAV vector containing an expression cassette with an eGFP transgene via intralacrimal injection. The lacrimal gland tissue was stained with anti-eGFP antibody to assess eGFP expression. The black arrows indicate staining indicating eGFP expression. [Figure 8C] Figures 8A-8K show images of lacrimal gland tissue stained with anti-eGFP antibody. The lacrimal gland was administered with an rAAV vector containing an expression cassette with an eGFP transgene via intralacrimal injection. The lacrimal gland tissue was stained with anti-eGFP antibody to assess eGFP expression. The black arrows indicate staining indicating eGFP expression. [Figure 8D] Figures 8A-8K show images of lacrimal gland tissue stained with anti-eGFP antibody. The lacrimal gland was administered with an rAAV vector containing an expression cassette with an eGFP transgene via intralacrimal injection. The lacrimal gland tissue was stained with anti-eGFP antibody to assess eGFP expression. The black arrows indicate staining indicating eGFP expression. [Figure 8E] Figures 8A-8K show images of lacrimal gland tissue stained with anti-eGFP antibody. The lacrimal gland was administered with an rAAV vector containing an expression cassette with an eGFP transgene via intralacrimal injection. The lacrimal gland tissue was stained with anti-eGFP antibody to assess eGFP expression. The black arrows indicate staining indicating eGFP expression. [Figure 8F] Figures 8A-8K show images of lacrimal gland tissue stained with anti-eGFP antibody. The lacrimal gland was administered with an rAAV vector containing an expression cassette with an eGFP transgene via intralacrimal injection. The lacrimal gland tissue was stained with anti-eGFP antibody to assess eGFP expression. The black arrows indicate staining indicating eGFP expression. [Figure 8G]Figures 8A-8K show images of lacrimal gland tissue stained with anti-eGFP antibody. The lacrimal gland was administered with an rAAV vector containing an expression cassette with an eGFP transgene via intralacrimal injection. The lacrimal gland tissue was stained with anti-eGFP antibody to assess eGFP expression. The black arrows indicate staining indicating eGFP expression. [Figure 8H] Figures 8A-8K show images of lacrimal gland tissue stained with anti-eGFP antibody. The lacrimal gland was administered with an rAAV vector containing an expression cassette with an eGFP transgene via intralacrimal injection. The lacrimal gland tissue was stained with anti-eGFP antibody to assess eGFP expression. The black arrows indicate staining indicating eGFP expression. [Figure 8I] Figures 8A-8K show images of lacrimal gland tissue stained with anti-eGFP antibody. The lacrimal gland was administered with an rAAV vector containing an expression cassette with an eGFP transgene via intralacrimal injection. The lacrimal gland tissue was stained with anti-eGFP antibody to assess eGFP expression. The black arrows indicate staining indicating eGFP expression. [Figure 8J] Figures 8A-8K show images of lacrimal gland tissue stained with anti-eGFP antibody. The lacrimal gland was administered with an rAAV vector containing an expression cassette with an eGFP transgene via intralacrimal injection. The lacrimal gland tissue was stained with anti-eGFP antibody to assess eGFP expression. The black arrows indicate staining indicating eGFP expression. [Figure 8K] Figures 8A-8K show images of lacrimal gland tissue stained with anti-eGFP antibody. The lacrimal gland was administered with an rAAV vector containing an expression cassette with an eGFP transgene via intralacrimal injection. The lacrimal gland tissue was stained with anti-eGFP antibody to assess eGFP expression. The black arrows indicate staining indicating eGFP expression. [Figure 9] Figure 9 provides images of pig lacrimal glands that received injections of AAV-secEGFP (AAV2 or AAV9 serotypes), were harvested on day 103, and were fixed in paraffin. IHC was performed on 5 μM sections using an anti-GFP antibody and DAPI (nuclear) counterstain. Images were captured using a confocal microscope at 100x magnification. Negative control animals received no injections. [Figure 10]Figure 10 provides images of pig lacrimal glands that received an injection of AAV9-secEGFP, were harvested on day 103, and were fixed in paraffin. IHC was performed on 5 μM sections using an anti-GFP antibody and DAPI (nuclear) counterstain. In addition to lacrimal acinar cells, ductal epithelial cells appear to have been transduced by AAV9 (white arrows). [Figure 11] Figure 11 provides images of pig lacrimal glands that were given an injection of AAV, harvested on day 103, and fixed in paraffin. H&E staining of 5 μM paraffin sections at 100x magnification reveals the absence of inflammatory infiltrates, macroscopic abnormalities, or microscopic abnormalities. [Figure 12] Figure 12 provides a schematic of the treatment schedule for administering AAV encoding a model protein via injection into the lacrimal gland of pigs in combination with administration of OC-01 (varenicline) via nasal spray. Time points for tear collection and termination of the study are indicated. [Figure 13] Figure 13 shows the results of Western blot analysis using anti-thioredoxin primary antibody (ThermoFisher catalog #14999-1-AP) and anti-rabbit IgG HRP secondary antibody (ProMega catalog #A5316). [Figure 14] Figure 14 provides an image of a Western blot for detecting TRX in cell lysates obtained from untransfected (293T) or transfected (AAV.TRX) 293T cells. Detection of actin was used as a dosage control. Whole cell extracts (30 μg / lane) were subjected to SDS-PAGE, and anti-TRX and anti-actin primary antibodies were used to detect the respective proteins. [Figure 15] Figure 15 is a graph showing the results of a TRX activity assay. 293T cells transfected with the thioredoxin gene plasmid exhibited a thioredoxin activity of 56.8 nM / min, while untransfected 293T cells exhibited an endogenous thioredoxin activity level of 16.90 nM / min. This functional assay confirmed the expression, secretion, and function of the gene product from the construct shown in Figure 1. DETAILED DESCRIPTION OF THE INVENTION

[0092] The present disclosure provides methods of treating an ocular condition in a subject in need thereof. Such methods may include, for example, inducing expression of oxidoreductase enzymes (such as TRX and / or PDI) in the eye. As disclosed herein, AAV-based gene delivery systems for ocular gene delivery include: This method provides a means to treat ocular diseases. rAAV vectors offer unique advantages for gene delivery. Genes delivered by rAAV vectors likely do not integrate into the genome of the transduced cells, thereby not supporting chronic expression and genomic inheritance of the gene product. rAAV vectors transport genes across the plasma membrane of the target cells and deliver them into the nucleus, where they exist episomal rather than integrated into the genome. Expression of the delivered gene depends on cellular turnover in the specific tissue of the transduced cells (BioDrugs. 2017; 31(4): 317-334). This advantage means that the gene expression period is relatively controlled, with no genomic inheritance, particularly in germ cells. Additionally, the rAAV vectors described here (e.g., rAAV virions) are less immunogenic than other viral delivery vectors, such as adenovirus. AAV-based delivery vectors that transduce cells in the posterior segment of the eye using subretinal and intravitreal injections have been described and shown to be effective 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)).

[0093] AAV serotypes used to deliver genes to the eye using AAV-based delivery include AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, and AAV9 (Lebherz et al. J Gene Med.; 10(4): 375-382 (2008)). AAV serotypes used to deliver genes to the lacrimal gland include AAV2, AAV4, AAV5, AAV5w8, AAV x5, AAV 9, AAV 12, and BAAV (Rocha et al., Invest Ophthalmol Vis Sci. 52:9567-9572 (2011)).

[0094] In some embodiments, the present disclosure provides rAAV vectors 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 disclosure provides an rAAV vector for expressing TRX (e.g., human TRX). The present disclosure provides rAAV vectors for expressing PDI (e.g., human PDI).

[0095] In some embodiments, cells of at least one eye and / or lacrimal gland are treated with an rAAV of the present disclosure. Transduced with vector. The lacrimal gland is the primary source of tears to promote a healthy ocular surface and maintain normal visual function. The main lacrimal gland consists of the palpebral and orbital lobes, which are continuous with each other at the lateral end of the aponeurosis of the levator palpebrae. The lobules contain many acini and intralobular ducts, which form excretory ducts that open into the fornix of the conjunctiva. The main lacrimal gland is composed of acinar cells, ductal cells, and / or myoepithelial cells. (Obata Cornea.; 25(10 Suppl 1):S82-9 (2006)). The main lacrimal gland secretes the aqueous layer and mucin of the tear film onto the surface of the eye (e.g., Paulsen, F., et al (2004) Cell and tissue research, 316(2), 167-177). The term "lacrimal gland" as used herein refers to the main lacrimal gland and the Wolff lacrimal gland. Also referred to as Ring glands and glands of Claus. The accessory glands, known as Wölfring's glands and Claus's glands, are located in the eyelids. The upper eyelid contains approximately 2-5 Wölfring's glands and 40 Claus's glands. The lower eyelid contains approximately 6-8 Claus's glands. The specific location and anatomy of the lacrimal functional unit are well known (Conrady et al., J Ophthalmol. Article ID 7542929 (2016)).

[0096] Aging-related oxidative stress is known to alter the structure and function of the lacrimal gland, leading to acinar atrophy and fibrosis (Rocha, E et. al., Ocul Surf. 2008 Oct.; 6(4)). Without being bound by theory, it is believed that alterations in lacrimal gland proteins, for example as a result of oxidation and the resulting disulfide bond formation, reduce nerve stimulation (afferent parasympathetic and sympathetic nerves) and the lacrimal gland's protein secretory function, resulting in reduced tear production in terms of quantity and quality. This decreased lacrimal gland function is thought to contribute to dry eye disease. Furthermore, a similar mechanism occurs in the Meibomian gland (another region of the lacrimal functional unit). The Meibomian gland is primarily responsible for producing the lipid layer of the tear film, and its loss of function leads to Meibomian gland dysfunction (MGD). Loss or shedding of Meybomian glands due to atrophy can be directly linked to MGD (Chhadya, P. et.al., Ophthalmology. 2017 Nov; 124(11)), while duct obstruction / atresia due to fibrosis can lead to atrophy and loss of function, further contributing to ocular surface disease.

[0097] The trabecular meshwork is the drainage network of the eye located at the iridocorneal angle (where the iris and cornea meet and where the sclera transitions into the cornea). The trabecular meshwork is a small, porous triangle (approximately 350 × 50-150 μm cross section) composed of beams and sheets (lamellae) of connective tissue covered by trabecular meshwork cells. (Abu-Hassan et al., J Ocul Biol. 2014 May;2(1)). Trabecular meshwork cells play an important role in maintaining intraocular pressure by regulating the outflow resistance of aqueous humor. Oxidative stress alters the structure of the trabecular meshwork, increasing outflow resistance and ultimately increasing intraocular pressure (IOP). and optic nerve damage (International Journal of Medicine Oct 2016; Vol 38 Issue 4; 995-1002). This damage may further lead to progressive visual field loss. Without being bound by theory, increased expression of TRX and / or PDI may result in the formation of additional disulfides. Reversing or preventing the formation of these bonds serves to reduce outflow resistance and associated optic nerve damage.

[0098] The retina is a light-sensitive layer of nerve tissue at the back of the eye that receives light impulses and transmits them as electrical signals through the optic nerve to the brain for visual perception. The survival of retinal photoreceptors and retinal ganglion cells requires an equilibrium between oxygen, reactive oxygen species, and antioxidant molecules that counteract oxidative stress. Oxidative stress can alter cellular homeostasis and trigger protective responses, especially in cells with high metabolic rates, such as photoreceptors and retinal ganglion cells, that are constantly exposed to light and oxidative stress insults (B. Domenech, E., & Marfany, G. (2020)). Oxidative stress is a key factor in retinal dystrophy. It is believed that these compounds contribute to the pathogenesis and treatment of rheumatoid arthritis. For example, reference).

[0099] Thioredoxin enzyme In one embodiment, the expression cassette of the present disclosure comprises a thioredoxin (TRX) enzyme or its The TRX enzyme is present in all living organisms. Contains a dithiol Cys-Gly-Pro-Cys (CGPC) active site motif that is widely conserved across kingdoms (Holmgren A. J Biochem. (1968) 6:475-484). In mammals, nycotinamide adenine It reduces oxidized cysteine ​​via a dinucleotide diphosphate (NADPH)-dependent reaction. There are at least two enzymes (TRX1 and TRX2) that can catalyze this process (Lee, et al (2013) Antioxidant Redox Signal 18:1165-1207). TRX1 is generally localized in the cytoplasm and is either translocated to the nucleus or cleaved. TRX2 is generally localized to mitochondria.

[0100] As used herein, the terms "thioredoxin," "TRX," and "TRX enzyme" refer to the TRX1 protein of any species. In one embodiment, the TRX1 protein is human. The term "functional variant" refers to a variant that has sequence substitutions, insertions, deletions, and / or N- or C-terminal alterations. Mutants that are truncated and retain one or more functions of the reference protein, e.g., the native TRX enzyme. Thioredoxins are proteins that form homodimers and function as oxidoreductases.

[0101] In some embodiments, the TRX enzyme is a human TRX enzyme. In some embodiments, the human TRX enzyme is identified in a public database. Known human TRX isoforms are generally identified through public databases. For example, the National Center for Biotechnology Information (NCBI) Gene Database at the U.S. National Library of Medicine (available on the web at ncbi.nlm.nih.gov / ) contains genetic information. A searchable gene database providing nomenclature, chromosomal location, gene product, gene attributes, associated markers, phenotypes, interactions, links to citations, sequence information, information on sequence variation, gene maps, expression reports, homologs, protein domain content, and access to external databases. As will be appreciated by those skilled in the art, sequence information for human TRX isoforms known in the art can be found by using the appropriate accession number in the NCBI Gene Enter sequence information into a database in a desired computer-readable format (e.g., FASTA). Such sequence information may include the complete sequence encoding TRX. The nucleotide sequence of the gene encoding TRX includes, but is not limited to, the nucleotide sequence of the pre-mRNA transcript encoding TRX, the nucleotide sequence of the mRNA encoding TRX, the nucleotide sequence of the open reading frame (ORF) encoding TRX, and the amino acid sequence of TRX. For example, one isoform of human TRX is identified in the NCBI gene database under Gene ID number 7295, and its sequence is: It has the amino acid sequence shown in sequence no. 25.

[0102] Exemplary amino acid sequences of human TRX and exemplary polynucleotide sequences (mRNA and ORF) encoding human TRX are shown in Table 1. In certain embodiments, the TRX enzyme comprises or consists of the amino acid sequence shown in Table 1. In certain embodiments, the TRX enzyme comprises or consists of the amino acid sequence encoded by the mRNA sequence shown in Table 1. In certain embodiments, the TRX enzyme comprises or consists of the amino acid sequence encoded by the ORF shown in Table 1.

[0103] 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 or consists of SEQ ID NO: 1. The enzyme may be 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% identical to SEQ ID NO:25. In one embodiment, the TRX enzyme comprises an amino acid sequence having the identity of SEQ ID NO: 25. or consisting of the amino acid sequence

[0104] In certain embodiments, the TRX enzyme is encoded by an ORF 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 one embodiment, the TRX enzyme is encoded by an ORF comprising SEQ ID NO:2. do.

[0105] In one embodiment, the TRX enzyme is at least 80% identical to SEQ ID NO:28, at least and / or by an ORF containing a nucleotide sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity. In one embodiment, the TRX enzyme is encoded by an ORF comprising SEQ ID NO:28.

[0106] In some embodiments, the TRX enzyme is a truncated version of any of the TRX enzyme sequences described herein (e.g., SEQ ID NOs: 1 or 25). For example, in some embodiments, the TRX enzyme is a truncated version of any of the sequences Sequence numbers 1 and 25 are fragments containing at least 20, 40, 60, 80, or 100 consecutive amino acids. In one embodiment, 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 substitutions compared to SEQ ID NO: 1 or 25. In one embodiment, the TRX enzyme has an insertion or deletion of SEQ ID NO: 1 or 25. A fragment containing at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 consecutive amino acids or a fragment having a length within a range selected from any pair of the aforementioned lengths. A truncated version of any TRX enzyme sequence described herein (e.g., SEQ ID NOs: 1 or 25) can contain one or more amino acid substitutions, insertions, or deletions and share at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NOs: 1 or 25. This may be the case.

[0107] Polynucleotides encoding thioredoxin enzymes In some embodiments, the expression cassette comprises a polynucleotide encoding a TRX enzyme described herein (e.g., a human TRX enzyme). In some embodiments, the polynucleotide comprises an ORF encoding a TRX enzyme described herein (e.g., a human TRX enzyme).

[0108] In some embodiments, the expression cassette increases expression of TRX in at least one eye and / or lacrimal gland. In some embodiments, the expression cassette increases expression of TRX in at least one Meibomian gland. In some embodiments, the expression cassette increases expression of TRX in the trabecular meshwork. In one embodiment, the expression cassette increases expression of TRX in the retina. In one embodiment, expression of TRX is increased in the eye of an untreated subject or a treated subject. The increase is 5%, 10%, 15%, 20%, or 25% compared to the contralateral eye. As used herein, "subject" refers to a mammal, such as a mouse, rabbit, primate (NHP), or human. In some embodiments, In this context, the subject is a human or NHP. Also, the terms "individual" and "patient" are used interchangeably with "subject." In one embodiment, TRX expression is detected in the contralateral eye of an untreated subject or a treated subject. In one embodiment, the expression level is increased by at least 1.5-fold, 2-fold, 3-fold, 4-fold, or 5-fold compared to Detectable levels of TRX expression in treated eyes and untreated or treated controls TRX was either not expressed or not expressed at detectable levels in the contralateral eye of the elephants. In other words, the eyes or lacrimal glands administered with the rAAV vectors described here are Decreased secretion of endogenous (i.e., native) expression only in the eye or lacrimal gland or endogenous TRX It may express more TRX than the eye.

[0109] The term "functional variant" refers to a variant that retains one or more functions of a reference protein, e.g., a native TRX, and does not include sequence substitutions, insertions, deletions, and / or N- or C-terminal truncations. This may include a breakdown.

[0110] In one embodiment, the expression cassette comprises a polypeptide encoding human TRX or a functional variant thereof. In one embodiment, the expression cassette comprises an amino acid sequence shown in Table 1. In one embodiment, the expression cassette comprises a polynucleotide comprising a nucleotide sequence encoding the sequence shown in Table 1.

[0111] In certain embodiments, the expression cassette encodes 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 an amino acid sequence shown in Table 1. In one embodiment, the expression cassette comprises a polynucleotide comprising a nucleotide sequence encoding an amino acid sequence set forth in Table 1. In one embodiment, the expression cassette is at least 80% identical to SEQ ID NO:1, In one embodiment, the expression cassette comprises a polynucleotide comprising a nucleotide sequence encoding a protein having 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to any of the sequences 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 SEQ ID NO:25. 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 SEQ ID NO:25. In one embodiment, 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 one embodiment, the expression cassette comprises a polynucleotide comprising a nucleotide sequence set forth in SEQ ID NO: 2. 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 one embodiment, the expression cassette comprises a polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 28.

[0112] In one embodiment, the expression cassette comprises a polynucleotide sequence encoding a TRX enzyme. 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 or consisting of SEQ ID NO: 1. In one embodiment, the TRX enzyme comprises or is comprised of SEQ ID NO: 1. In one embodiment, the expression cassette comprises a polynucleotide comprising a nucleotide sequence encoding the TRX enzyme. The TRX enzyme may be at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or less similar to SEQ ID NO:25. In one embodiment, the TRX enzyme comprises or consists of an amino acid sequence having at least 99% identity to SEQ ID NO: 25. In one embodiment, the TRX enzyme comprises or consists of SEQ ID NO: 25. The present cassette comprises a polynucleotide comprising a nucleotide sequence encoding a TRX enzyme, wherein 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 or consists of SEQ ID NO: 24. In one embodiment, the TRX enzyme comprises or consists of SEQ ID NO: 24.

[0113] Percent identity can be determined using any suitable method known in the art. For example, a method for determining percent identity of a nucleotide sequence is genome sequencing. Methods for determining percent identity of a nucleotide sequence or amino acid sequence are known to those skilled in the art.

[0114] Protein disulfide isomerase In certain embodiments, the expression cassette of the present disclosure comprises a polynucleotide sequence encoding a protein disulfide isomerase (PDI) or a functional variant thereof. The ability of PDI to reduce binding is a key factor in the progression of oxidative stress and normal aging, such as cataract formation and presbyopia. Important protection against diseases associated with disulfide bond formation in an oxidative environment due to disease, environmental, or pharmacological events, including dry eye disease, Meybomian gland dysfunction, and elevated intraocular pressure / glaucoma PDI contains a thioredoxin active site with a CXXC motif. This will highlight the potential of PDI in disulfide bond reduction to reduce disease states in various tissues. Protein disulfide isomerase (gene name: P4HB) is a 508 amino acid protein with a mass of approximately 57.1 kDa (also known as ERBA2L, PDI, PDIA1, and PO4DB).

[0115] As used herein, "protein disulfide isomerase" or "PDI" refers to any The term "functional variant" refers to a PDI protein of a species. refers to variants that retain one or more functions of native PDI and contain sequence substitutions, insertions, deletions, and / or deletions. or may include N- or C-terminal truncations.

[0116] In one embodiment, the PDI is a human PDI enzyme. In one embodiment, the human PDI is a PDI enzyme from public data. Known human PDI isoforms are generally identified from public databases. As understood by a skilled technician, human PDI known in the art Isoform sequence information can be obtained by entering the appropriate accession number into the NCBI gene database and selecting the sequence information in the desired computer-readable format (e.g., FASTA). Such sequence information includes the nucleotide sequence of the complete gene encoding TRX. The nucleotide sequence of a PDI gene includes, but is not limited to, the nucleotide sequence of a pre-mRNA transcript encoding PDI, the nucleotide sequence of an mRNA encoding PDI, the nucleotide sequence of an open reading frame (ORF) encoding PDI, and the amino acid sequence of PDI. For example, one isoform of human PDI can be identified in the NCBI gene database under Gene ID number 5034, and its sequence can be found at: It has the amino acid sequence shown in column no. 26.

[0117] Exemplary amino acid sequences of human PDI and exemplary polynucleotide sequences (mRNA and ORF) encoding human PDI are shown in Table 2. In some embodiments, PDI comprises or consists of the amino acid sequence shown in Table 2. In some embodiments, PDI comprises or consists of the amino acid sequence encoded by the mRNA sequence shown in Table 2. In some embodiments, PDI comprises or consists of the amino acid sequence encoded by the nucleotide sequence shown in Table 2.

[0118] In one embodiment, the PDI has a sequence similar to SEQ ID NO: 26, such as at least 80%, at least 85%, At least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or In one embodiment, the PDI comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or identical to the amino acid sequence spanning positions 24-474 of SEQ ID NO:26. In one embodiment, the nucleotide sequence encoding PDI comprises or consists of SEQ ID NO:30.

[0119] In one embodiment, the PDI is a truncated version of any of the PDI sequences described herein (e.g., For example, in one embodiment, the PDI comprises at least one of SEQ ID NO: 26. or a fragment containing 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. In some embodiments, the 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 a sequence similar to or different from SEQ ID NO:26. , 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. In one embodiment, 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 selected from any pair of the foregoing lengths. Any truncated version of the amino acid sequence of the present invention may contain 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 the sequence spanning positions 24-474 of SEQ ID NO:26.

[0120] Polynucleotides encoding PDI In some embodiments, the expression cassette comprises a polynucleotide encoding a PDI enzyme described herein (e.g., a human PDI enzyme). In some embodiments, the polynucleotide comprises an ORF encoding a PDI enzyme described herein (e.g., a human PDI enzyme).

[0121] In certain embodiments, the expression cassette increases expression of PDI in at least one eye and / or lacrimal gland. In certain embodiments, the expression cassette increases expression of PDI in at least one Meibomian gland. In certain embodiments, the expression cassette increases expression of PDI in a thyroid gland. The expression of PDI in the trabecular meshwork is increased. In one embodiment, the expression of PDI is increased by 5%, 10%, 15%, 20%, or 25% in the untreated or treated subject's eye compared to the contralateral eye. In one embodiment, the expression of PDI is increased by 5%, 10%, 15%, 20%, or 25% in the untreated or treated subject's eye compared to the contralateral eye. In some embodiments, PDI is expressed at detectable levels in the treated eye, and PDI is not expressed or not expressed at detectable levels in the contralateral eye of an untreated subject or a treated subject. In other words, the eyes or lacrimal glands administered with the rAAV vectors described here are the first to undergo PDI. Decreased secretion of endogenous (i.e., native) expression only in the eye or lacrimal gland or endogenous PDI The eye may express more PDI than the brain.

[0122] The term "functional variant" refers to a variant that retains one or more functions of a reference protein, e.g., a native PDI, and does not include sequence substitutions, insertions, deletions, and / or N- or C-terminal truncations. This may include a breakdown.

[0123] In one embodiment, the expression cassette encodes human PDI or a functional variant thereof. In one embodiment, the expression cassette comprises a polynucleotide comprising an amino acid sequence shown in Table 2. In one embodiment, the expression cassette comprises a polynucleotide comprising a nucleotide sequence encoding a nucleotide sequence shown in Table 2. include.

[0124] In certain embodiments, the expression cassette has a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 100%, at least 120%, at least 140%, at least 160%, at least 180%, at least 190%, at least 200%, at least 210%, at least 220%, at least 230%, at least 240%, at least 250%, at least 260%, at least 270%, at least 280%, at least 290%, at least 300%, at least 310%, at least 320%, at least 330%, at least 340%, at least 350%, at least 360%, at least 370%, at least 380%, at least 390%, at least 400%, at least 410%, at least 420%, at least 430%, at least encode proteins with at least 97%, at least 98%, or at least 99% identity In one embodiment, the expression cassette comprises a polynucleotide comprising a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 26. In one embodiment, the expression cassette comprises a polynucleotide comprising a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 30. at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least contain nucleotide sequences that share 97%, at least 98%, or at least 99% identity with In one embodiment, the expression cassette comprises a polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO:30.

[0125] In one embodiment, the expression cassette comprises a nucleotide sequence encoding a PDI enzyme. The PDI comprises a polynucleotide having at least 80%, at least 85%, or , at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or or an amino acid sequence with at least 99% identity to SEQ ID NO: 26, or consisting of SEQ ID NO: 26 In one embodiment, the PDI comprises or consists of SEQ ID NO: 26. In the present invention, the expression cassette is a polynucleotide comprising a nucleotide sequence encoding a PDI enzyme. The PDI contains 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% of the sequence of SEQ ID NO: 30. or consisting of an amino acid sequence having identity to SEQ ID NO: 30.

[0126] Codon optimization 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 mouse cell, or a non-human primate (NHP) cell.

[0127] A codon-optimized nucleotide sequence, e.g., a codon-optimized nucleotide sequence encoding a TRX enzyme or a PDI, typically contains at least one synonymous nucleotide 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 can be partially or completely different from the reference sequence. For example, polyserine, which is uniformly encoded by the TCT codon, can be The coding reference sequence is sequence optimized by substituting all nucleotides to convert it into a sequence that uniformly encodes polyserine with the AGC codon. In this case, the reference polyserine nucleotide Although the global pairwise alignment of the sequence with a sequence-optimized polyserine nucleotide sequence yields 0% sequence identity, the protein product generated from both sequences are 100% identical.

[0128] Methods of codon optimization are known to those skilled in the art and can be useful for achieving desired results, such as increasing expression of a target gene. In one embodiment, the expression cassette comprises a nucleotide sequence whose sequence has been optimized relative to a reference sequence using sequence optimization methods. Methods of sequence optimization are known to those skilled in the art and include known sequence optimization tools, algorithms, and services. Non-limiting examples include the services of GeneArt (Life Technologies), DNA2.0 (Menlo Park, CA), Geneious®, and GeneGPS® (Atum, Newark, CA).

[0129] In certain embodiments, an expression cassette of the present disclosure comprises a polynucleotide encoding a TRX or PDI enzyme that has been sequence-optimized relative to a reference sequence using sequence optimization methods (e.g., GeneGPS®, Geneious®). In certain embodiments, the reference sequence encoding a TRX is set forth in SEQ ID NO: 28. In certain embodiments, an expression cassette comprises a polynucleotide that has been sequence-optimized relative to a reference sequence encoding a TRX. In 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 one embodiment, the reference sequence encoding PDI is set forth in SEQ ID NO: 30. In one embodiment, the expression cassette comprises a polynucleotide whose sequence is optimized relative to the reference sequence encoding PDI.

[0130] In one embodiment, the method of sequence optimization comprises the codon optimization algorithms described in U.S. Patents 7,561,972; 7,561,973; 8,126,653; and 8,401,798, each of which is incorporated herein by reference. In one embodiment, the polynucleotide sequence is optimized using sequence optimization methods known to those skilled in the art (e.g., GeneGPS®, Geneious®). The sequence is optimized relative to the reference sequence based on the codon usage bias of the host cell (e.g., mammalian cell, human cell, mouse cell, non-human primate cell). In some embodiments, the polynucleotide sequence comprises or consists of a nucleotide sequence that has been codon-optimized relative to the reference sequence using sequence optimization methods. In some embodiments, the polynucleotide sequence comprises or consists of a nucleotide sequence that has been 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.

[0131] In certain embodiments, the present disclosure provides a polynucleotide comprising or consisting of a nucleotide sequence that is at least 80%, at least 85%, at least 90% similar to SEQ ID NO:2. In one embodiment, the polynucleotide comprises or consists of SEQ ID NO:2. [Table 1] [Table 2]

[0132] Expression cassette The vectors of the present disclosure comprise an expression cassette. As used herein, the term "expression cassette" refers to a polynucleotide comprising at least one polynucleotide sequence, The nucleotide sequence encodes a protein of interest (e.g., TRX or PDI). In one embodiment (e.g., when the vector is a rAAV virion), the expression cassette comprises inverted terminal repeats flanking the polynucleotide sequence encoding at least one 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 enzyme. In some embodiments, the protein of interest is TRX. In this embodiment, the target protein is protein disulfide isomerase (PDI). In some embodiments, the expression cassette contains other polynucleotide sequences, e.g., promoter sequences. a promoter, regulatory elements (e.g., one or more promoters), a translation initiation sequence, a coding sequence, and and termination sequences.

[0133] In some embodiments, the expression cassette of the present disclosure comprises a polynucleotide sequence encoding an oxidoreductase enzyme (e.g., TRX or PDI) or a functional variant thereof. In some embodiments, the expression cassette increases expression of TRX in at least one eye, lacrimal gland, and / or trabecular meshwork. In some embodiments, TRX expression is increased by 5%, 10%, 15%, 20%, or 25% compared to the contralateral eye of an untreated or treated subject. In some embodiments, TRX expression is 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 contralateral eye of an untreated or treated subject. In some embodiments, TRX is expressed at detectable levels in treated eyes and not in untreated or treated subjects. In the contralateral eye, TRX expression may be absent or not detectable. In other words, the rAAV vector-injected eye, lacrimal gland, Meibomian gland, and / or The eye, lacrimal gland, Meibomian gland, and / or trabecular meshwork may express higher concentrations of oxidoreductase enzymes (e.g., TRX or PDI), whereas the eye, lacrimal gland, Meibomian gland, and / or trabecular meshwork, which only have endogenous (i.e., native) expression of oxidoreductase enzymes, may express higher concentrations of oxidoreductase enzymes (e.g., TRX or PDI). Enzymes (e.g., TRX or PDI) may be expressed at higher concentrations compared to eyes with reduced secretion.

[0134] The term "functional variant" refers to a variant that retains one or more functions of a reference protein, e.g., a native TRX or PDI, and that includes sequence substitutions, insertions, deletions, and / or N-terminal or or C-terminal truncation.

[0135] In one embodiment, the expression cassette provided herein comprises a polynucleotide encoding TRX. Exemplary amino acid sequences of TRX and exemplary polynucleotide sequences encoding TRX are shown in Table 3. [Table 3]

[0136] In one embodiment, the polynucleotide is at least 80% identical to SEQ ID NO:1, and at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 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 to SEQ ID NO:25. In some embodiments, the polynucleotide encodes human TRX or a functional variant thereof.

[0137] In certain embodiments, the polynucleotide encoding TRX has a sequence similar to that of SEQ ID NO:2, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, or at least The term "sequence" includes sequences that share 97%, at least 98%, at least 99%, or 100% identity with any of the sequences. In one embodiment, the polynucleotide encoding TRX has a sequence similar to that of SEQ ID NO:28. at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least The term "sequence" includes sequences that share 97%, at least 98%, at least 99%, or 100% identity with any of the sequences. In certain embodiments, the protein is human TRX or a functional variant thereof.

[0138] In one embodiment, the polynucleotide encoding TRX also encodes a signal peptide. The signal peptide serves to facilitate protein expression and subcellular localization. In certain embodiments, the signal peptide is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, or at least 100% identical to SEQ ID NO: 3. In certain embodiments, the polynucleotide shares at least 80%, at least 85%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:4. Also includes sequences that share 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity.

[0139] In one embodiment, the expression cassettes provided herein express an oxidoreductase other than TRX. It contains a transgene encoding a lyase enzyme.

[0140] In one embodiment, the expression cassettes provided herein comprise bicistronic expression cassettes that allow for the simultaneous expression of two proteins using a single mRNA transcript. The bicistronic expression cassette may be constructed in a manner that allows for the expression of both a TRX enzyme sequence and a PDI sequence selected from any of the sequences or embodiments described herein (e.g., by incorporation of an internal ribosome entry site "IRES"). For example, a bicistronic expression cassette may contain both a TRX enzyme sequence and a PDI sequence selected from any of the sequences or embodiments described herein. In certain embodiments, simultaneous expression of TRX and PDI in cells of the eye can provide a synergistic therapeutic effect.

[0141] In some embodiments, the expression cassette increases expression of PDI in at least one eye, lacrimal gland, at least one Meibomian gland, and / or trabecular meshwork. In some embodiments, PDI expression may be increased by 5%, 10%, 15%, 20%, or 25% compared to the contralateral eye of an untreated or treated subject. In some embodiments, PDI expression 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 contralateral eye of an untreated or treated subject. In some embodiments, PDI is expressed at detectable levels in the treated eye and is absent or increased five-fold in the contralateral eye of the untreated or treated subject. In other words, the eyes, lacrimal glands, Meibomian glands, and / or trabecular meshwork to which the rAAV vector was administered may express oxidoreductases Enzymes (e.g., PDI) may be expressed at higher concentrations, and oxidoreductase enzymes Eye, lacrimal gland, Meibomian gland, retina, and / or retina with only endogenous (i.e., native) expression of the gene or decreased secretion of trabecular meshwork or endogenous oxidoreductase enzymes (e.g., PDI) May occur at higher concentrations than in the eye.

[0142] The term "functional variant" refers to a variant that retains one or more functions of a reference protein, e.g., a native PDI, and that does not include sequence substitutions, insertions, deletions, and / or N- or C-terminal This includes cutting.

[0143] In one embodiment, the polynucleotide has at least 80% identity to SEQ ID NO: 26 At least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least In one embodiment, the polynucleotide encodes a protein that shares at least 98%, at least 99%, or 100% identity with human PDI or a functional variant thereof. .

[0144] In one embodiment, the polynucleotide encoding PDI has the sequence shown in SEQ ID NO: 30. At least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least sequences that share at least 97%, at least 98%, at least 99%, or 100% identity In one embodiment, the protein is human PDI or a functional variant thereof.

[0145] In one embodiment, the polynucleotide encoding PDI also encodes a signal peptide. The signal peptide serves to facilitate protein expression and subcellular localization. In certain embodiments, the signal peptide is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, or at least 100% identical to SEQ ID NO: 3. In certain embodiments, the polynucleotide shares at least 80%, at least 85%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:4. Also includes sequences that share 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity.

[0146] The expression cassettes of the present disclosure include a promoter. As used herein, the term "promoter" refers to a DNA molecule that directs the binding of RNA polymerase, thereby promoting RNA synthesis. The term "enhancing sequence" refers to a sequence, i.e., a minimal sequence sufficient to direct transcription. Expression of a promoter and the corresponding protein or polypeptide may be cell-type-, tissue-, or species-specific, or may be strongly activated or strongly activated across a wide range of cells, tissues, and species. Promoters may be "constitutive," meaning continuously activated, or "inducible," meaning activated or deactivated by the presence or absence of a biotic or abiotic factor. The nucleic acid constructs or vectors of the present disclosure also include enhancing sequences, whether or not they are contiguous with the promoter sequence. Enhancement sequences affect promoter-dependent gene expression and may be located in the 5' or 3' regions of the native gene.

[0147] The polynucleotide cassettes described herein include a promoter region that drives expression of a polynucleotide sequence encoding an oxidoreductase enzyme (e.g., TRX or PDI) in one or more of the eye, lacrimal gland, Meibomian gland, and / or trabecular meshwork, provided that an appropriate promoter region or promoter sequence is used. In one embodiment, the promoter drives gene expression in the mammalian eye, lacrimal gland, Meibomian gland, and / or trabecular meshwork. In certain embodiments, the expression cassette includes a cell-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 certain embodiments, the promoter is a corneal stroma-specific promoter (e.g., collagen peptide 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)), corneal epithelium / limbal Stem cell-specific promoters (e.g., keratin-12 or Pax-6 promoters (Wang, I., et al. "Cis-regulatory elements of the mouse Krt1.12 gene." Molecular vision, 8, 94-101 (2002); 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 (e.g., chitin 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.” Invest Ophthalmol Vis Sci. 2005 Jan;46(1):183-90)), or iris cells. These may include cell-specific promoters (e.g., 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)).

[0148] In some embodiments, the promoter is a CAG promoter. The promoter may be at least 90%, 95%, 96%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 310 In some embodiments, the nucleotide sequence has 6%, 97%, 98%, or 99% identity. and the promoter comprises SEQ ID NO:5.

[0149] In one embodiment, the promoter is a CMV promoter. Thus, the promoter comprises a nucleotide sequence having at least 95%, 96%, 97%, 98%, or 99% identity to the nucleotide sequence set forth in SEQ ID NO: 17. In one embodiment, the promoter The promoter comprises SEQ ID NO:17.

[0150] In one embodiment, the enhancer is a CMV enhancer. The CMV enhancer has at least 95%, 96%, or more identical sequence to the nucleotide sequence shown in SEQ ID NO: 18. In one embodiment, the promoter comprises a sequence having 97%, 98%, or 99% identity to the promoter. includes SEQ ID NO: 18.

[0151] In one embodiment, the expression cassette comprises a promoter and an enhancer. In one embodiment, the CMV enhancer and promoter are the nucleotide sequence set forth in SEQ ID NO: 19. The sequences include sequences that have at least 95%, 96%, 97%, 98%, or 99% identity to the nucleic acid sequence. In one embodiment, the enhancer and promoter comprise SEQ ID NO:19.

[0152] The expression cassette may include a polyadenylation (polyA) sequence. In certain embodiments, the expression cassettes described herein contain transcription termination signals. Elements that direct efficient termination and polyadenylation of exogenous nucleotide transcripts increase exogenous gene expression. Transcription termination signals are generally located downstream of polyadenylation signals. In certain embodiments, the vector contains a polyadenylation sequence 3' to the polynucleotide encoding the polypeptide. As used herein, the term "polyA site" or "polyA sequence" refers to a sequence that initiates the RNA polymerase. This figure shows the DNA sequence that directs both the termination and polyadenylation of the nascent RNA transcript by ribosomal enzyme II. The polyadenylation sequence increases mRNA stability and adds a polyA tail to the 3' end of the coding region. This improves translation efficiency. Polyadenylation and cleavage are directed by the polyA sequence in the RNA. The core polyA sequence of mammalian pre-mRNA contains two recognition elements, one on either side of the cleavage-polyadenylation site. Typically, the nearly invariant AAUAAA hexamer is replaced by a more variable one rich in U or GU residues. The 5' cleavage product is located 20-50 nucleotides upstream of the nucleotide sequence. Cleavage of the nascent transcript occurs between these two elements, with the addition of up to 250 adenosines to the 5' cleavage product. In certain embodiments, In certain embodiments, the core polyA sequence is an ideal polyA sequence (e.g., AATAAA, ATTAAA, AGTAAA). In certain embodiments, the polyA sequence is an SV40 polyA sequence, a bovine growth hormone polyA sequence (BGH polyA), a rabbit β-globin polyA sequence (rβgpA), a variant thereof, or any other suitable exogenous or endogenous polyA sequence known in the art. In certain embodiments, the polyA sequences described herein are In one embodiment, the expression cassette comprises a polyA sequence. In one embodiment, the BGH polyA sequence is the nucleotide sequence set forth in SEQ ID NO: 20. Containing or consisting of at least 95%, 96%, 97%, 98%, or 99% identity to the sequence In one embodiment, the BGH polyA sequence consists of SEQ ID NO:20.

[0153] In some embodiments, the rAAV vector of the present disclosure comprises a woodchuck post-transcriptional regulatory element (WPRE). In some embodiments, the rAAV vector comprises a WPRE comprising SEQ ID NO: 21.

[0154] Recombinant AAV vectors In some embodiments, the expression cassettes described herein are used to deliver an oxidoreductase enzyme (e.g., TRX or PDI) or a functional variant thereof to at least one eye and / or lacrimal gland of a subject, e.g., for the treatment of ocular diseases. In some embodiments, the expression cassettes described herein are used to deliver an oxidoreductase enzyme (e.g., TRX or PDI) or a functional variant thereof to at least the Meibomian gland of a subject. Thus, in one embodiment, the composition that provides expression of an oxidoreductase enzyme (e.g., TRX or PDI) or a functional variant thereof in at least one eye and / or lacrimal gland of a subject is a gene delivery vector that includes an expression cassette described herein.

[0155] In one embodiment, the gene delivery vector is a rAAV vector (e.g., a rAAV virion). In one embodiment, the expression cassette is flanked on both the 5' and 3' ends by functional AAV inverted terminal repeat (ITR) sequences. By "functional AAV ITR sequences" is meant ITR sequences that function as intended for the recovery, replication, and packaging of the AAV vector. Therefore, the AAV ITRs used in the gene delivery vectors of the present disclosure contain wild-type nucleotides. The AAV ITRs need not have the same sequence as the AAV ITRs, but may be altered by nucleotide insertions, deletions, or substitutions, or may be derived from any of several AAV serotypes (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10). In one embodiment, the AAV ITRs are derived from AAV2. In some embodiments, the AAV ITRs are derived from AAV3. In some embodiments, the AAV ITRs are derived from AAV4. In some embodiments, the AAV ITRs are derived from AAV5. In some embodiments, the AAV ITRs are derived from AAV6. In some embodiments, the AAV ITRs are derived from AAV7. In one embodiment, the AAV ITRs are derived from AAV9. In one embodiment, the AAV ITRs are derived from AAV10. In one embodiment, the rAAV vector has the wild-type REP and CAP genes deleted, in whole or in part, but retains a functional IRT sequence. In one embodiment, the AAV ITRs are shown in Table 4. [Table 4]

[0156] In one embodiment, the rAAV vector comprises an AAV capsid of any adeno-associated virus serotype known in the art, or discovered in the future. Non-exclusive examples include AAV1, AAV2, , AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, etc. For example, the AAV capsid may be a wild-type (or "native") capsid. In some embodiments, the rAAV vector comprises an AAV capsid derived from AAV1. In some embodiments, the rAAV vector In some embodiments, the rAAV vector comprises an AAV capsid derived from AAV2. In some embodiments, the rAAV vector comprises an AAV capsid derived from AAV3. In some embodiments, the rAAV vector comprises an AAV capsid derived from AAV4. In one embodiment, the rAAV vector comprises an AAV capsid derived from AAV5. In one embodiment, the rAAV vector comprises an AAV capsid derived from AAV6. The vector comprises an AAV capsid derived from AAV7. In one embodiment, the rAAV vector is AAV8. In one embodiment, the rAAV vector comprises an AAV capsid derived from AAV9. In one embodiment, the rAAV vector comprises an AAV capsid derived from AAV10. AAV capsids of particular interest include AAV2, AAV5, AAV8, and AAV9 (Table 5). However, like the ITRs, the capsid need not have the wild-type nucleotide sequence but may be altered by the insertion, deletion, or substitution of nucleotides in the VP1, VP2, or VP3 sequences. However, the capsid must be capable of gene transfer into cells of the eye and / or lacrimal gland. In other words, In some embodiments, the AAV capsid may be a mutant AAV capsid. A "pseudotyped" AAV is made using the capsid (cap) gene of one AAV and the rep gene and ITRs of a different AAV. For example, pseudotyped AAV2s are made with rep inherited from AAV2 and cap inherited from AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, or AAV9, along with 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.

[0157] In one embodiment, the rAAV is replication-deficient, meaning that the rAAV vector cannot independently replicate and package its genome further. For example, the rAAV vector can be used to infect the eye and / or Or if the lacrimal gland is transgenic, the gene is expressed in the transgenic eye and / or lacrimal gland. Although transfected eyes and / or lacrimal glands express AAV rep and cap genes and Lacking accessory genes, rAAV is unable to replicate.

[0158] In certain embodiments, rAAV vectors of the present disclosure encapsulating the expression cassettes described herein can be produced using helper-free production. rAAV is a replication-deficient virus that typically requires the components of a live helper virus (e.g., adenovirus) to package infectious rAAV vectors within host cells. The rAAV helper-free production system allows for the production of infectious rAAV vectors without the use of a live helper virus. In the helper-free system, a host packaging cell line is co-transfected with three plasmids: The first plasmid contains the adenoviral genes required for packaging the rAAV vector. The first plasmid contains the necessary AAV genes (i.e., the E2A, E4, and VA RNA genes). The second plasmid contains the necessary AAV genes (i.e., the REP and CAP genes). The third plasmid contains a polynucleotide sequence encoding an oxidoreductase enzyme (e.g., TRX or PDI) or a functional variant thereof and a promoter flanked by ITRs. The host packaging cell line is For example, it can be an AAV-293 host cell. Suitable host cells can be The rAAV vector may contain additional components necessary for packaging an infectious rAAV vector that are not supplied. In some embodiments, the CAP gene may encode an AAV capsid protein, for example, as described herein. In some embodiments, the promoter may be a promoter sequence described herein. In some embodiments, the promoter sequence is CAG In certain embodiments, the polynucleotide is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least In certain embodiments, the polynucleotide encodes a protein that shares at least 99%, or 100% identity with SEQ ID NO: 1. In certain 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 certain embodiments, the polynucleotide 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.

[0159] AAV serotypes that have been shown to infect the eye and / or lacrimal gland include AAV2, AAV5, AAV 5w8, and AAV9 (Rocha et al., supra). Exemplary AAV capsid proteins include: The amino acid and nucleotide sequences are shown in Table 5.

[0160] In some embodiments, the AAV capsid protein shares at least 95%, 98%, or 100% identity to the AAV2 VP1 protein (SEQ ID NO: 6). The polynucleotide sequence encoding the VP1 protein is at least 95%, 98% identical to SEQ ID NO: 7. In some embodiments, the AAV capsid protein shares at least 95%, 98%, or 100% identity to 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 to SEQ ID NO: 9. In some embodiments, the polynucleotide sequence encoding the AAV2 VP3 protein shares at least 95%, 98%, or 100% identity to SEQ ID NO: 9. In one embodiment, the AAV capsid protein shares at least 95%, 98%, or 100% identity with the AAV5 capsid protein (SEQ ID NO: 10). The polynucleotide sequence encoding shares at least 95%, 98%, or 100% identity with SEQ ID NO: 11. In one embodiment, the AAV capsid protein is the AAV8 capsid protein. In one embodiment, the polynucleotide sequence encoding the AAV8 capsid protein shares at least 95%, 98%, or 100% identity with SEQ ID NO: 13. In one embodiment, the AAV capsid protein shares at least 95%, 98%, or 100% identity with SEQ ID NO: 14. In one embodiment, the polynucleotide sequence encoding the AAV9 capsid protein shares at least 95%, 98%, or 100% identity with SEQ ID NO:15. [Table 5]

[0161] Exemplary rAAV Vectors In one embodiment, the rAAV vector comprises an AAV capsid. The rAAV vectors described herein comprise a polynucleotide sequence encoding TRX. In one embodiment, the rAAV vectors described herein comprise an expression cassette comprising a polynucleotide comprising a nucleotide sequence encoding a PDI. In some embodiments, the rAAV vectors described herein comprise a bicistronic expression cassette comprising 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 capsid and an expression cassette, and the expression The cassette links a polynucleotide to an operably linked promoter, and the polynucleotide comprises a nucleotide sequence encoding TRX. The target contains an AAV capsid and an expression cassette, and the expression cassette can manipulate polynucleotides. The polynucleotide is linked to a promoter connected to a nucleotide sequence encoding PDI. The promoter may be any of those described herein or known in the art. In one embodiment, the promoter is a CAG promoter. In one embodiment, 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. Expression Cassette may comprise, from 5' to 3', a nucleotide sequence including: a 5' ITR (e.g., AAV2 5' ITR), a promoter (e.g., a CMV promoter), a 5' untranslated region (5' UTR), a TRX In one embodiment, the expression cassette comprises, in a 5' to 3' direction, the following: a polynucleotide sequence comprising a nucleotide sequence encoding an enzyme, a polyA sequence, and a 3' ITR (e.g., AAV2 3' ITR). nucleotide sequences including: 5'ITR (e.g., AAV2 5'ITR), enhancer In one embodiment, the 5' UTR comprises a polynucleotide sequence comprising a nucleotide sequence encoding a TRX enzyme, a WPRE sequence, a polyA sequence, and a 3' ITR (e.g., AAV2 3' ITR). The codon is located immediately adjacent to and immediately before the start codon of the nucleotide sequence encoding the TRX enzyme. Contains a sequence.

[0162] In one embodiment, the rAAV vector comprises an AAV capsid and an expression cassette. The kit links a polynucleotide to an operably connected promoter, the polynucleotide comprising a nucleotide sequence encoding a human TRX enzyme as described herein. In embodiments, the rAAV vector comprises an AAV capsid and an expression cassette, the polynucleotide is operably linked to a CAG promoter or a CMV promoter, and the polynucleotide comprises a nucleotide sequence encoding a TRX enzyme as described herein. In one embodiment, the polynucleotide comprises, in a 5' to 3' direction: (i) a signal peptide; and (ii) a nucleotide sequence encoding a TRX enzyme described herein. It includes a nucleotide sequence.

[0163] In one embodiment, the rAAV vector comprises an AAV capsid and an expression cassette. The kit includes a promoter (e.g., a CAG promoter) operably linked to the polynucleotide. The polynucleotide is linked to a promoter (e.g., a CMV promoter) and comprises, in a 5' to 3' direction, (i) a nucleotide sequence encoding a signal peptide, and (ii) a nucleotide sequence encoding a TRX enzyme. and the TRX enzyme has a sequence identical to SEQ ID NO:1, which sequence is 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 In one embodiment, the TRX enzyme comprises or consists of the amino acid sequence of SEQ ID NO: 1. In one embodiment, the rAAV vector comprises an AAV capsid and an expression cassette. the expression cassette links the polynucleotide to an operably connected promoter (e.g., a CAG promoter or a CMV promoter), the polynucleotide comprising, in 5' to 3' direction, (i) a nucleoid sequence encoding a signal peptide, and (ii) a nucleoid sequence encoding a TRX enzyme, the TRX enzyme having a sequence similar to SEQ ID NO:25 by 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 Contains amino acid sequences that share at least 97%, at least 98%, or at least 99% identity In one embodiment, the TRX enzyme has the amino acid sequence of SEQ ID NO:25. In one embodiment, the rAAV vector comprises or consists of an AAV capsid. and an expression cassette, the expression cassette links the polynucleotide to an operably connected promoter (e.g., a CAG promoter), and the polynucleotide comprises, in a 5' to 3' direction, (i) a nucleotide sequence encoding a signal peptide, and (ii) a nucleotide sequence encoding a TRX enzyme. The nucleotide sequence may be at least as long as SEQ ID NO: 2 or SEQ ID NO: 28. At least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least In one embodiment, the nucleoid sequence comprises a nucleoid sequence sharing 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:2. In one embodiment, the nucleoid sequence comprises SEQ ID NO:28.

[0164] In some embodiments, the rAAV vector comprises an AAV capsid and an expression cassette, The polynucleotide is operably linked to a promoter (e.g., a CAG promoter or a CMV promoter). The polynucleotide comprises, in the 5' to 3' direction, (i) a signal peptide; and (ii) a nucleotide sequence encoding a TRX enzyme. The TRX enzyme may be 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 or an amino acid sequence that shares 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with In some embodiments, the TRX enzyme is set forth in SEQ ID NO:25. It is composed of an amino acid sequence.

[0165] In some embodiments, the rAAV vector comprises an AAV2 capsid and an expression cassette, wherein a polynucleotide is operably linked to a promoter. The polynucleotide comprises a nucleotide sequence encoding a human TRX enzyme described herein. In some embodiments, the rAAV The vector contains an AAV2 capsid and an expression cassette, and the polynucleotide is a CAG promoter. The polynucleotide is operably linked to a 5' to 3' promoter. In this direction, (i) a nucleotide sequence encoding a signal peptide, and (ii) a nucleotide sequence as described herein. The disclosed TRX enzymes include nucleotide sequences encoding the enzymes.

[0166] In some embodiments, the rAAV vector comprises an AAV2 capsid and an expression cassette, wherein the polynucleotide is operably linked to a promoter (e.g., a CAG promoter or a CMV promoter). The polynucleotide comprises, in the 5' to 3' direction, (i) a signal peptide; and (ii) a nucleotide sequence encoding a TRX enzyme. The TRX enzyme has at least 85% identity to the amino acid sequence shown in SEQ ID NO: 1, 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%, amino acid sequences that share at least 97%, at least 98%, or at least 99% identity In some embodiments, the TRX enzyme may comprise or consist of the amino acid sequence thereof. The element 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, wherein the polynucleotide is a promoter The polynucleotide is operably linked to a promoter (e.g., a CAG promoter or a CMV promoter). The polynucleotide comprises, in the 5' to 3' direction, (i) a nucleotide sequence encoding a signal peptide; and (ii) a nucleotide sequence encoding a TRX enzyme, the TRX enzyme having at least 85%, at least 86%, at least 87%, 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%, at least 99%, at least 100%, at least 101%, at least 102%, at least at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least or comprises an amino acid sequence that shares at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence. In some embodiments, the TRX enzyme is set forth in SEQ ID NO:25. In some embodiments, the rAAV vector comprises an AAV2 capsid and an expression cassette, wherein the polynucleotide is a promoter (e.g., a CAG promoter). The polynucleotide is operably linked to a promoter (e.g., a CMV promoter or a CMV promoter). , in the 5' to 3' direction, (i) a nucleotide sequence encoding a signal peptide, and (ii ) A nucleotide sequence encoding a TRX enzyme, the nucleotide sequence being 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%, at least 99%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 108%, at least 109%, at least 110%, at least 111%, at least 112%, at least 113%, at least 114%, at least 115%, at least 116%, at least 117%, at least 118%, at least 119%, at least 120%, at at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or In some embodiments, the nucleotide sequence of the present invention may include sequences that share at least 99% identity with the target gene. In some embodiments, the nucleotide sequence comprises SEQ ID NO: 2. comprises SEQ ID NO: 28.

[0167] In some embodiments, the rAAV vector comprises an AAV5 capsid and an expression cassette, the expression cassette comprising a polynucleotide operably linked to a promoter, the polynucleotide comprising, in the 5' to 3' direction, (i) a nucleotide sequence encoding a signal peptide, and (ii) comprises a nucleotide sequence encoding the human TRX enzyme described herein. In one embodiment, the rAAV vector comprises an AAV5 capsid and an expression cassette comprising a polynucleotide operably linked to a promoter. In one embodiment, the promoter is a CAG promoter. The polynucleotide may include, in the 5' to 3' direction: (i) a nucleic acid encoding a signal peptide; and (ii) a nucleotide sequence encoding a TRX enzyme described herein. Includes:

[0168] In some embodiments, the rAAV vector comprises an AAV5 capsid and an expression cassette, the expression cassette comprising a polynucleotide operably linked to a promoter (e.g., a CAG promoter or a CMV promoter). The polynucleotide comprises, from 5' to 3', (i) a nucleotide sequence encoding a signal peptide, and (ii) a nucleotide sequence encoding a TRX enzyme. The TRX enzyme has a sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, or at least 100% similar to the amino acid sequence set forth in SEQ ID NO: 1. The rAAV vector comprises an amino acid sequence that shares 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 the TRX enzyme. In some embodiments, the TRX enzyme comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the rAAV vector comprises an AAV5 capsid and an expression cassette, wherein the expression cassette comprises a polynucleotide operably linked to a promoter (e.g., a CAG promoter or a CMV promoter). The polynucleotide comprises, from 5' to 3', (i) a nucleotide sequence encoding a signal peptide, and (ii) a nucleotide sequence encoding the TRX enzyme. The TRX enzyme comprises the amino acid sequence set forth in SEQ ID NO: 25. vs. 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% In some embodiments, the TRX enzyme comprises an amino acid sequence that shares identity with the TRX enzyme of SEQ ID NO: 25. In some embodiments, the rAAV vector comprises an AAV5 capsid and an expression cassette, wherein the expression cassette comprises a polynucleotide operably linked to a promoter (e.g., a CAG promoter or a CMV promoter). The polynucleotide comprises, in the 5' to 3' direction, (i) a nucleotide sequence encoding a signal peptide, and (ii) ) A nucleotide sequence encoding a TRX enzyme, the nucleotide sequence being 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%, at least 99%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 108%, at least 109%, at least 110%, at least 111%, at least 112%, at least 113%, at least 114%, at least 115%, at least 116%, at least 117%, at least 118%, at least 119%, at least 120%, at at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or In some embodiments, the nucleotide sequence of the present invention may include sequences that share at least 99% identity with the target gene. In some embodiments, the nucleotide sequence comprises SEQ ID NO: 2. comprises SEQ ID NO: 28.

[0169] In some embodiments, the rAAV vector comprises an AAV9 capsid and an expression cassette comprising a polynucleotide operably linked to a promoter, the polynucleotide comprising, in the 5' to 3' direction, (i) a nucleotide sequence encoding a signal peptide, and (ii) a nucleotide sequence encoding a nucleotide sequence encoding a nucleotide sequence described herein. In some embodiments, the rAAV vector comprises a nucleotide sequence encoding a human TRX enzyme. The vector comprises an AAV9 capsid and an expression cassette comprising a polynucleotide operably linked to a promoter. The promoter may be a CAG promoter. A nucleotide sequence includes, in the 5' to 3' direction: (i) a nucleotide sequence encoding a signal peptide; and (ii) a nucleotide sequence encoding a TRX enzyme described herein.

[0170] In some embodiments, the rAAV vector comprises an AAV9 capsid and an expression cassette, wherein the polynucleotide is operably linked to a promoter (e.g., a CAG promoter or a CMV promoter). Specifically, the polynucleotide is operably linked in the 5' to 3' direction to a signal (ii) a nucleotide sequence encoding a peptide, and (iii) a nucleotide sequence encoding a TRX enzyme. The TRX enzyme contains a sequence that is at least 85%, at least 86%, or At least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least If the amino acid sequence shares at least 97%, at least 98%, or at least 99% identity with the In some embodiments, the TRX enzyme comprises the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the TRX enzyme has an amino acid sequence that is at least 85%, at least 86%, or At least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least If the amino acid sequence shares at least 97%, at least 98%, or at least 99% identity with the In some embodiments, the TRX enzyme comprises the amino acid sequence set forth in SEQ ID NO:25. In some embodiments, the nucleotide sequence comprises a nucleotide sequence encoding a TRX enzyme. and has a sequence similar to SEQ ID NO: 2 or SEQ ID NO: 28, at least 85%, at least 86%, or at least 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%, In some embodiments, the nucleotide sequence comprises SEQ ID NO: 2. In some embodiments, the nucleotide sequence comprises SEQ ID NO: 2. Includes SEQ ID NO: 28.

[0171] In some embodiments, the promoter may be any of those 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 the present invention, the expression cassette may comprise a nucleotide sequence having the following components: from 5' to 3', a 5' ITR (e.g., AAV2 5' ITR), a promoter (e.g., CMV promoter), a 5' untranslated region (5' UTR), a polynucleotide comprising a nucleotide sequence encoding PDI, a polyA sequence, and a nucleotide sequence encoding PDI. and a 3' ITR (e.g., AAV2 3' ITR). In some embodiments, the expression cassette has the following structure: The nucleotide sequence may comprise, from 5' to 3', a 5' ITR (e.g., AAV2 5' ITR), an enhancer (e.g., CMV enhancer), a promoter (e.g., CMV promoter), a In some embodiments, the 5'UTR comprises a Kozak sequence located immediately before the start codon of the nucleotide sequence encoding the PDI.

[0172] In some embodiments, the rAAV vector comprises an AAV capsid and an expression cassette, The nucleotide is operably linked to a promoter. The polynucleotide comprises a nucleotide sequence encoding a human PDI as described herein. In some embodiments, the rAAV vector The vector comprises an AAV capsid and an expression cassette, wherein the polynucleotide is operably linked to a CAG promoter or a CMV promoter. The polynucleotide can be any of the vectors described herein. It comprises a nucleotide sequence encoding a PDI.

[0173] In some embodiments, the rAAV vector comprises an AAV capsid and an expression cassette operably linked to a promoter (e.g., a CAG promoter or a CMV promoter). The nucleotides include a nucleotide sequence encoding a PDI, wherein the PDI is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, or at least 90% identical to SEQ ID NO: 26. At least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% similarity In some embodiments, the PDI comprises or consists of the amino acid sequence set forth in SEQ ID NO:26. In some embodiments, the rAAV vector may be comprised of an AAV capsule. An expression cassette operably linked to a promoter (e.g., a CAG promoter) The polynucleotide comprises a nucleotide sequence encoding PDI, The nucleotide sequence is at least 85%, at least 86%, at least 87%, or at least 89% of SEQ ID NO: 30. at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least In some embodiments, the nucleic acid sequence may share 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the nucleic acid sequence. The octide sequence comprises SEQ ID NO:30.

[0174] In some embodiments, the rAAV vector comprises an AAV capsid and an expression cassette operably linked to a promoter (e.g., a CAG promoter or a CMV promoter). The nucleotides include a nucleotide sequence encoding a PDI, wherein the PDI is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 160%, at least 170%, at least 180%, at least 190%, at least 200%, at least 210%, at least 220%, at least 230%, at least 240%, at least 250%, at least 260%, at least 270%, at least 280%, at least 290%, at least 300%, at least 310%, at least 320%, at least 330%, at least 340%, at least 350%, at least 360%, at least 370%, at least 380%, at least 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% identical In some embodiments, the PDI comprises or consists of the amino acid sequence set forth in SEQ ID NO: 26. It may consist of an array.

[0175] In some embodiments, the rAAV vector comprises an AAV2 capsid and an expression cassette, wherein the polynucleotide is operably linked to a promoter. The polynucleotide comprises a nucleotide sequence encoding a human PDI as described herein. In some embodiments, the rAAV vector The polynucleotide comprises an AAV2 capsid and an expression cassette, and the polynucleotide is a CAG promoter or is operably linked to a CMV promoter. The polynucleotide comprises a nucleotide sequence encoding a PDI as described herein.

[0176] In some embodiments, the rAAV vector comprises an AAV2 capsid and a promoter (e.g., CAG The expression cassette is operably linked to a promoter (e.g., a CMV promoter). The oligonucleotide comprises a nucleotide sequence encoding PDI, wherein the PDI has a sequence similar to SEQ ID NO: 26, such as 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 at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% similarity In some embodiments, the 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 operably linked to a promoter (e.g., a CAG promoter or a CMV promoter). The polynucleotide may comprise a nucleotide sequence encoding PDI. the nucleotide sequence of which is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, or at least In some embodiments, the nucleotide sequence comprises SEQ ID NO: 30.

[0177] In some embodiments, the rAAV vector comprises an AAV5 capsid and an expression cassette operably linked to a promoter. The polynucleotide encodes a human PDI as described herein. In some embodiments, the rAAV vector comprises an AAV5 capsid and an expression cassette operably linked to a CAG promoter. The present invention includes nucleotide sequences encoding the PDIs described herein.

[0178] In some embodiments, the rAAV vector comprises an AAV5 capsid and a promoter (e.g., CAG The expression cassette is operably linked to a promoter (e.g., a CMV promoter). The oligonucleotide comprises a nucleotide sequence encoding PDI, wherein the PDI has at least 85%, at least 86%, at least 87%, at least 88%, at least 89% similarity to SEQ ID NO: 26. , 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% In some embodiments, the PDI comprises an amino acid sequence that shares identity with the amino acid sequence of SEQ ID NO: 26. In some embodiments, the rAAV vector comprises an AAV5 capsid and an expression cassette operably linked to a promoter (e.g., a CAG promoter or a CMV promoter). The polynucleotide comprises a nucleotide sequence encoding PDI, and The nucleotide sequence is at least 85%, at least 86%, at least 87%, or at least 89% of SEQ ID NO: 30. at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least In some embodiments, the nucleic acid sequence may share 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the nucleic acid sequence. The octide sequence comprises SEQ ID NO:30.

[0179] In some embodiments, the rAAV vector comprises an AAV9 capsid and an expression cassette operably linked to a promoter. The polynucleotide encodes a human PDI as described herein. In some embodiments, the rAAV vector comprises an AAV9 capsid and an expression cassette operably linked to a CAG promoter. The present invention includes nucleotide sequences encoding the PDIs described herein.

[0180] In some embodiments, the rAAV vector comprises an AAV9 capsid and a promoter (e.g., CAG The expression cassette is operably linked to a promoter (e.g., a CMV promoter). The nucleotides include a nucleotide sequence encoding a PDI, wherein the PDI is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, or at least 90% identical to SEQ ID NO: 26. At least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% similarity In some embodiments, the PDI comprises an amino acid sequence that shares identity with the amino acid sequence of SEQ ID NO: 26. In some embodiments, the rAAV vector comprises an AAV9 capsid and an expression cassette operably linked to a promoter (e.g., a CAG promoter or a CMV promoter). The polynucleotide comprises a nucleotide sequence encoding PDI, and the nucleotide sequence The sequence of the peptide is at least 85%, at least 86%, at least 87%, or at least 89% similar to SEQ ID NO: 30. at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least In some embodiments, the nucleoside may share at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity. The sequence of the peptide comprises SEQ ID NO: 30.

[0181] In some embodiments, the rAAV vector comprises an AAV capsid and an expression cassette operably linked to a promoter (e.g., a CAG promoter or a CMV promoter). The nucleotides are arranged in the 5' to 3' direction as follows: (i) a nucleotide sequence encoding a signal peptide; and (ii) a nucleotide sequence encoding a PDI, wherein the PDI is 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%, at least 99%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 108%, at least 109%, at least 1109%, at least 1111%, at least 112%, at least 113%, at least 114%, at least 115%, at least 116%, at least 117%, at least 118%, at least 119%, at least 12 at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least or at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity In some embodiments, the PDI comprises or consists of the amino acid sequence of SEQ ID NO: 26. In some embodiments, the rAAV vector comprises an AAV capsid and a promoter. The polynucleotide comprises an expression cassette operably linked to a promoter (e.g., a CAG promoter). The nucleotides are arranged in the 5' to 3' direction as follows: (i) a nucleotide sequence encoding a signal peptide; and (ii) a nucleotide sequence encoding PDI, the nucleotide sequence being the sequence Number: 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 at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or less In some embodiments, the nucleotide sequence may share 99% identity with the sequence Number: 30 inclusive.

[0182] In some embodiments, the rAAV vector comprises an AAV capsid and an expression cassette operably linked to a promoter (e.g., a CAG promoter or a CMV promoter). The nucleotides are arranged in the 5' to 3' direction as follows: (i) a nucleotide sequence encoding a signal peptide; and (ii) a nucleotide sequence encoding a PDI, which PDI is 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%, at least 99%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 108%, at least 109%, at least 1109%, at least 1111%, at least 112%, at least 113%, at least 114%, at least 115%, at least 116%, at least 117%, at least 118%, at least 119%, at least 120 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% identical In some embodiments, the PDI comprises or consists of the amino acid sequence of SEQ ID NO:26. This may have been done.

[0183] In some embodiments, the rAAV vector comprises an AAV2 capsid and a promoter (e.g., CAG The expression cassette is operably linked to a promoter (e.g., a CMV promoter). The oligonucleotide comprises, in a 5' to 3' direction, (i) a nucleotide sequence encoding a signal peptide; and (ii) a nucleotide sequence encoding a PDI, wherein the PDI has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, or At least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% similarity In some embodiments, the PDI comprises or consists of the amino acid sequence of SEQ ID NO: 26. In some embodiments, the rAAV vector comprises an AAV2 capsid and an expression cassette operably linked to a promoter (e.g., a CAG promoter or a CMV promoter). The polynucleotide comprises a nucleotide sequence encoding PDI, The nucleotide sequence is 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%, at least 99%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 108%, at least 109%, at least 110%, at least 111%, at least 112%, at least 113%, at least 114%, at least 115%, at least 116%, at least 117%, at least 118%, at least 119%, at least 119%, at least 120%, at least 121 In some embodiments, the nucleotide sequence comprises SEQ ID NO: 30.

[0184] In some embodiments, the rAAV vector comprises an AAV5 capsid and a promoter (e.g., CAG The expression cassette is operably linked to a promoter (e.g., a CMV promoter). The oligonucleotide comprises, in the 5' to 3' direction, (i) a nucleotide sequence encoding a signal peptide and (ii) a nucleotide sequence encoding a PDI, which PDI is 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%, at least 99%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 108%, at least 109%, at least 1109%, at least 1111%, at least 112%, at least 113%, at least 114%, at least 115%, at least 116%, at least 117%, at least 118%, at least 119%, at least 120 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% identical In some embodiments, the PDI may comprise an amino acid sequence that shares a common amino acid sequence. In some embodiments, the rAAV vector comprises an AAV5 capsid and an expression cassette operably linked to a promoter (e.g., a CAG promoter or a CMV promoter). The polynucleotide may comprise a nucleotide sequence encoding PDI. the nucleotide sequence of which is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, or at least In some embodiments, the nucleotide sequence comprises SEQ ID NO: 30.

[0185] In some embodiments, the rAAV vector comprises an AAV9 capsid and a promoter (e.g., CAG The expression cassette is operably linked to a promoter (e.g., a CMV promoter). The oligonucleotide comprises, in a 5' to 3' direction, (i) a nucleotide sequence encoding a signal peptide; and (ii) a nucleotide sequence encoding a PDI, wherein the PDI has at least 85%, at least 86%, at least 87%, at least 88%, at least 89% similar to SEQ ID NO: 26; At least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% similarity In some embodiments, the PDI may comprise an amino acid sequence that shares identity with the AAV9 capsid. In some embodiments, the PDI may comprise the amino acid sequence of SEQ ID NO: 26. In some embodiments, the rAAV vector comprises an AAV9 capsid. and an expression cassette operably linked to a promoter (e.g., a CAG promoter or a CMV promoter). The polynucleotide comprises a nucleotide sequence encoding PDI and the nucleotide sequence is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least may also share 97%, at least 98%, or at least 99% identity. I got it.

[0186] How to use The methods and compositions described herein can be used to treat ocular conditions and alleviate their symptoms. The terms "treatment," "treating," and the like refer to the intended pharmacological and / or "Treatment" refers to the treatment of a disease in a mammal, including, but not limited to: (a) inhibiting the progression of the disease; (b) alleviating, reducing, or suppressing one or more symptoms of the disease; (c) alleviating, reducing, or suppressing one or more signs of the disease; or (d) causing regression of the disease. Therapeutic agents can be administered before, during, or after the onset of disease or injury. Of particular interest are treatments that stabilize or reduce the patient's clinical symptoms. Such treatments are preferably administered before complete loss of function of the affected tissue. Targeted treatments are preferably administered during the symptomatic phase of the disease, and in some cases may be administered after the symptomatic phase.

[0187] As used herein, the terms "administration," "administering," "administration method," and the like refer to providing a substance (e.g., an rAAV vector) to a subject in a pharmacologically useful manner (e.g., to treat a disease, disorder, or condition in the subject).

[0188] In some embodiments, methods are provided for treating an ocular condition in a subject by administering to the subject an rAAV vector or pharmaceutical composition provided herein. Herein, the terms "subject" and "patient" are used interchangeably to refer to mammals. This includes humans and non-human primates, including monkeys and humans. It also includes mammalian sport animals (e.g., horses), mammalian farm animals (e.g., sheep, goats, etc.), mammalian pets (e.g., dogs, cats, etc.), and rodents (e.g., mice, rats, etc.). A subject may, for example, have any of the conditions or ocular disorders described herein.

[0189] In some embodiments, the subject is an adult human, e.g., an adult aged 40 or older, or an adult aged 45 to 55. In some embodiments, the subject is an adult aged 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90 or older.

[0190] In some embodiments, the ocular condition treated according to the method is a disorder associated with increased oxidative stress. In some embodiments, the condition is associated with loss of expression or function of one or more oxidoreductase enzymes. In some embodiments, the condition is associated with loss of expression and / or function of protein disulfide isomerase (PDI). In embodiments, the condition is associated with loss of expression and / or function of TRX. Without wishing to be limited by theory, it is believed that expression of TRX, PDI, and / or other oxidoreductase enzymes is It is hypothesized that an increase in ATP can reduce oxidative stress in tissues and delay the onset of conditions associated with or caused by aging. In some embodiments, the condition is associated with loss of myopia, i.e., the gradual loss of the eye's ability to focus on nearby objects. In some embodiments, the condition is cataract formation. In some embodiments, the condition is loss of accommodation. In some embodiments, the condition is presbyopia. In some embodiments, the condition is meibomian gland dysfunction (MGD). In some embodiments, the condition is elevated intraocular pressure. In some embodiments, In some embodiments, the condition is glaucoma. In some embodiments, neuroprotection is provided by the methods described herein.

[0191] In some embodiments, the described methods of treatment further comprise administering to the subject 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 agent. In some embodiments, the additional therapeutic agent is another AAV-based gene therapy construct. do.

[0192] Tear-producing drugs 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 one described herein.

[0193] nAChRs are pentameric receptors with high affinity and selectivity for both nicotine and acetylcholine. nAChRs are a class of ligand-gated ion channels that contain a combination of alpha and beta subunits. Examples of nAChR subtypes include, but are not limited to, alpha3beta4, alpha4beta2, alpha3alpha5beta4, and alpha4alpha6beta2.

[0194] nAChR agonists may be characterized as full or partial agonists depending on their ability to activate a particular receptor relative to the response to acetylcholine (ACh). In general, an nAChR agonist is considered a full agonist if it binds to a particular receptor and elicits a response that is equal to or greater than that of ACh. nAChR agonists ACh is a partial agonist if it binds to the receptor and evokes a lower response than ACh. It is said that.

[0195] The response of nAChR agonists may be used to determine receptor activation. For example, cell-based assays that express specific nAChR receptor subtypes and generate a current response when bound and activated by an nAChR agonist can be used to characterize a compound's agonist profile and degree of receptor activation.

[0196] In some embodiments, the nAChR agonist, or a pharmaceutically acceptable salt thereof, is at least one selected from alpha3beta4, alpha3alpha5beta4, alpha4beta2, and alpha4alpha6beta2. As used herein, "selectively binds" or "selective for" means that a compound has a higher affinity for a particular nAChR subtype, or a lower half-maximal effective concentration, relative to at least one reference nAChR subtype. Selectivity means that there is at least a 5-fold difference in affinity, at least In some embodiments, the affinity of a nAChR agonist or a pharmaceutical composition thereof may be increased by at least 10-fold, at least 20-fold, or at least 50-fold. In some embodiments, the nAChR agonist, or a pharmaceutically acceptable salt thereof, selectively binds to the nAChR subtype alpha3beta4. In some embodiments, the nAChR agonist, or a pharmaceutically acceptable salt thereof, selectively binds to the nAChR subtype alpha3alpha5beta4. In some embodiments, the nAChR agonist, or a pharmaceutically acceptable salt thereof, selectively binds to the nAChR subtype alpha3alpha5beta4. In some embodiments, the nAChR agonist, or a pharmaceutically acceptable salt thereof, selectively binds to the nAChR subtype alpha4beta2. In some embodiments, the nAChR agonist, or a pharmaceutically acceptable salt thereof, selectively binds to the nAChR subtype alpha4alpha6beta2. In some embodiments, the nAChR agonist, or a pharmaceutically acceptable salt thereof, selectively binds to the nAChR subtype alpha7. In some embodiments, the nAChR agonist, or a pharmaceutically acceptable salt thereof, selectively binds to the nAChR subtype alpha4alpha6beta2. , nAChR agonists, or pharmaceutically acceptable salts thereof, do not selectively bind to nAChR subtype alpha7.

[0197] nAChR agonists contemplated in this disclosure include varenicline, its pharmaceutically acceptable salts, and Compound 1 or a pharmaceutically acceptable salt thereof. However, varenicline is not a nAChR agonist.

[0198] Varenicline is characterized as a full agonist of nAChR subtype alpha7 and a partial agonist of subtypes alpha3beta4, alpha4beta2, alpha6beta2, alpha3alpha5beta4, and alpha4alpha6beta2. In some embodiments, the nAChR agonist is varenicline. or a pharmaceutically acceptable salt thereof. Pharmaceutically acceptable salts include varenicline tartrate, etc. Additional information related to varenicline can be found in, for example, 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. Patent Application Publication No. 2022 / 0233528.

[0199] Compound 1 described therein has the following structure: [ka]

[0200] An alternative structural representation of compound 1 is shown below: [ka]

[0201] Compound 1 may also be referred to by its chemical name. For example, Compound 1 is (R)-5-((E)-2-pyrrolidone). It is also called 5-(({E)-2-[(3R)-pyrrolidin-3-yl]vinyl}pyrimidine, or similar names, or sympanicline, 5-(({E)-2-[(3R)-pyrrolidin-3-yl]vinyl}pyrimidine, and (R,E)-5-((2- Also known as pyrrolidin-3-yl)vinyl)pyrimidine.

[0202] Compound 1 is a full agonist of nAChR subtypes alpha4beta2, alpha3beta4, alpha3alpha5beta4, and alpha4alpha6beta2. and alpha3beta4 full agonist.

[0203] Compound 1 is a partial agonist of the subtype alpha3beta2.

[0204] Compound 1 is a weak partial agonist of the alpha7 subtype. For example, 300 micromolar concentrations of Compound 1 citrate evoked only 25% of the maximal ACh-evoked current.

[0205] In some embodiments, the nAChR agonist is Compound 1 or a pharmaceutically acceptable salt thereof. Pharmaceutically acceptable salts of Compound 1 include galactosates (e.g., hemigalactosides). hydrate) and citrate salts (e.g., monocitrate). The information may be found in, for example, U.S. Patent 7,098,331, U.S. Patent 7,714,001, U.S. Patent 8,063,068, U.S. Patent 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.

[0206] In some embodiments, the nAChR agonist is (R)-5-((E)-2-pyrrolidin-3-ylvinyl)pyrimidine or a pharmaceutically acceptable salt thereof. is (R)-5-((E)-2-pyrrolidin-3-ylvinyl)pyrimidine hemigalactoate dihydrate In some embodiments, the nAChR agonist is (R)-5-((E)-2-pyrrolidin-3-ylvinyl)pyrimidine monocitrate.

[0207] In some embodiments, the nAChR agonist is used to treat tear production in subjects with impaired tear production. Increase production.

[0208] Treatment method In some embodiments, the present disclosure provides a method of treating an ocular disease or disorder in a subject, comprising administering to the subject an rAAV comprising nucleotides encoding human TRX. of human TRX in the subject compared to the contralateral eye of an untreated subject or a treated subject Expression increases.

[0209] In some embodiments, the present disclosure provides a rAAV vector for use in a method for treating an ocular condition in a subject, the method comprising administering a recombinant adeno-associated virus (rAAV) vector operably linked to an AAV capsid and promoter. The vector contains an expression cassette containing a polynucleotide encoding human TRX. The rAAV vector is administered to at least one eye, at least one lacrimal gland, or at least one meibomian gland. In some embodiments, the rAAV vector is administered to the lacrimal gland of a subject. In some embodiments, the rAAV vector comprises an expression cassette encoding human TRX.

[0210] In some embodiments, the present disclosure provides rAAV vectors that are used or can be used to treat a subject with an ocular disease, disorder, or condition. In some embodiments, the rAAV vectors that are used or can be used include an AAV capsid and a human avian ... The vector contains an expression cassette comprising a polynucleotide encoding a TRX.

[0211] In some embodiments, the present disclosure provides an rAAV vector for use in a method of treating an ocular condition in a subject, the method comprising administering a recombinant adeno-associated virus (rAAV) vector described herein to at least one eye of the subject or at least one lacrimal gland of the subject.

[0212] In some embodiments, the disclosure provides an rAAV vector for use in a method of treating an ocular condition in a subject, the method comprising administering a recombinant adeno-associated virus (rAAV) vector having an expression cassette comprising the nucleic acid sequence of SEQ ID NO: 16 to at least one eye of the subject or at least one lacrimal gland of the subject.

[0213] In some embodiments, 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 contralateral eye of an untreated or treated subject. In some embodiments, expression of human TRX is increased by at least 1.5-fold, 2-fold, 3-fold, 4-fold, or 5-fold, 6-fold, 7-fold, 8-fold, or 9-fold compared to the contralateral eye of an untreated or treated subject.

[0214] In some embodiments, the 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 week, 2 weeks, 3 weeks, 4 weeks, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 32 days, 33 days, 34 days, 35 days, 36 days, 37 days, 38 days, 39 days, 40 days, 41 days, 42 days, 43 days, 44 days, 45 days, 46 days, 47 days, 48 ​​days, 49 days, 50 days, 51 days, 52 days, 53 days, 54 days, 55 days, 56 days, 57 days, 58 days, 59 days, 60 days, 61 days, 62 days, 63 days, 64 days, 65 days, 66 days, 67 days, 68 days, 69 days, 70 days, 71 days, 72 days, 73 days, 74 days, 75 days, 76 days, 77 days, 78 days, 79 days, 80 days, 81 days, 82 days, 83 days, 84 days, 85 days, 86 days, 87 days, 88 days, 89 days, 90 days, 91 days, 92 days, 9 Increase by months, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months or 1 year.

[0215] As used herein, "approximately" or "about" applies to one or more values ​​of interest. and refers to a value similar to the stated reference value. In certain embodiments, "about" refers to a value that is greater than or less than the stated reference value, such as 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, or 25%. , 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less. (unless this number exceeds 100% of the possible values).

[0216] In some embodiments, the subject is administered an rAAV comprising nucleotides encoding a human TRX enzyme. and increases expression of the human TRX enzyme in the subject compared to the contralateral eye of an untreated subject or a treated subject.

[0217] In some embodiments, the expression of the human TRX enzyme is measured in untreated and treated subjects. In some embodiments, the expression of the human TRX enzyme is increased by at least 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, or 9-fold compared to the contralateral eye of an untreated or treated subject.

[0218] In some embodiments, the expression of the human TRX enzyme is measured in untreated and treated subjects. Compared to the contralateral eye, approximately 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 increase for one year.

[0219] In some embodiments, the present disclosure provides a method for administering to a subject an rAAV comprising nucleotides encoding a human TRX enzyme, and detecting a human TRX enzyme in the subject compared to the contralateral eye of an untreated subject or a treated subject. The present invention provides a method for increasing expression of the human TRX enzyme.

[0220] In some embodiments, the present disclosure provides an rAAV comprising nucleotides encoding a human PDI in a subject. and increasing expression of human PDI in the subject relative to an untreated subject or the contralateral eye of the treated subject.

[0221] In some embodiments, the present disclosure provides a rAAV vector for use in a method for treating an ocular condition in a subject, the method comprising administering a recombinant adeno-associated virus (rAAV) vector operably linked to an AAV capsid and promoter. The vector contains an expression cassette containing a polynucleotide encoding human PDI. In some embodiments, the rAAV vector is administered to at least one eye or at least one lacrimal gland of a subject. In some embodiments, the rAAV vector encodes human PDI. It contains an expression cassette that encodes the

[0222] In some embodiments, the present disclosure provides rAAV vectors that are used or usable to treat a subject having an ocular disease, disorder, or condition. In some embodiments, the rAAV vectors that are used or usable comprise an AAV capsid and a human avian fusion protein linked to a promoter. The vector includes an expression cassette containing a polynucleotide encoding a target PDI.

[0223] In some embodiments, the present disclosure provides a rAAV vector for use in a method of treating an ocular condition in a subject, the method comprising administering to a subject a recombinant adeno-associated virus (rAAV) vector described herein. The method includes administering a medicament to at least one eye of a subject or at least one lacrimal gland of a subject.

[0224] In some embodiments, the disclosure provides an rAAV vector for use in a method of treating an ocular condition in a subject, the method comprising administering a recombinant adeno-associated virus (rAAV) vector having an expression cassette comprising the nucleic acid sequence of SEQ ID NO: 16 to at least one eye of the subject or at least one lacrimal gland of the subject.

[0225] In some embodiments, the expression of human PDI is measured in a control group of untreated and treated subjects. 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% compared to the side eye In some embodiments, the expression of human PDI is increased by 75%, 80%, 85%, 90%, 95%, or 99%. at least 1.5, 2, or 3 times greater than the contralateral eye of untreated or treated subjects It may increase by a factor of two, four, five, six, seven, eight, or nine.

[0226] In some embodiments, the expression of human PDI is measured in a control group of untreated and treated subjects. Increased by approximately 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 the lateral eye.

[0227] In some embodiments, an rAAV comprising nucleotides encoding human PDI is administered to a subject; The expression of human PDI in the treated subjects compared to the contralateral eyes of untreated subjects and treated subjects Increase.

[0228] In some embodiments, the expression of human PDI is measured in a control group of untreated and treated subjects. 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% compared to the side eye In some embodiments, the expression of human PDI is increased by 75%, 80%, 85%, 90%, 95%, or 99%. at least 1.5, 2, or 3 times greater than the contralateral eye of untreated or treated subjects It may increase by a factor of two, four, five, six, seven, eight, or nine.

[0229] In some embodiments, the expression of human PDI is measured in a control group of untreated and treated subjects. Increased by approximately 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 the lateral eye.

[0230] In some embodiments, the subject is administered an rAAV comprising nucleotides encoding human PDI; The expression of human PDI in the treated subjects compared to the contralateral eyes of untreated subjects and treated subjects Provides a way to increase

[0231] In some embodiments, the method includes administering rAAV to a subject having a condition associated with cataract formation, where, if the condition is characterized by one or more symptoms, the administration occurs before the onset of the 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 includes administering rAAV to a subject having one or more symptoms characterized by cataract formation (e.g., blurred vision, double vision, faded color vision, night vision problems), where the administration prevents or reduces the severity of the symptoms. In some embodiments, the administration occurs after the onset of one or more symptoms and reduces the severity of the symptoms.

[0232] In some embodiments, the methods include a schedule of administering rAAV to a subject having a condition associated with cataract formation, where the condition is characterized by one or more symptoms (e.g., blurred vision, double vision, faded color vision, night vision problems), and the administration schedule includes an initial administration followed by at least one administration. The administration schedule may include one booster dose, where the initial rAAV administration is administered before or after the onset of symptoms, and at least one booster dose is administered after the initial administration, thereby preventing or reducing the severity of the symptoms. In some embodiments, the administration schedule selects the administration frequency and / or dosage based on the pharmacokinetic parameters of the rAAV. In some embodiments, the clinician may administer the desired effect. Or, the rAAV is administered at a specific frequency and / or dosage for maintenance. In some embodiments, the one or more desired effects are prevention of one or more symptoms associated with the condition in the subject. In some embodiments, the one or more desired effects are reduction in severity of one or more symptoms associated with the condition in the subject. In some embodiments, the severity of the one or more symptoms is assessed in a method for assessing a condition associated with cataract formation. In some embodiments, the one or more desired effects are achieved shortly after the initial rAAV administration. In some embodiments, the one or more desired effects occur at any time after the initial rAAV administration. In some embodiments, the one or more desired effects are achieved after at least one additional rAAV administration. In some embodiments, the one or more desired effects are achieved at any time in the dosing schedule. In some embodiments, the one or more desired effects are achieved after the initial rAAV administration, and the subject receives at least one additional rAAV administration to prevent reversal of the one or more desired effects. In some embodiments, the method includes administering an initial rAAV administration to a subject before, shortly after, or during the onset of one or more symptoms, and administering the initial rAAV administration to a subject at 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, or about 2 weeks after the initial administration. A booster dose is administered after approximately 1.5 or 2 years.

[0233] Some embodiments include methods of reversing cataract formation, including administering rAAV to a subject with cataract formation, whereby the administration reverses cataract formation.

[0234] In some embodiments, the method includes administering rAAV to a subject having a condition associated with presbyopia, where the condition is characterized by one or more symptoms, and the administration occurs before the onset of the 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 includes administering rAAV to a subject having one or more symptoms characterized by presbyopia (e.g., blurred vision, difficulty reading, headaches), where the administration prevents or reduces the severity of the symptoms. In some embodiments, the administration occurs after the onset of one or more symptoms and reduces the severity of the symptoms.

[0235] In some embodiments, the methods include an administration schedule of rAAV to a subject having a presbyopia-related condition, where the condition is characterized by one or more symptoms (e.g., blurred vision, difficulty reading, headaches), the administration schedule includes an initial administration and at least one follow-up administration, where the initial administration of rAAV is administered before or after the onset of the symptoms and at least one follow-up administration is administered after the initial administration, thereby preventing or reducing the severity of the symptoms. In some embodiments, the administration schedule selects the administration frequency and / or dosage based on pharmacokinetic parameters of the rAAV. In some embodiments, the clinician administers the rAAV at a specific frequency and / or dosage to achieve or maintain a desired effect. In some embodiments, the one or more desired effects are prevention of one or more symptoms associated with the subject's condition. In some embodiments, the one or more desired effects are reduction in severity of one or more symptoms associated with the subject's condition. In some embodiments, the severity of the one or more symptoms is assessed in a method for assessing a pathology associated with presbyopia. In some embodiments, the one or more desired effects are achieved shortly after the first rAAV administration. In some embodiments, the one or more desired effects occur at any time after the first rAAV administration. In some embodiments, the one or more desired effects are achieved after at least one additional rAAV administration. In some embodiments, the one or more desired effects are achieved at any time in the dosing schedule. In some embodiments, the one or more desired effects are achieved after the first rAAV administration, and the subject receives at least one additional rAAV administration to reduce the severity of one or more presbyopia-related symptoms. In some embodiments, the method includes administering an initial rAAV administration to a subject before, shortly after, or during the onset of one or more symptoms, and administering the initial rAAV administration to a subject at 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, or about 9 months after the initial administration. A booster dose is administered after approximately 1 month, approximately 9 months, approximately 10 months, approximately 11 months, approximately 1 year, approximately 1.5 years, or approximately 2 years.

[0236] In some embodiments, the method includes administering rAAV to a subject having a condition associated with loss of accommodation, and when the condition is characterized by one or more symptoms, the administration occurs before the onset of the 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 includes administering rAAV to a subject having one or more symptoms characterized by loss of accommodation (e.g., blurred vision, difficulty reading, headaches), and the administration prevents or reduces the severity of the symptoms. In some embodiments, the administration occurs after the onset of one or more symptoms and reduces the severity of the symptoms.

[0237] In some embodiments, the methods include administering an rAAV to a subject having a condition associated with loss of accommodation, where the condition is characterized by one or more symptoms (e.g., blurred vision, difficulty reading, headaches), the administration schedule includes an initial administration and at least one follow-up administration, where the initial administration of rAAV is administered before or after the onset of the symptoms, and the at least one follow-up administration is administered after the initial administration, thereby preventing or reducing the severity of the symptoms. In some embodiments, the administration schedule selects the administration frequency and / or dosage based on pharmacokinetic parameters of the rAAV. In some embodiments, the rAAV is administered at a specific frequency and / or dosage to achieve or maintain a desired effect as determined by the clinician. The desired effect is to prevent one or more symptoms associated with the subject's condition. In some embodiments, the one or more desired effects are to reduce the severity of one or more symptoms associated with the subject's condition. In some embodiments, the severity of the one or more symptoms is assessed in a method for assessing a condition associated with loss of regulation. In some embodiments, the one or more desired effects are achieved shortly after a first rAAV administration. In some embodiments, the one or more desired effects occur at any time after a first rAAV administration. In some embodiments, the one or more desired effects are achieved after at least one additional rAAV administration. In some embodiments, the one or more desired effects are achieved at any time in a dosing schedule. In some embodiments, the one or more desired effects are achieved after a first rAAV administration, and the subject receives at least one additional rAAV administration to prevent reversal of the one or more desired effects. In some embodiments, the method comprises administering a first rAAV administration to the subject before, shortly after, or during the onset of one or more symptoms, and administering the first rAAV administration to the subject about 1 day after the first administration. A booster dose is administered 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.

[0238] In some embodiments, the method includes administering rAAV to a subject having a condition associated with elevated intraocular pressure, where the condition is characterized by one or more symptoms, and the administration occurs before the onset of the 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 includes administering rAAV to a subject having one or more symptoms characterized by elevated intraocular pressure (e.g., eye pain, nausea, vomiting), where the administration prevents or reduces the severity of the symptoms. In some embodiments, the administration occurs after the onset of one or more symptoms and reduces the severity of the symptoms.

[0239] In some embodiments, the method includes a dosing schedule of rAAV to a subject having a condition associated with elevated intraocular pressure, where the condition is characterized by one or more symptoms (e.g., eye pain, nausea, vomiting), and the dosing schedule includes an initial dose and at least one follow-up dose. The initial dose of rAAV is administered before or after the onset of the symptoms, and at least one follow-up dose is administered after the initial dose to prevent or reduce the severity of the symptoms. In some embodiments, the dosing schedule select the administration frequency and / or dose based on the pharmacokinetic parameters of rAAV. In some embodiments, the clinician administers the rAAV at a specific frequency and / or dosage to achieve or maintain a desired effect. The effect may be to prevent one or more symptoms associated with the subject's condition. In some embodiments, the one or more desired effects may include reducing the severity of one or more symptoms associated with the subject's condition. In some embodiments, the severity of the one or more symptoms is assessed using a method for assessing a condition associated with elevated intraocular pressure. In some embodiments, the one or more desired effects are achieved shortly after a first rAAV administration. In some embodiments, the one or more desired effects occur at any time after a first rAAV administration. In some embodiments, the one or more desired effects are achieved after at least one additional rAAV administration. In some embodiments, the one or more desired effects are achieved at any time in a dosing schedule. In some embodiments, the one or more desired effects are achieved after a first rAAV administration, and the subject receives at least one additional rAAV administration to prevent reversal of the one or more desired effects. In some embodiments, the method includes administering a first rAAV administration to the subject before, shortly after, or during the onset of one or more symptoms, and administering the first rAAV administration at 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, or about 7 months after the first administration. A booster dose is administered at 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.

[0240] Some embodiments involve administering rAAV to a subject having a condition associated with meibomian gland dysfunction (MGD). When the condition is characterized by one or more symptoms, the administration is effective to treat the onset of the 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 administration reduces the severity of one or more symptoms characterized by meibomian gland dysfunction (MGD) (e.g., dry eyes, burning sensation in the eyes). In some embodiments, the administration of rAAV to a subject suffering from one or more of the following symptoms (soreness, itching, redness, discharge, blurred vision) prevents or reduces the severity of the symptoms. In some embodiments, the administration occurs after the onset of one or more symptoms and reduces the severity of the symptoms.

[0241] In some embodiments, the method includes a schedule of administering rAAV to a subject having a condition associated with meibomian gland dysfunction (MGD), where the condition is characterized by one or more symptoms (e.g., e.g., dryness, burning, itching, redness, discharge, blurred vision) of the eye, the dosing schedule includes an initial dose and at least one follow-up dose, where the initial rAAV dose is administered before or after the onset of symptoms, and at least one follow-up dose is administered after the initial dose to prevent or reduce the severity of the symptoms. In some embodiments, the dosing schedule selects the dosing frequency and / or dosage based on pharmacokinetic parameters of the rAAV. In some embodiments, the clinician selects the desired dose. rAAV is administered at a specific frequency and / or dose to achieve or maintain the desired effect. In some embodiments, the one or more desired effects are prevention of one or more symptoms associated with the subject's condition. In some embodiments, the one or more desired effects are reduction in the severity of one or more symptoms associated with the subject's condition. In some embodiments, the severity of one or more symptoms is reduced using the methods described herein or known for assessing conditions associated with meibomian gland dysfunction (MGD). In some embodiments, the one or more desired effects are achieved shortly after the initial rAAV administration. In some embodiments, the one or more desired effects occur at any time after the initial rAAV administration. In some embodiments, the one or more desired effects are achieved after at least one booster rAAV administration. In some embodiments, the one or more desired effects are achieved at any time during the administration schedule. In some embodiments, the one or more desired effects are achieved after the initial rAAV administration, and the subject receives at least one booster rAAV administration to prevent reversal of the one or more desired effects. In some embodiments, the method comprises administering a first rAAV administration to the subject before, shortly after, or during the onset of one or more symptoms, and administering the first rAAV administration to the subject at about 1 week, about 2 weeks, about 3 weeks, about 1 month, about 2 months, or about 3 months after the first administration. 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 A booster dose is administered after approximately 1 month, 1 year, 1.5 years, or 2 years.

[0242] In some embodiments, the method comprises administering rAAV to a subject having a condition associated with oxidative stress, where the condition is characterized by one or more symptoms, the administration occurs before the onset of the 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 rAAV to a subject having oxidative stress, where the condition has one or more symptoms characterized by oxidative stress (e.g., retinal degeneration, cataracts, macular degeneration due to oxidative stress), the administration prevents or reduces the severity of the symptoms. In some embodiments, the administration occurs after the onset of one or more symptoms and reduces the severity of the symptoms.

[0243] In some embodiments, the method includes a dosing schedule of rAAV to a subject having a condition associated with oxidative stress, where the condition is characterized by one or more symptoms (e.g., retinal degeneration, cataract, or macular degeneration due to oxidative stress), and the dosing schedule includes an initial dose and at least one follow-up dose. The initial rAAV dose is administered before or after the onset of symptoms, and at least one follow-up dose is administered after the initial dose to prevent or reduce the severity of the symptoms. In some embodiments, the dosing schedule selects the dosing frequency and / or dosage based on pharmacokinetic parameters of the rAAV. In some embodiments, the dosing schedule is administered to a subject to achieve a desired effect or to provide a therapeutically effective treatment. The rAAV is administered at a specific frequency and / or dosage for maintenance. In some embodiments, the one or more desired effects may include preventing one or more symptoms associated with a condition in a subject. In some embodiments, the one or more desired effects may include reducing the severity of oxidative stress associated with a condition in a subject. In some embodiments, the severity of the one or more symptoms is assessed using a method described herein or a method known for assessing a pathology associated with oxidative stress. In some embodiments, the one or more desired effects are achieved shortly after a first rAAV administration. In some embodiments, the one or more desired effects occur at any time after a first rAAV administration. In some embodiments, the one or more desired effects are achieved after at least one booster rAAV administration. In some embodiments, the one or more desired effects are achieved at any time in a dosing schedule. In some embodiments, the one or more desired effects are achieved after a first rAAV administration, and the subject receives at least one booster rAAV administration to prevent reversal of the one or more desired effects. In some embodiments, the method includes administering a first rAAV administration to a subject before, shortly after, or during the onset of one or more symptoms, and administering the first rAAV administration to the subject about one week after the first administration. Approximately 2 weeks, approximately 3 weeks, approximately 1 month, approximately 2 months, approximately 3 months, approximately 4 months, approximately 5 months, approximately 6 months, approximately 7 months , approximately 8 months, approximately 9 months, approximately 10 months, approximately 11 months, approximately 1 year, approximately 1.5 years, or approximately 2 years later Do the following.

[0244] In some embodiments, a method of treating cataract formation in a subject in need thereof is provided, the method comprising administering to the subject a therapeutically effective amount of one or more of the rAAVs described herein.

[0245] In some embodiments, a method of treating presbyopia in a subject in need thereof is provided, the method comprising administering to the subject a therapeutically effective amount of one or more of the rAAVs described herein.

[0246] In some embodiments, a method of treating oxidative stress in a subject in need thereof is provided, the method comprising therapeutically administering to the subject one or more of the rAAVs described herein.

[0247] In some embodiments, a method of treating loss of regulation in a subject in need thereof is provided, the method comprising administering to the subject a therapeutic effect of one or more of the rAAVs described herein.

[0248] In some embodiments, a method of treating elevated intraocular pressure in a subject in need thereof is provided, the method comprising: This includes administering to a subject one or more therapeutically effective doses of the rAAVs described herein.

[0249] In some embodiments, a method of treating meibomian gland dysfunction in a subject in need thereof is provided, the method comprising administering to the subject a therapeutically effective amount of one or more of the rAAVs described herein.

[0250] In some embodiments, there is provided an rAAV as described herein for use in a method of treating cataract formation in a subject in need thereof, the method comprising administering an effective amount of the rAAV to the subject.

[0251] In some embodiments, there is provided an rAAV described herein for use in the manufacture of a medicament for the treatment of cataract formation in a subject in need thereof.

[0252] In some embodiments, there is provided an rAAV as described herein for use in a method of treating presbyopia in a subject in need thereof, the method comprising administering to the subject an effective amount of the rAAV.

[0253] In some embodiments, there is provided an rAAV described herein for use in manufacturing a medicament for treating presbyopia in a subject in need thereof.

[0254] In some embodiments, there is provided an rAAV as described herein for use in a method of treating a loss of regulation in a subject in need thereof, the method comprising administering to the subject an effective amount of the rAAV.

[0255] In some embodiments, there is provided an rAAV described herein for use in manufacturing a medicament for treating a loss of regulation in a subject in need thereof.

[0256] In some embodiments, there is provided an rAAV as described herein for use in a method of treating oxidative stress in a subject in need thereof, the method comprising administering to the subject an effective amount of the rAAV.

[0257] In some embodiments, there is provided an rAAV described herein for use in manufacturing a medicament for treating oxidative stress in a subject in need thereof.

[0258] In some embodiments, there is provided an rAAV as described herein for use in a method of treating elevated intraocular pressure in a subject in need thereof, the method comprising administering to the subject an effective amount of the rAAV.

[0259] In some embodiments, there is provided an rAAV described herein for use in the manufacture of a medicament for the treatment of elevated intraocular pressure in a subject in need thereof.

[0260] In some embodiments, there is provided an rAAV as described herein for use in a method of treating meibomian gland dysfunction in a subject in need thereof, the method comprising administering to the subject an effective amount of the rAAV.

[0261] In some embodiments, there is provided an rAAV as described herein for use in the manufacture of a medicament for treating meibomian gland dysfunction in a subject in need thereof.

[0262] Administration method In some embodiments, the present disclosure provides a method of administering an rAAV vector, wherein the rAAV vector comprises an AAV capsid and an expression cassette comprising a polynucleotide encoding TRX operably linked to a promoter, and is administered to the eye of a subject, the lacrimal gland of a subject's eye, or the trabecular meshwork of a subject's eye. In some embodiments, the present disclosure provides a method of administering an rAAV vector, wherein the rAAV vector comprises an AAV capsid and an expression cassette comprising a polynucleotide encoding PDI operably linked to a promoter, and is administered to the eye of a subject, the lacrimal gland of a subject's eye, or the trabecular meshwork of a subject's eye. It is administered into the trabecular meshwork.

[0263] As mentioned above, the lacrimal functional unit consists of the main and accessory lacrimal glands, the ocular surface, and their interconnected innervation. For each eye, the main lacrimal gland is located on the superior lateral side within the lacrimal fossa of the frontal bone of the orbit. The accessory lacrimal glands, known as Wölfring's glands and Krause's glands, are located in the eyelids. The upper eyelid contains approximately 2-5 Wölfring's glands and approximately 40 Krause's glands. The lower eyelid contains approximately 6 There are approximately eight Krause's glands. The specific location and anatomy of the lacrimal functional unit are not well known. (Conrady et al. J Ophthalmol.; 2016: 7542929 (2016)). Together, the lacrimal glands secrete a tear film onto the ocular surface through the lacrimal duct. The lacrimal glands also secrete proteins and products necessary for the tear film, and provide transformative growth factor-β and retinol, which are necessary for corneal regeneration and promoting transparency. (Conrady et al. J Ophthalmol.; 2016: 7542929 (2016); Pan et al. Optom Vis Sci.; 95:27-31 (2018)). In addition to secreting tears into the eye, the lacrimal ducts also drain tears into the nasal cavity.

[0264] Delivery of rAAV vectors to the lacrimal gland can be achieved by topical administration to the ocular surface, by direct injection into the lacrimal gland, and / or by topical administration to the lacrimal gland, which can be surgically accessed. The rAAV vector can be injected directly into the lacrimal gland or accessible by manipulation of the eyelid. Manipulation of the eyelid provides access for topical administration (e.g., by irrigating the tissue with a pharmaceutical composition containing the rAAV vector). Direct injection into the lacrimal gland can be achieved by penetrating the skin over the lacrimal gland (FIG. 2A) or by manipulating the eyelid to access the lacrimal gland (FIG. 2B). Administration of the rAAV vector to the lacrimal gland can also be achieved by subconjunctival injection. In some embodiments, the rAAV vector is injected directly into the lacrimal gland as shown in FIG. 2A. In some embodiments, the rAAV vector is administered to the lacrimal gland by manipulating the eyelid as shown in FIG. 2B.

[0265] In some embodiments, cells within the eye, lacrimal gland, or / and lacrimal nasal duct are transfected with an rAAV vector. Cells in the eye, lacrimal gland, and / or nasal duct are transduced or transformed by In some embodiments, the transduced or transformed cells in the eye, lacrimal gland, and / or lacrimal nasal duct include, but are not limited to, acinar cells, luminal cells, and / or myoepithelial cells, cells of the iris and ciliary body (ICB), lens epithelial cells, cells of the meibomian gland, and cells of the trabecular meshwork. In some embodiments, the transduced or transformed cells in the eye, lacrimal gland, and / or lacrimal nasal duct express a therapeutically effective amount of TRX. In some embodiments, cells within the meibomian gland are transduced or transformed with an rAAV vector. In some embodiments, cells within the trabecular meshwork are transduced or transformed with an rAAV vector. In some embodiments, a therapeutically effective amount of TRX In some embodiments, a therapeutically effective amount of TRX is administered to the eye surface of the subject. secreted onto the surface.

[0266] In some embodiments, transformed or transduced cells in the eye, lacrimal gland, and / or lacrimal nasal duct are In some embodiments, the cells express a therapeutically effective amount of PDI. The transformed or transduced cells in the nasal and / or lacrimal 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 the subject. In some embodiments, a therapeutically effective amount of the PDI is secreted onto the ocular surface of the subject.

[0267] Delivery of rAAV vectors to the eye and / or lacrimal gland results in gene expression in the tear film in vivo The primary lacrimal gland of mice was transfected with serotypes AAV2, AAV4, AAV5, and AAV 5w8. AAVx5, AAV9, AAV12, and bovine AAV (BAAV) were directly injected with rAAV vectors encoding the luciferase gene. AAV9, AAV5w8, AAV5, and AAV2 were injected with the tear film. The luminal and acinar cells of the gland can be transduced (Rocha et al., supra).

[0268] In some embodiments, the rAAV vector is administered to the lacrimal gland of a subject. The lacrimal gland is the main lacrimal gland. In some embodiments, the lacrimal gland is either a Wolffring's gland or a Krause's gland in a subject.

[0269] The compositions and rAAV vectors of the present disclosure are administered to the subject's lacrimal gland in any suitable manner, for example, the subject compositions may be administered by direct injection into the main or accessory lacrimal gland.

[0270] Access to the lacrimal gland in a human subject can be achieved, for example, by manually lifting the upper eyelid to expose the palmar lobe of the lacrimal gland and administering the therapeutic agent with a syringe using a 30G needle.

[0271] In some embodiments, the rAAV vector is administered to the trabecular meshwork of the subject's eye.

[0272] In some embodiments, the rAAV vector is administered to corneal endothelial cells of a subject.

[0273] The rAAV vectors of the present disclosure are typically administered to a subject as a pharmaceutical composition. The pharmaceutical composition comprises a pharmaceutically acceptable solvent (e.g., water) and one or more excipients. In some embodiments, the pharmaceutical composition comprises a buffer at approximately 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 include a pharmaceutically acceptable salt. The concentration of the salt may be selected to make the pharmaceutical composition isotonic or nearly isotonic with the target tissue.

[0274] The pharmaceutical compositions of the present disclosure, in various embodiments, contain about 1 × 10 8 Genome copies (GC / mL), Approximately 5 x 10 8 GC / mL, approximately 1 × 10 9 GC / mL, approximately 5 × 10 9 GC / mL, approximately 1 × 10 10 GC / mL, approximately 5 × 10 10 GC / mL, approximately 1 × 10 11 GC / mL, approximately 5 × 10 11 GC / mL, approximately 1 × 10 12 GC / mL, approximately 5 × 10 12 GC / mL, approximately 5 × 10 13 GC / mL, or approximately 1 × 10 14 GC / mL of rAAV vector. × 10 8 GC / mL to approximately 5 × 10 8 GC / mL, approximately 5 × 10 8 GC / mL to approximately 1 × 10 9 GC / mL, approximately 1 × 10 9 GC / mL to approximately 5 × 10 9 GC / mL, approximately 5 × 10 9 GC / mL to approximately 1 × 10 9 GC / mL, approximately 1 × 10 10 GC / mL to approximately 5 × 10 10 GC / mL, approximately 5 × 10 10 GC / mL to approximately 1 × 10 11GC / mL, approximately 1 × 10 11 GC / mL to approximately 5 × 10 11 GC / mL, approximately 5 × 10 11 GC / mL to approximately 1 × 10 12 GC / mL, approximately 1 × 10 12 GC / mL to approximately 5 × 10 12 GC / mL, approximately 5 × 10 12 GC / mL to approximately 5 × 10 13 GC / mL, or approximately 5 × 10 13 GC / mL to approximately 1 × 10 14 Pharmaceutical compositions may be designed in the range of about 1 × 10 GC / mL. 8 GC / mL to approximately 5 × 10 8 GC / mL, approximately 5 × 10 8 GC / mL to approximately 5 × 10 9 GC / mL, approximately 5 × 10 9 GC / mL to approximately 5 × 10 10 GC / mL, approximately 5 × 10 10 GC / mL to approximately 5 × 10 11 GC / mL, approximately 5 × 10 11 GC / mL to approximately 5 × 10 12 GC / mL, or approximately 5 × 10 12 GC / mL to approximately 1 × 10 14 In some embodiments, pharmaceutical compositions may be formulated in the range of about 5 x 10 GC / mL. 8 Approximately 5 × GC / mL 10 10 GC / mL, approximately 5 × 10 10 GC / mL to approximately 5 × 10 12 GC / mL, or approximately 5 × 10 12 GC / mL to approximately 1 × 10 14 Pharmaceutical compositions may be designed in the range of GC / mL.

[0275] In some embodiments, the pharmaceutical compositions of the present disclosure comprise about 1 × 10 12 GC / mL to approximately 6.2 × 10 12In some embodiments, the pharmaceutical compositions of the present disclosure may comprise about 1 GC / mL of rAAV vector. × 10 12 GC / mL or approximately 6.2 × 10 12 May contain GC / mL of rAAV vector.

[0276] In some embodiments, the pharmaceutical compositions of the present disclosure may be 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, about 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. The compositions may be 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 about 110 μL, about 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. may be administered in μL, about 180 μL to about 190 μL, or about 190 μL to about 200 μL .

[0277] The genome copy number per milliliter can be determined by quantitative polymerase chain reaction (qPCR) using a standard curve, which is generated using a reference sample containing a known concentration of the viral polynucleotide genome. In the case of AAV, the reference sample used for production The reference sample is typically the transfer plasmid used to generate the rAAV vector, although other reference samples can also be used.

[0278] Alternatively or additionally, the concentration of the viral vector can be determined by titrating the vector against the cell line. Viral titers are typically expressed as vp / mL. In various embodiments, the pharmaceutical compositions of the present disclosure are expressed as about 1 x 10 viral particles (vp). 8 Virus particles (vp / mL), approximately 5 × 10 8 vp / mL, approximately 1 × 10 9 vp / mL, approximately 5 × 10 9 vp / mL, approximately 1 × 10 10 vp / mL, approximately 5 × 10 10 vp / mL, approximately 1 × 10 11 vp / mL, approximately 5 × 10 11 vp / mL, approximately 1 × 10 12 vp / mL, approximately 5 × 10 12 vp / mL, approximately 5 × 10 13 vp / mL, or approximately 1 × 10 14 vp / mL of rAAV vector. In addition, approximately 1 × 10 8 vp / mL to approximately 5 × 10 8 vp / mL, approximately 5 × 10 8 vp / mL to approximately 1 × 10 9 vp / mL, approximately 1 × 10 9 vp / mL to approximately 5 × 10 9 vp / mL, approximately 5 × 10 9 vp / mL to approximately 1 × 10 10 vp / mL, approximately 1 × 10 10 vp / mL to approximately 5 × 10 10 vp / mL, approximately 5 × 10 10 vp / mL to approximately 1 × 10 11 vp / mL, approximately 1 × 10 11 vp / mL to approximately 5 × 10 11 vp / mL, approximately 5 × 10 11 vp / mL to approximately 1 × 10 12 vp / mL, approximately 1 × 10 12 vp / mL to approximately 5 × 10 12 vp / mL, approximately 5 × 10 12 vp / mL to approximately 5 × 1013 vp / mL, or approximately 5 × 10 13 vp / mL to approximately 1 × 10 14 Pharmaceutical compositions may be designed in the vp / mL range.

[0279] Evaluation of effectiveness There are a variety of tests available to assess the condition of a subject's eye before and after treatment with any of the disclosed methods or compositions. In some disclosed embodiments, effective treatment is deemed by any of the following tests: a) dry eye syndrome a) Core test (visual analog scale), b) Schirmer test, c) Corneal fluorescein staining test, d) Ocular Surface Disease Index test. Also included are the Dry Eye Questionnaire Evaluation (SANDE), meibomian gland atrophy or dropout, gland secretion, meibography / meibometry, and tear film lipids. stromal layer thickness, corneal fluorescein staining, tear break-up time (TBUT), hyperopia-corrected near visual acuity activity (DCNVA), intraocular pressure measurement (e.g., tonometry), visual acuity testing, slit lamp examination, or fundus In some embodiments, tests, including examinations, are provided for assessing a subject's condition. Assessing signs and symptoms of an eye condition can include administering the test under standardized or reproducible conditions and obtaining a subject's test score. Conditions include those in which an adverse environment (temperature, humidity, airflow) is artificially created or carefully controlled.

[0280] The efficacy of the methods described herein can be assessed using any suitable method known in the art.

[0281] In some embodiments, the methods described herein result in a reduction in one or more symptoms of an ocular condition in a subject compared to before administration of the rAAV vector. The diagnostic criteria or symptoms associated with certain ocular conditions, as well as those described herein, include, but are not limited to, symptoms that may be alleviated by treatment, such as worsening of vision and the need for corrective lenses, such as glasses or contact lenses, and surgery.

[0282] In some embodiments, the methods of treatment described herein result in a delay in the onset of the ocular condition. In some embodiments, the onset of the ocular condition is delayed compared to a control subject. In some embodiments, the onset of the ocular condition is delayed compared to the contralateral eye. In some embodiments, the methods of treatment described herein result in a delay in the progression of the ocular condition. In some embodiments, the progression of the ocular condition is delayed compared to a control subject. In some embodiments, the progression of the ocular condition is delayed compared to the contralateral eye. A "control subject" may be, for example, a control subject not receiving treatment. In some embodiments, the control subject is an age-matched subject not treated with an rAAV vector containing an expression cassette. The expression cassette comprises a polynucleotide encoding TRX or PDI. In some embodiments, the control subject is an age-matched subject not receiving treatment. The "contralateral eye" refers to the eye opposite the eye treated with the disclosed compositions. The contralateral eye can be used as a treatment control when a subject has bilateral disease or when a model animal undergoes an experimental protocol on both eyes.

[0283] The methods of treatment described herein may slow the progression of an ocular condition, in some embodiments, the progression of the condition 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 95% compared to a control subject.

[0284] In some embodiments, the methods of treatment described herein result in a delay in onset of the condition in a subject from about 6 months to about 9 months, from about 9 months to about 12 months, from about 12 months to about 15 months, from about 15 months to about 18 months, from about 18 months to about 21 months, from about 21 months to about 24 months, from about 2 years to about 3 years, from about 3 years to about 4 years, from about 4 years to about 5 years, from about 5 years to about 6 years, from about 6 months to about 9 months, from about 9 months to about 12 months, from about 12 months to about 15 months, from about 15 months to about 18 months, from about 18 months to about 21 months, from about 21 months to about 24 months, from about 2 years to about 3 years, from about 3 years to about 4 years, from about 5 years to about 6 years, from about 6 months years, about 4 to about 5 years, about 5 to about 6 years, about 6 to about 7 years, about 7 to about 8 years, about 8 to about 9 years, about 9 to about 10 years, about 10 to about 15 years, about 15 to about 20 years, or more than 20 years late.

[0285] In some embodiments, the treatment methods described herein improve the subject's visual acuity. In some embodiments, the treatment methods described herein delay the decline in visual acuity compared to the contralateral eye or control subjects. Visual acuity can be measured using a chart requiring identification of letters from a specific distance, including, for example, the Snellen visual acuity chart or the Early Treatment Diabetic Retinopathy Study (ETDRS) chart (Bailey and Kitchin, Vision Research 90 (2013) 2-9; Bennet et al., Semin Pediatr Neurol. 2019 October; 31: 30-40). Visual acuity may be assessed using binocular distance-corrected near visual acuity (DCNVA).

[0286] In some embodiments, the methods of treatment described herein provide a survival benefit of 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 after administration of the rAAV vector. 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 The subject's vision remains unchanged for about 4 years, about 4 to about 5 years, about 5 to about 6 years, about 6 to about 7 years, about 7 to about 8 years, about 8 to about 9 years, about 9 to about 10 years, about 10 to about 15 years, about 15 to about 20 years, or more than 20 years.

[0287] In some embodiments, the methods of treatment described herein provide a survival benefit of 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 after administration of the rAAV vector. 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 In some embodiments, the subject's vision does not decrease by 5% or more for about 4 years, about 4 to about 5 years, about 5 to about 6 years, about 6 to about 7 years, about 7 to about 8 years, about 8 to about 9 years, about 9 to about 10 years, about 10 to about 15 years, about 15 to about 20 years, or more than 20 years. at least about 6 months to about 9 months, about 9 months to about 12 months after administration of the rAAV vector, 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 or more The subject's vision does not decrease by 10% or more for about 7 years, about 7 to about 8 years, about 8 to about 9 years, about 9 to about 10 years, about 10 to about 15 years, about 15 to about 20 years, or more than 20 years.

[0288] Another indicator of visual acuity is the need for corrective lenses (e.g., contact lenses or glasses). Typically, the strength increases as the condition progresses. In some embodiments, a subject treated by the methods described herein required corrective lenses prior to administration of the rAAV vector, and the treatment method extends that strength requirement by 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 30 months, or about 30 months to about 40 months. Months to about 24 months, about 2 to about 3 years, about 3 to about 4 years, about 4 to about 5 years, about 5 to about 6 years, about 6 to about 7 years, about 7 to about 8 years, about 8 to about 9 years, about 9 to about 10 years, about 10 to about 15 years It has remained unchanged for about 15 to 20 years, or more than 20 years.

[0289] The treatment methods described herein may delay cataract formation in a subject compared to the contralateral eye or a control subject. Cataract formation can be assessed using any method known in the art, including, for example, assessing cataract severity based on the Lens Opacities Classification System III.

[0290] In some embodiments, cataract formation in a subject is delayed 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 95% compared to a control subject.

[0291] In some embodiments, the methods of treatment described herein may 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, or about 15 months. From about 1 month 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 Delayed by about 4 years, about 4 to about 5 years, about 5 to about 6 years, about 6 to about 7 years, about 7 to about 8 years, about 8 to about 9 years, about 9 to about 10 years, about 10 to about 15 years, about 15 to about 20 years, or more than 20 years.

[0292] In some embodiments, the methods of treatment described herein are effective in treating a subject who had a cataract prior to administration of the rAAV vector, and the methods of treatment reduce the severity of the cataract by at least about 6 months. From about 9 months, from about 9 months to about 12 months, from about 12 months to about 15 months, from about 15 months to about 18 months, from about 18 months to about 21 months, from about 21 months to about 24 months, from about 2 years to about 3 years, from about 3 years to about 4 years, from about 4 years It has remained unchanged for about 5 years, about 5 to about 6 years, about 6 to about 7 years, about 7 to about 8 years, about 8 to about 9 years, about 9 to about 10 years, about 10 to about 15 years, about 15 to about 20 years, or more than 20 years.

[0293] In some embodiments, effective treatment of a subject is indicated by the Dry Eye Symptom Assessment Questionnaire (SANDE). In some embodiments, effective treatment of a subject is indicated by meibomian gland atrophy. In some embodiments, effective treatment of a subject is indicated by glandular shrinkage / dropout. In addition, effective treatment of the subject is indicated by meibography / meibometolysis. tear film lipid layer thickness, corneal fluorescein staining, tear break-up time (TBUT), hyperopia-corrected near visual acuity activity (DCNVA), intraocular pressure measurement, visual acuity testing, slit lamp examination, and fundus examination. The methods described herein for assessing the effectiveness of treatment of a subject are known to those of skill in the art.

[0294] Maintenance of long-term therapeutic effects The efficacy of the treatment methods described herein can be assessed at any suitable time point after administration, for example, 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. Evaluated after about 5 years. In some embodiments, efficacy is evaluated after each administration, for example, about every 3 months, about every 6 months, or about every 12 months.

[0295] The present disclosure may provide effective treatments over a period of time during which a measured effect (e.g., visual acuity) is maintained. As used herein, "maintained" means that the effect measure (e.g., visual acuity) is statistically A measure of efficacy after treatment according to the methods described herein may be maintained without additional administration or may be maintained after one or more additional administrations.

[0296] Timing and method of administration The dosage schedule administered to a subject depends on various considerations, including the duration of effect of each dose, the transduction (or transfection) efficiency of the rAAV vector, and the impact of the administration on the body. For example, if a patient's condition does not improve, the healthcare provider may, at their discretion, adjust the methods for treating eye conditions described herein, modifying the dosage or administering multiple doses to improve, control, or limit symptoms. For example, the period between multiple doses may be extended, or the number of days between doses may be extended. For example, the administration schedule may be adjusted after measuring symptoms of the eye condition.

[0297] As used herein, "dosage" refers to the dosage of a pharmaceutical composition of the present disclosure or the dosage of a treatment that alleviates the symptoms of an ocular condition.

[0298] In some embodiments, the dose of the rAAV vector may be the dose of the rAAV vector carrying the expression cassette. In such cases, to achieve an appropriate dose (e.g., an effective amount) of the gene product, an appropriate amount / potency of the rAAV vector is administered to the target site to allow for the effective administration of the gene product. The onset of efficacy is achieved over a period of time. An "effective amount" refers to an amount or dosage of a rAAV, treatment, or composition described herein administered to a subject that is sufficient to alleviate the symptoms or signs of an ocular condition. As used herein, "amount" can refer to an absolute amount (e.g., absolute amount of protein or rAAV particles) or a concentration (e.g., concentration of protein in solution), and it can be understood without confusion whether the absolute amount, concentration, or both are being referred to depending on the specific context.

[0299] In some embodiments, the rAAV vector is an rAAV viral particle. 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, administration of the rAAV vector results in stable production of a gene product 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 more. In embodiments, administration of the rAAV vector results in stable production of the gene product for about one week. In some embodiments, administration of the rAAV vector results in stable production of the gene product for about two weeks. In some embodiments, administration of the rAAV vector results in stable production of the gene product for up to about 3 weeks. In some embodiments, administration of the rAAV vector results in stable production of the gene product for approximately 4 weeks. In some embodiments, administration of the rAAV vector results in stable production of the gene product for about one month. In some embodiments, administration of the rAAV vector results in stable production of the gene product for about one month. In some embodiments, administration of the rAAV vector results in a stable production of the gene product for about two months. In some embodiments, administration of the rAAV vector results in stable production of the gene product for approximately three months. In some embodiments, administration of the rAAV vector results in sustained production of the gene product for approximately four months. In some embodiments, administration of the rAAV vector results in stable production of the gene for approximately 5 months. In some embodiments, administration of the rAAV vector results in stable production of the gene for approximately six months. In some embodiments, administration of the rAAV vector results in stable production of a progeny product for approximately 9 months. It results in stable production of the gene product for approximately 12 months.

[0300] In some embodiments, the methods described herein involve the analysis of at least one gene product (e.g., an effective amount of a rAAV vector containing a polynucleotide encoding TRX and / or PDI In some embodiments, the method includes administering to the subject an initial dose of the rAAV vector. and delivering one or more subsequent doses. The one or more subsequent doses are administered a period of time after the first dose. In some embodiments, the period between the first dose and the 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 more. In some embodiments, the period between the first and subsequent doses ranges from 1 to 7 days, 1 week to 4 weeks, 1 month to 2 months, 1 month to 3 months, 2 weeks to 6 months, or 4 months to 12 months. In some embodiments, the 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 5 days, at least 6 weeks, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days, at least 16 At least 1 month, At least 2 months, At least 4 months, At least 6 months, At least 9 months In some embodiments, the period is between 1 and 7 days. , 1 week to 4 weeks, 4 weeks to 8 weeks, 1 month to 3 months, 2 months to 4 months, 3 months to 6 months, 4 months to 12 months, and 6 months to 24 months.

[0301] In some embodiments, the method includes an initial dose and one or more subsequent doses. In some embodiments, one or more subsequent doses are administered after a period of time. The period between doses should be at least 1 hour, at least 2 hours, or at least 3 hours. The period between one or more subsequent doses may be 1 to 3 hours, 2 to 4 hours, 3 to 5 hours, 4 to 5 hours, 5 to 6 hours, 6 to 7 hours, or 8 to 9 hours. The period between one or more subsequent doses is at least 1 hour, 4 hours, 8 hours, or 12 hours. The period between one or more subsequent doses is 1 to 3 hours, 2 to 4 hours, 3 to 6 hours, 4 to 8 hours, or 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.

[0302] Pharmaceutical Compositions and Kits In some embodiments, the present disclosure provides pharmaceutical compositions comprising the described rAAV vectors. The pharmaceutical compositions may include the described rAAV vectors and a pharmaceutically acceptable carrier, delivery agent, or excipient.

[0303] In some embodiments, the present disclosure provides for the use of a described rAAV vector or pharmaceutical composition in the manufacture of a medicament for the treatment of an ophthalmic disease, disorder, or condition, hi some embodiments, the disclosure provides for the use of a rAAV vector or pharmaceutical composition used or adapted for use in the treatment of an ophthalmic disease, disorder, or condition.

[0304] In some embodiments, the pharmaceutically acceptable carrier comprises phosphate buffered saline. The pharmaceutical composition may be formulated to be compatible with the intended route of administration (e.g., intralacrimal administration). The pharmaceutical composition may be formulated for administration to the lacrimal gland or for administration to the ocular surface.

[0305] The present disclosure provides pharmaceutical compositions for treating an ocular condition in a subject, the pharmaceutical compositions comprising a vector (e.g., a described rAAV vector) encoding a described oxidoreductase and a pharmaceutically acceptable carrier. Any concentration of rAAV vector suitable for effectively transducing or transforming cells of the eye, lacrimal gland, meibomian gland, or trabecular meshwork can be used. For example, rAAV can be administered at a concentration of 10 8 Vector genomes / mL or more, e.g., 5 x 10 8 Vector genomes / mL, 10 9 Vector genomes / mL, 5 x 10 9 Vector genomes / mL, 10 10 Vector genomes / mL, 5 x 10 10 Vector genomes / mL, 10 11 Vector genomes / mL, 5 x 10 11 Vector genomes / mL, 10 12 Vector genomes / mL, 5 x 10 12 Vector genomes / mL, 10 13 Vector Geno m / mL, 1.5 x 10 13 Vector genomes / mL, 3 x 10 13 Vector genomes / mL, 5 x 10 13 vector Genomes / mL, 7.5 x 10 13 Vector genomes / mL, 9 x 10 13 Vector genomes / mL, 1 x 10 14 Baek Turgenomes / mL, 5 x 10 14 vector genomes / mL, or 1 x 10 15 The vector is prepared at a concentration of 1000 vector genomes / mL or less. If necessary, the vector can be prepared at any concentration within this range.

[0306] For example, an appropriate concentration of rAAV vector in a therapeutic formulation is an effective concentration that results in expression of 100 pg / mL to 50 μg / mL of TRX and / or PDI in the tear film after treatment.

[0307] The rAAV vector may be prepared in any suitable unit dose, e.g., 1x10 8 Vectorge Nom or higher, 1x10 9 , 1x10 10 , 1x10 11 , 1x10 12 , or 1x10 13 Vector genome And in some cases 1x10 14 The vector genome is also prepared.

[0308] In some embodiments, a vector containing a plasmid expressing TRX and / or PDI is administered at a concentration of 1x10 7 , 1x10 8 , 1x10 9 , 1x10 10 , 1x10 11 , 1x10 12 , 1x10 13 , 1x10 14 , or 1x10 15 It is prepared at a concentration of

[0309] In some embodiments, the unit dose of the pharmaceutical composition is measured using a multiplicity of infection (MOI). The MOI is the number of cells in the vector or viral genome of the provided rAAV vector. In some embodiments, the MOI is 1x10 6 In other cases it may be 1x10 5 ~1x10 7 It may be in the range of.

[0310] In some embodiments, the amount of the pharmaceutical composition is about 1x10 8 From about 1x10 15 rAAV vector, approximately 1x10 9 From about 1x10 14 rAAV vector, approximately 1x10 10 From about 1x10 13 of rAAV vector, or approximately 1x10 11 From about 3x10 12 Contains rAAV vectors.

[0311] In preparing the subject rAAV compositions, any host cell suitable for producing rAAV vectors can be used, including mammalian cells (e.g., 293 cells), insect cells (e.g., SF9 cells), microorganisms, yeast, etc. Host cells are those that stably maintain the AAV rep and cap genes. The rAAV vector genome may be stably maintained and packaged in a packaging cell or a producer cell. Exemplary packaging and producer cells include SF-9, 293, A549, or HeLa cell-derived. rAAV vectors are purified and prepared using standard techniques known in the art.

[0312] In some embodiments, the present disclosure provides for the use of an rAAV vector in the manufacture of a medicament for use in the methods described herein.

[0313] In some embodiments, the present disclosure provides a kit containing the rAAV and instructions for use. In some embodiments, the kit includes a package insert containing the rAAV and instructions for use of the kit. In some embodiments, the kit includes a pharmaceutically acceptable carrier or pharmaceutical composition for treating or slowing the progression of a disease, disorder, or condition, and includes instructions for treating a subject in need thereof.

[0314] Illustrative Embodiments Embodiment I-1. Recombinant adeno-associated virus (rAAV) containing an AAV capsid and an expression cassette A vector, wherein the expression cassette comprises a polynucleotide encoding an oxidoreductase, operably linked to a promoter.

[0315] Embodiment I-2. The rAAV vector of embodiment I-1, wherein the oxidoreductase is thioredoxin (TRX).

[0316] Embodiment I-3. The rAAV vector of embodiment I-1, wherein the oxidoreductase is a protein disulfide. The enzyme is propidoisomerase (PDI).

[0317] Embodiment I-4. The rAAV vector of embodiment I-3, wherein the polynucleotide comprises a sequence encoding a protein at least 95% identical to SEQ ID NO:26.

[0318] 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.

[0319] Embodiment I-6. The rAAV vector of embodiment I-2 or I-5, wherein the polynucleotide is Contains sequences that are at least 95% identical to sequence number 2.

[0320] Embodiment I-7. The rAAV vector of embodiment I-3 or I-4, wherein the polynucleotide is Contains sequences that are at least 95% identical to sequence no. 30.

[0321] Embodiment I-8. The rAAV vector of any one of embodiments I-1 to I-7, wherein the promoter The promoter is a CMV promoter comprising the nucleotide sequence set forth in SEQ ID NO:17.

[0322] Embodiment I-9. The rAAV vector of any of embodiments I-8, wherein the expression cassette comprises a CMV promoter and a CMV enhancer.

[0323] Embodiment I-10. The rAAV vector according to any one of embodiments I-1 to I-9, wherein the expression The set includes polyadenylation (polyA) sequences.

[0324] Embodiment I-11. The rAAV vector of embodiment I-10, wherein the polyA sequence is a BGH polyA sequence. do.

[0325] Embodiment I-12. The rAAV vector of any of embodiments I-1 to I-11, wherein the expression cassette comprises a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE).

[0326] Embodiment I-13. The rAAV vector of any of embodiments I-1 to I-12, wherein the expression cassette comprises a Kozak sequence.

[0327] Embodiment I-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 nucleotide sequence comprising SEQ ID NO:2 and at least one The polynucleotide comprises a polynucleotide that shares at least 95% identity with the promoter, and the polynucleotide is linked to a promoter.

[0328] Embodiment I-15. The rAAV vector of any of embodiments I-1 to I-11, wherein the expression cassette is flanked by two inverted terminal repeats (ITRs).

[0329] Embodiment I-16. The rAAV vector of embodiment I-12, wherein the ITRs are AAV2 ITRs.

[0330] Embodiment I-17. The rAAV vector of any of embodiments I-1 to I-2, and I-5 to I-13, or the composition of any of embodiments I-14 to I-16, wherein the expression cassette is Shares at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with column number 16 The present invention includes a nucleotide sequence having the following structure:

[0331] Embodiment I-18. The rAAV vector according to any one of embodiments I-1 to I-17, wherein the AAV vector The VP3 sequences are AAV2 VP3 (SEQ ID NO: 8), AAV5 VP3 (SEQ ID NO: 10), and AAV8 VP3 (SEQ ID NO: 12). , a VP3 that shares at least 95%, 98%, or 100% identity with AAV9 VP3 (SEQ ID NO: 14) Includes:

[0332] Embodiment I-19. The rAAV vector according to any one of embodiments I-1 to I-17, wherein the AAV vector The VP3 comprises a VP3 that shares at least 95%, 98%, or 100% identity with AAV9 (SEQ ID NO: 14).

[0333] Embodiment I-20. A composition comprising a rAAV vector, wherein the rAAV vector comprises: (a) an AAV2, AAV5, AAV8, or AAV9 capsid, and (b) an expression cassette, wherein the expression cassette comprises a nucleotide sequence comprising SEQ ID NO:2 and at least one The polynucleotide comprises a polynucleotide that shares at least 95% identity with the promoter, and the polynucleotide is linked to a promoter.

[0334] Embodiment I-21. A composition comprising a rAAV vector, wherein the rAAV vector comprises: (a) an AAV2, AAV5, AAV8, or AAV9 capsid, and (b) an expression cassette, wherein the expression cassette shares at least 95% identity with SEQ ID NO: 2. The polynucleotide sequence includes:

[0335] Embodiment I-22. The composition of embodiment I-20 or I-21, wherein the AAV capsid is AAV2.

[0336] Embodiment I-23. The composition of embodiment I-20 or I-21, wherein the AAV capsid is AAV5.

[0337] Embodiment I-24. The composition of embodiment I-20 or I-21, wherein the AAV capsid is AAV9.

[0338] Embodiment I-25. An rAAV vector or composition according to any of embodiments I-1 to I-24, wherein the polynucleotide comprises a sequence encoding a signal peptide.

[0339] Embodiment I-26. A pharmaceutical composition comprising the rAAV vector or composition of any of embodiments I-1 to I-25 and a pharmaceutically acceptable carrier.

[0340] Embodiment I-27. The pharmaceutical composition of embodiment I-26, wherein the composition is about 1 x 10 7 From approximately 1 x 10 14 Contains genome copies / milliliter of rAAV vector.

[0341] Embodiment I-28. The pharmaceutical composition of embodiment I-26, wherein the composition is about 1 x 10 12 From approximately 6.2 x 10 12 Contains genome copies / milliliter of rAAV vector.

[0342] EMBODIMENT I-29 A method of treating an ophthalmic condition in a subject, comprising administering to the eye of the subject a therapeutically effective amount of the pharmaceutical composition of any of Embodiments I-26 to I-28.

[0343] Embodiment I-30. The method of embodiment I-29, wherein the pharmaceutical composition is delivered to an ocular gland of the subject. can be.

[0344] Embodiment I-31. The method of embodiment I-29 or I-30, wherein the pharmaceutical composition is delivered to the lacrimal gland.

[0345] Embodiment I-32. The method of any of embodiments I-29 to I-31, wherein the pharmaceutical composition is an accessory lacrimal agent. delivered to the gland.

[0346] Embodiment I-33. The method of embodiment I-32, wherein the accessory lacrimal gland is a meibomian gland.

[0347] Embodiment I-34. The method of any of Embodiments I-29 to I-33, wherein the pharmaceutical composition comprises a fiber. Delivered to the trabecular meshwork.

[0348] Embodiment I-35. The method of any of Embodiments I-29 to I-34, wherein about 1×10 9 to approximately 1 x 10 10 , about 1×10 10 to approximately 1 x 10 11 , from about 1 × 10 to about 1 × 10 12 , about 1×10 12 to approximately 1 x 10 13 , or approximately 1 × 10 13 to approximately 1 x 10 15 of genome copies is administered.

[0349] Embodiment I-36. The method of any of Embodiments I-29 through I-35, wherein the ocular condition is an oxidative stress disorder. Associated with increased stress.

[0350] Embodiment I-37. The method of any of embodiments I-29 through I-35, wherein the ocular condition is associated with loss of expression and / or function of one or more oxidoreductase enzymes.

[0351] Embodiment I-38. The method of any of Embodiments I-29 through I-35, wherein the ocular condition is associated with loss of TRX expression and / or function.

[0352] Embodiment I-39. The method of any of embodiments I-29 through I-35, wherein the ocular condition is associated with loss of PDI expression and / or function.

[0353] Embodiment I-40. The method of any of embodiments I-29 through I-35, wherein the ocular condition is myopia. Characterized by loss.

[0354] Embodiment I-41. The method of any of embodiments I-29 to I-40, wherein the ocular condition is presbyopia. be.

[0355] Embodiment I-42. The method of any of Embodiments I-29 through I-40, wherein the ocular condition is cataract. The formation of

[0356] Embodiment I-43. The method of any of Embodiments I-29 to I-40, wherein the ocular condition is intraocular hypertension. It is a progressive disease.

[0357] Embodiment I-44. The method of any of embodiments I-29 to I-40, wherein the ocular condition is myocardial infarction. It is thyroid dysfunction (MDI).

[0358] Embodiment I-45. The method of any of Embodiments I-29 to I-40, wherein the ocular condition is glaucoma. is.

[0359] Embodiment I-46. The method of any of Embodiments I-29 to I-45, wherein the method comprises: This results in expression in cells of the primary and / or accessory lacrimal glands, and / or trabecular meshwork.

[0360] Embodiment I-47. The method of any of Embodiments I-29 through I-46, wherein the method results in expression of TRX in cells of the lacrimal gland and / or accessory lacrimal gland, and / or trabecular meshwork.

[0361] Embodiment I-48. The method of any of Embodiments I-29 through I-47, wherein the method results in secretion of TRX into the tears and / or ocular surface.

[0362] Embodiment I-49. The method of any of embodiments I-29 through I-48, wherein the method results in amelioration of one or more symptoms of the ocular condition.

[0363] Embodiment I-50. The method of any of embodiments I-29 through I-49, wherein the method comprises improving vision. results.

[0364] Embodiment I-51. The method of any of embodiments I-29 through I-50, wherein the method comprises: resulting in a reduction in need.

[0365] Embodiment I-52. The method of any of Embodiments I-29 through I-51, wherein the method results in a delay in progression of the condition.

[0366] Embodiment I-53. The method of embodiment I-52, wherein the method slows 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 95%.

[0367] Embodiment I-54. The method of embodiment I-53 or I-54, wherein the method delays onset of the condition from about 6 months to about 12 months, from about 12 months to about 18 months, or from about 18 months to about 24 months relative to a control subject. , about 2 to about 3 years, about 3 to about 4 years, about 4 to about 5 years, about 5 to about 6 years, about 6 to about 7 years, about 7 to about 8 years, about 8 to about 9 years, about 9 to about 10 years, about 10 to about 15 years, about 15 years Delay by about 20 years or more.

[0368] Embodiment I-55. The method of embodiment I-53 or I-54, wherein the control subject has not been treated with an rAAV vector containing an expression cassette comprising a polynucleotide encoding TRX. .

[0369] Embodiment I-56. The method of any of embodiments I-29 to I-55, wherein the subject is administered rAAV. have previously required corrective lenses and the administration will result in the strength of the corrective lenses remaining constant for at least 6 to 12 months, 12 to 18 months, 18 to 24 months, 2 to 3 years, 3 to 4 years, 4 to 5 years, 5 to 6 years, 6 to 7 years, 7 to 8 years, 8 to 9 years, 9 to 10 years, 10 to 15 years, 15 to 20 years, or 20 years or more.

[0370] Embodiment I-57. The method of any of embodiments I-29 through I-56, wherein the subject's visual acuity is determined by administering At least about 6 to about 9 months, about 9 to about 12 months, about 12 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 The above remains unchanged.

[0371] Embodiment I-58. The method of any of Embodiments I-29 through I-57, wherein the method further comprises administration of one or more other therapeutic agents.

[0372] Embodiment I-59. The method of any of embodiments I-29 to I-58, wherein the subject is a human. .

[0373] Embodiment I-60. The use of the pharmaceutical composition of any of embodiments I-26 to I-28, wherein the subject The present invention relates to a method for treating an ocular condition in a subject by administering an effective amount of the pharmaceutical composition to the eye of the subject.

[0374] Embodiment I-61. Use of the pharmaceutical composition of any of embodiments I-26 to I-28, wherein the subject for use in the manufacture of a medicament for treating an eye condition.

[0375] Embodiment I-62. A compound, composition or embodiment for treating an ocular condition in a subject, The rAAV of any of embodiments I-1 to I-25 or any of embodiments I-26 to I-28. The pharmaceutical composition comprises a pharmaceutical agent and a pharmaceutically acceptable carrier.

[0376] Embodiment I-63. The rAAV vector or composition of any of embodiments I-1 to I-25, or the pharmaceutical composition of any of embodiments I-26 to I-28, and a pharmaceutically acceptable carrier. A kit comprising instructions for use in treating an ocular condition in a subject, wherein the pharmaceutical composition is administered to the eye of the subject.

[0377] Embodiment I-64. The rAAV vector or composition of any of embodiments I-1 to I-25, or the pharmaceutical composition of any of embodiments I-26 to I-28, and a pharmaceutically acceptable carrier. A. A kit comprising instructions for use for treating presbyopia, wherein the pharmaceutical composition is administered to the eye of a subject.

[0378] Embodiment I-65. The rAAV vector or composition of any of embodiments I-1 to I-25, or the pharmaceutical composition of any of embodiments I-26 to I-28, and a pharmaceutically acceptable carrier. A. A kit comprising instructions for use to treat cataract formation, wherein a pharmaceutical composition is administered to the eye of a subject.

[0379] Embodiment I-66. The rAAV vector or composition of any of embodiments I-1 to I-25, or the pharmaceutical composition of any of embodiments I-26 to I-28, and a pharmaceutically acceptable carrier. A. A kit comprising instructions for use to treat loss of accommodation, wherein the pharmaceutical composition is administered to the eye of a subject.

[0380] Embodiment I-67. The rAAV vector or composition of any of embodiments I-1 to I-25, or the pharmaceutical composition of any of embodiments I-26 to I-28, and a pharmaceutically acceptable carrier. A. A kit comprising instructions for use for treating elevated intraocular pressure, wherein the pharmaceutical composition is administered to the eye of a subject.

[0381] Embodiment I-68. The rAAV vector or composition of any of embodiments I-1 to I-25, or the pharmaceutical composition of any of embodiments I-26 to I-28, and a pharmaceutically acceptable carrier. A kit including instructions for use for treating meibomian gland dysfunction (MGD), wherein the pharmaceutical The composition is administered to the eye of the subject.

[0382] Embodiment I-69. The method of any of Embodiments I-29 through I-59, further comprising administering one or more other therapeutic agents.

[0383] Embodiment I-70. The method of embodiment I-69, wherein the one or more other therapeutic agents increase tear production. To make.

[0384] Embodiment I-71. The method of embodiment I-70, wherein the one or more other therapeutic agents are administered topically intranasally. by.

[0385] Embodiment I-72. The method of Embodiment I-71, wherein the topical intranasal administration is by nasal spray.

[0386] Embodiment I-73. The method of any of Embodiments I-70 through I-72, wherein the one or more tear production-increasing therapeutic agents are nicotinic acetylcholine receptor (nAChR) agonists, or is a pharmaceutically acceptable salt of

[0387] EMBODIMENT I-74. The method of embodiment I-73, wherein the nAChR agonist is a full agonist of a nAChR subtype selected from α4β2, α3β4, α3α5β4, α4α6β2, or a combination thereof. It is a combination.

[0388] Embodiment I-75. The method of embodiment I-73 or I-74, wherein the nAChR agonist is varenicline. or a pharmaceutically acceptable salt thereof.

[0389] Embodiment I-76. The method of 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. do.

[0390] Embodiment I-77. The method of any of Embodiments I-70 through I-76, wherein one or more other tear production-increasing therapeutic agents are administered before or after administration of the pharmaceutical composition.

[0391] Embodiment I-78. The method of Embodiments I-70 through I-77, wherein the one or more other tear production-increasing therapeutic agents are administered about one week after administration of the pharmaceutical composition.

[0392] Embodiment I-79. The method of any of embodiments I-70 to I-78, wherein the method results in expression of TRX in tears and / or the cornea, and wherein the expression of TRX in tears and / or the cornea is increased in the subject after administration of the pharmaceutical composition. Add.

[0393] Embodiment I-80. The method of embodiment I-79, wherein expression of TRX in tears and / or the cornea is increased by a given amount compared to the absence of one or more other therapeutic agents that increase tear production. Increases in time.

[0394] Embodiment I-81. The method of embodiment I-80, wherein the predetermined time period is about 5 minutes.

[0395] Embodiment I-82. The method of embodiment I-80, wherein the predetermined period of time is about 1 hour.

[0396] Embodiment I-83. The method of any of Embodiments I-29 to I-82, wherein the subject is a human. .

[0397] Example The following specific examples are provided for illustrative purposes only, and not limiting of the remainder of the disclosure in any way whatsoever.

[0398] Example 1A: Expression of thioredoxin rAAV vector containing an expression cassette containing a transgene encoding thioredoxin (TRX) Thioredoxin (TRX) expression in cells transduced with the AAV transfer plasmid was measured by ELISA. The AAV transfer plasmid contained the 5' and 3' AAV2 ITRs (SEQ ID NOs: 22 and 23, respectively). Between the 5' and 3' ITRs was a sequence encoding the human TRX polypeptide. The cDNAs included those that provide optimal expression levels of TRX polypeptides and those that provide optimal expression levels of TRX polypeptides. The amino acid sequence of the encoded human TRX polypeptide is shown in SEQ ID NO: 1. The cDNA encoding the human TRX polypeptide has been codon-optimized to improve expression in human cells. The nucleotide sequence is shown in SEQ ID NO: 2. From 5' ITR to 3' ITR The nucleotide sequence is shown in SEQ ID NO: 16. Table 6 shows the standard curve and ELISA results for samples taken from two separate cell lines sampled in triplicate. [Table 6]

[0399] Next, we evaluated the expression of AAV-thioredoxin plasmids in 293T cells. 293T cells were grown in a 6-well plate at 6.5x10 cells per well. 5 Cells were seeded and transduced with 2.5 μg of AAV.TRX plasmid DNA using Lipofectamine 3000. Cells were grown and then plated onto 35 mm glass-bottom tissue plates. 5.5x10 cells per well of a culture plate 5 Cells were plated. 24 hours after transduction (without mediated exchange), transduced and non-transduced cells were stained with a primary polyclonal antibody against thioredoxin (immunogen: MVKQIESKTAFQEALDAAGDKLVVVDFSATWCGPCKMIKPFFHSLSEKYSNVIFLEVDVDDCQDVASECEVKCMPTFQFFKKGQKVGEFSGANKEKLEATINELV; SEQ ID NO: 1) and an AlexaFluor 488 fluorescently tagged secondary antibody. Cells were counterstained with 4',6-diamidino-2-phenylindole (DAPI) and observed under a fluorescent microscope at 20x and 40x magnification. The results, shown in Figure 4, demonstrate bright, granular signals in AAV-thioredoxin-transduced cells and minimal signals in non-transduced control cells, indicating thioredoxin protein expression. 293T cells express some thioredoxin, although to a lesser extent than AAV.TRX plasmid-transduced cells. It is being expressed.

[0400] Considering the expression level of endogenous thioredoxin in 293T cells, a study comparing the expression and secretion levels of thioredoxin in untransduced 293T cells and AAV.TRX plasmid DNA-transduced cells was performed in four biological replicates. Cells were cultured and the medium was collected. ELISA results for transduced and transduced 293T cells are shown in FIGS. 5A-5B, respectively. Conditioned medium from non-transduced cells was collected 4 days after transduction to allow adequate thioredoxin accumulation to enable detection by ELISA, whereas conditioned medium from AAV.TRX-transduced cells Conditioned medium was collected 24 hours post-transduction. Furthermore, the y-axis in FIGS. 5A-5B shows a significant difference in expression levels. Untransduced cells expressed and secreted an average of 19.86 nanograms / mL of thioredoxin in the conditioned medium, while the level in AAV.TRX-transduced cells was 14.3 μg / mL. The R of the thioredoxin standard curve (data not shown) generated for ELISA analysis was 0.01. 2 The value was 0.999 .

[0401] Western blot analysis of whole cell extracts of 293T cells and AAV.TRX-transduced 293T cells Blot analysis was performed. Whole cell extracts 48 hours after transduction were analyzed by Western blot, and Western blots for thioredoxin and GAPDH were performed using a GAPDH protein loading control for comparison. SDS-PAGE was performed using 30 μg of protein per lane. To standardize protein loading, a primary polyclonal antibody against thioredoxin and a secondary anti-rabbit IgG antibody tagged with horseradish peroxidase (HRP) were used. A loading control Western blot was also performed using an antibody against GAPDH to Band density analysis was performed using Image J software developed by the National Institutes of Health. Based on band density analysis, AAV.TRX plasmid DNA-transduced whole-cell extracts produced 3.5-fold more thioredoxin than non-transduced 293T cells.

[0402] Example 1B: Thioredoxin Expression, Further Studies A second Western blot analysis was performed using an anti-thioredoxin primary antibody, ThermoFisher catalog #14999-1-AP, and an anti-rabbit IgG HRP secondary antibody, ProMega catalog #A5316. The results are shown in Figure 13 and demonstrate that thioredoxin was expressed at 2, 4, and 6 hours in transduced 293T cells. The presence of syn transcripts was confirmed in transduced cells, but not in non-transduced cells. Furthermore, whole cell extracts were collected 24 hours after transduction and subjected to Western blot analysis using actin as a loading control. Transduced cells (AAV.TXN) reacted with anti-chillin antibody at the relevant molecular weight. The abundant protein detected with the oredoxin primary antibody is shown (shown in Figure 14).

[0403] To assess the functional activity of the expressed transgene product, a thioredoxin activity assay (Cayman Chemical Thioredoxin Fluorometric Activity Assay Catalog #500228) was performed. The assay was performed on conditioned medium and whole cell extracts of transduced cells, with non-transduced cells used as controls. The principle of the assay is to assess the ability of the transgene product to reduce disulfide bonds. Using a substrate consisting of insulin and eosin linked by a disulfide bond, reduction of this bond releases eosin from insulin, which can be detected at a wavelength of 560 nm. The increase in thioredoxin activity is directly proportional to the rate of increase in fluorescence (i.e., release of eosin from the insulin-eosin substrate). Thioredoxin activity is measured as follows: It is calculated according to the manufacturer's recommendations using the formula:

number

[0404] For this assay, 293T cells were transduced with AAV-TXN and washed 18 hours later, and the medium was replaced with OPTImem cell culture medium. The medium was collected 6 hours after replenishment to perform the assay. Recombinant thioredoxin was used as a positive control, and eosin was used to generate a standard curve of fluorescence at 560 nm. Figure 15 shows that the thioredoxin transcript in the transduced conditioned medium was active and eosin was expressed. Recombinant thioredoxin (Trx) was loaded into the assay at 120 ng per well, demonstrating that it can reduce the disulfide bond of the thioredoxin-labeled insulin substrate. Thioredoxin activity was 62.45 nM / min. Transduced 293T cells had a thioredoxin activity of 56.8 nM / min, while untransduced 293T cells had an endogenous thioredoxin activity level of 16.90 nM / min. This functional assay confirmed the expression, secretion, and function of the transgene product from the construct shown in FIG. 1, demonstrating its ability to reduce disulfide bonds after secretion from target cells. This finding supports the ability of the transgene product to diffuse out of cells expressing the transgene with the ability to reduce disulfide bonds formed by oxidative stress.

[0405] Example 2: Intralacrimal gene therapy using rAAV vectors This example demonstrates a single dose of rAAV vector administered as an intralacrimal injection followed by varenicline. A 9-day pilot study was conducted in Dutch-Belted rabbits to demonstrate the efficacy of a panel of rAAV vectors administered intranasally in a single injection into the lacrimal gland. Each rAAV vector composition was administered at two concentrations (1 × 10 12 GC / mL and 6.2 x 10 12 The rAAV vector panel includes AAV2, AAV5, AAV8, and AAV9 vectors. The expression cassette delivered by the rAAV vector includes an enhanced green fluorescent protein (ENFP) operably linked to a CMV promoter. The transgene encoding the protein (eGFP) was inserted into the rAAV vector (FIG. 7). The animals were then intranasally administered varenicline. Varenicline induced tear production in the animals, and the eGFP delivered to the lacrimal gland by the rAAV vector was expressed under the control of the CMV promoter in the lacrimal gland cells. This approach is used to achieve two main goals: (1) the capsid proteins of AAV2, AAV5, AAV8, and AAV9 serotypes are expressed and secreted into the tear film and ocular surface of animals; delivers transgenes to lacrimal gland cells and induces measurable CMV promoter-driven expression in the cells. and (2) assessing their potential to increase the relative amount of the transgene encoded by the expression cassette in the tear film and ocular surface of the animal.

[0406] Animal experiments were performed at Charles River Laboratories (CRL) facilities by CRL scientific staff.

[0407] Animal Testing Systems, Housing, and In-Life Monitoring The animals used in this study were male Dutch-Belted rats, 4–5 months old and weighing 1.3–2.3 kg. Rabbits were allowed to acclimate for 10 days before treatment initiation, and each animal was housed individually and cared for according to standard animal husbandry protocols (including normal environmental conditions, feeding schedules, and veterinary care).

[0408] rAAV Vector Compositions and Formulations In this study, we used a gene encoding an eGFP transgene operatively linked to a CMV promoter. A panel of compositions containing rAAV vectors containing the present cassettes are proposed for intralacrimal injection under the conditions shown in Table 7. Each composition contained a rAAV vector of a different AAV capsid protein serotype. The compositions are labeled OC-100a-d, each corresponding to a different AAV capsid protein serotype. The dosage formulations for intralacrimal injection are: It was prepared in a clean procedure by dilution with phosphate buffered saline solution. [Table 7]

[0409] Intralacrimal injection of rAAV composition Animals were dosed by intralacrimal injection on day 1 of the study. The formulation concentrations used for each composition tested were: The dose, frequency of administration, number of animals and lacrimal glands are outlined in Table 8. Before injection, animals were Anesthesia was initiated with an intramuscular injection of detomidine (0.25 mg / kg) and maintained using an isoflurane / oxygen mix via a mask as needed. Topical antibiotics were applied to each eye after administration. On day 9 of the study, animals were administered varenicline tartrate (50 μL of 1.2 mg / mL varenicline in each nostril) intranasally to induce tear production. [Table 8]

[0410] biological analysis Blood samples were collected from all animals via an ear vein on Day 1 prior to dosing and on Days 8 and 9 (approximately 1 hour after intranasal dosing). Blood samples were placed on ice and chilled until plasma was isolated by centrifugation. Plasma samples were divided into 250 μL aliquots and stored frozen at -80°C for later analysis.

[0411] On days 8 and 9, animals were subjected to a Schirmer test to collect ocular moisture. Test strips were inserted into the inside of the lower eyelid for approximately 1 minute. The test strips were removed and placed in separate tubes for later analysis. The samples were stored frozen at −80°C for analysis.

[0412] Plasma samples and Schirmer test strips were analyzed for eGFP and eGFP mRNA concentrations according to established procedures at the Syneos analytical laboratory.

[0413] Immunohistochemistry of lacrimal gland tissue On the 9th day after administration, the animals were treated with intravenous sodium pentobarbital after blood and eye examination. Sections of the left eye and both lacrimal glands were used for immunohistochemistry (IHC). IHC was performed according to the laboratory's standard operating procedures. Lacrimal gland IHC samples were stained for eGFP and subjected to microscopic evaluation.

[0414] result Microscopic evaluation demonstrated the efficacy of in vivo administered rAAV compositions in eGFP expression in lacrimal gland tissue. The positive acinar cells isolated in the IHC samples had pink to red cytoplasmic staining indicative of GFP expression (FIGS. 8A-8K; exemplary staining indicated by black arrows). eGFP expression was 6.2 x 10 12 rAAV composition (OC-100a) containing GC / mL of AAV2 capsid protein (Figure 8A), 1 × 10 12 GC / mL (Figure 8B) and 6.2 × 10 12 AAV5 capsids in both GC / mL (Figure 8C-8H) rAAV composition containing 6.2 × 10 12 Contains GC / mL of AAV9 capsid protein This was observed in all rAAV compositions (Figures 8I-8K).

[0415] conclusion The results of this example demonstrate that rAAV vectors can deliver expression cassettes to the lacrimal gland by direct injection. We also demonstrate that rAAV vectors carrying capsid proteins of AAV2, AAV5, or AAV9 serotypes can deliver expression cassettes to cells within the lacrimal gland. Furthermore, we demonstrate that delivery of expression cassettes containing transgenes operatively linked to the CAG promoter sequence can be achieved. have shown that transgene expression in lacrimal gland cells is achieved.

[0416] Example 3: Expression of EGFP transgene in porcine lacrimal glands by rAAV via intralacrimal injection The goal of this study was to evaluate whether the lacrimal gland could be utilized as a method to modify or enrich the tear film of pigs with proteins of interest. Subsequently, in vivo studies were performed to test whether eGFP could be produced within the acinar cells of the lacrimal gland and subsequently secreted into the tear film after delivery of an adenoviral vector containing a plasmid encoding eGFP. cDNA One approach to introduce eGFP into acinar cells is to use cDNA encoding secreted eGFP (secEGFP). The first step was to inject adeno-associated viruses (AAV) containing secEGFP into the lacrimal gland. To generate AAVs of different serotypes (2 and 9), AAV transfer plasmids containing the elements necessary for secEGFP expression were generated, and the DNA sequence located between the ITRs was packaged into the produced AAV (Fig. 7 ).

[0417] Design Analysis and Methodology Research-grade AAV containing secreted EGFP (serotypes 2 and 9) was synthesized at Sirion and 5x10 12 No. The AAV was provided at a specific concentration. In vitro testing to confirm the ability of the produced AAV to transduce cells was performed at CJ Solutions using HEK 293T cells and ELISA. At Texas A&M University, eight domestic pigs were injected once with a low dose of EGFP in the right lacrimal gland (OD; oculus dexter) and a high dose in the left lacrimal gland (OS; oculus sinister). Six weeks after the first injection, the high dose of AAV2 The study was terminated 8 weeks after the second injection, after which the presence of EGFP in the lacrimal gland and potential inflammation or glandular abnormalities were assessed (Tables 9 and 10). Administration was carried out (Table 11). [Table 9] [Table 10] [Table 11]

[0418] On day 82 after the second AAV-secEGFP injection, tears were collected from each eye using Schirmer tear test strips. The tear test strips were inserted into the inferior conjunctival sac and held in place for 2 minutes. Tear proteins were extracted from the paper and mesoscale discovery (MSD) analysis was performed to detect the presence of EGFP protein in the tears.

[0419] On day 103, lacrimal glands were collected for ocular histopathology, and samples were sent to Zyagen, Inc. (San Diego, CA) for EGFP immunohistochemistry (IHC).

[0420] ELISA results showed that the produced AAV serotypes could transduce HEK 293T cells and produce secreted EGFP in vitro. EGFP expression in tear samples was consistent with AAV transduction. 82 days after transduction, MSD analysis confirmed the presence of HIV-1 markers with some levels >400 pg / mL, and this was also confirmed by IHC (Figure 9). IHC demonstrated a higher infectivity of acinar cells with AAV2 compared to AAV9. The results showed EGFP expression in acinar cells. Furthermore, transduction of ductal epithelial cells was observed in AAV9-injected lacrimal glands (Figure 10). Hematoxylin-eosin staining of pig lacrimal glands after repeated AAV injections. Staining showed no inflammatory infiltrate, atrophy, or edema (Figure 11).

[0421] Pig lacrimal glands injected with AAV2-secEGFP or AAV9-secEGFP expressed the EGFP transgene product in acinar cells and ductal epithelial cells. The EGFP expressed in the lacrimal glands was confirmed to be secreted into the tear film. Furthermore, both animals primed with a low or high dose of AAV in the initial injection showed a significant increase in the EGFP transgene product. However, no safety signals or inflammatory infiltrates were observed after repeated injections of AAV2 or AAV9. The results of this study suggest that lacrimal acinar cells may be a potential target for gene therapy approaches to modify and / or enrich the tear film.

[0422] Example 4: rAAV transgene expression in porcine lacrimal glands in combination with OC-1 nasal spray This example evaluates the expression of rAAV encoding a model protein after a single intralacrimal injection. This study describes a study conducted in pigs to confirm the expression of mRNA encoding a model protein (hereafter referred to as "Protein_A") in the lacrimal gland and the transgene protein levels in tears after a single injection of AAV protein_A (hereafter referred to as "AAV-Protein_A"). The study was further designed to confirm the expression and secretion of Protein_A after intralacrimal injection and to assess the relative amount of protein present on the ocular surface upon stimulation of tear production with varenicline nasal spray. Varenicline ("OC-1") is the following compound: [ka]

[0423] The AAV-protein_A plasmid contains, from 5' to 3', the AAV2 5'ITR, the CMV enhancer / promoter, an intron sequence containing Kozak, an open reading frame encoding protein_A, and a Woodchuck Encoding the hepatitis virus posttranscriptional regulatory element (WPRE), polyA sequence, and AAV2 3'ITR do.

[0424] Details of study parameters are shown in Table 12. Treatment and dosage are shown in Table 13. [Table 12] [Table 13]

[0425] The timeline of the pig study is shown in Figure 12. For all pigs (N=14), day 0 was the day of injection. On days 7, 14, 21, 28, 35, 42, 60 / 61, and 90, tears were collected from each eye using Schirmer test strips (applied for approximately 2 minutes and then removed). Immediately cut the tear-soaked Schirmer test strip with scissors above the fluid or dye line, and place the lower part (the tear-saturated part) in a microcentrifuge tube and store it on ice or in a freezer (-80°C). On days 14 and 42, tears were collected first, followed by administration of the nasal spray. Tears were collected again approximately two minutes after administration. From days 22 to 28, OC-01 nasal spray was administered twice daily (at least six hours between doses) into each nostril, with one dose administered on day 28. On day 28, tears were collected two minutes after administration of the nasal spray.

[0426] On day 90, gross pathology examinations are performed and body weights are assessed. In addition, one stool sample from each animal is taken. The lacrimal glands are harvested for ocular pathology using IHC. After harvesting, the lacrimal glands are fixed in 10% formalin for 24-48 hours (room temperature), then transferred to 70% EtOH and stored at 4°C. The volume in the tube is Keep 5x the tissue so that the tissue is completely submerged.

[0427] The second lacrimal gland was washed with phosphate-buffered saline and immediately transferred to RNA-Later. The samples were then rapidly frozen in liquid nitrogen. Approximately 0.5-1 cm square pieces of tissue were collected from the apex of the heart, kidney, and liver, and immediately placed in 2 mL cryotubes and rapidly frozen in liquid nitrogen. The samples are then stored at -80°C and sent for mRNA analysis.

[0428] mRNA analysis to analyze gene expression of AAV-derived protein A in the lacrimal gland of domestic pigs The assay is performed using a one-step duplex RT-qPCR method developed. The lysate was then loaded onto a silica-based QIASymphony system for automated RNA extraction. The AAV-protein A vector contains a bovine growth hormone (bGH) polyA sequence in the 3' untranslated region of the transgene. Protein A mRNA was analyzed using primers and probes targeting the bGH polyA sequence. The sequence is shown in Table 14. 76 bp of the bGH sequence is amplified. The samples were analyzed in 96-well plates for both bGH mRNA copy number and Ct value of porcine endogenous Hprt1 mRNA using a quantitative real-time PCR system (QuantStudio 7 Flex). Each plate contains a standard curve, negative control, and quality control samples, which are prepared separately to avoid cross-contamination. Each standard curve contains 1000 ribonucleotides per well. 8 , 10 7 , 10 5 , 10 4 , 10 3 , 10 2 bGH standard DNA levels of 50, 25, and 0 copies are included. RT-qPCR of RNA samples is performed in duplicate wells up to 100 ng per well. The bGH mRNA copy number for each RT-qPCR well is interpolated from the bGH DNA standard curve (acceptance criteria: R 2 ≥ 0.980). A 2-fold multiplication step was used to interpolate single-stranded (ss) mRNA from the double-stranded (ds) standard curve, and the average copy number of two replicate wells is reported as the ss bGH mRNA copy number per 100 ng of RNA sample. Additionally, each RNA sample was tested using qScript XLT One-step RT-qPCR without additional reverse transcriptase to monitor for potential vector DNA contamination. [Table 14] [Table 15]

[0429] Finally, while aspects of this specification have been emphasized with reference to particular embodiments, it should be understood that these are illustrative only of the principles of the disclosure. For that reason, the subject matter described herein is not limited to specific compounds, compositions, articles, devices, methods, protocols, and / or reagents, etc. Furthermore, it should be understood that certain changes, modifications, variations, additions, deletions, and subcombinations of the described embodiments may be made in accordance with the teachings of the present invention.

[0430] When the term "may" or "can" is used in referring to an embodiment or aspect thereof, it carries the alternative meaning of "may not" or "cannot." In such cases, the embodiment or When an aspect is indicated as possibly being included as part of the subject matter of the invention, any corresponding negative restriction or exclusion is also expressly intended. Similarly, when the term "optionally" is used when referring to an embodiment or aspect thereof, it means that such embodiment or element thereof is or is not included as part of the subject matter of the invention.

[0431] Although the ranges and values ​​are approximations within the broad scope of the invention, they are reported as precisely as possible in the specific examples. Any range or value contains a certain amount of error. When a range of values ​​is recited, it is merely a shorthand method of referring individually to each value falling within the range.

[0432] In the description, terms such as "a," "an," "the," etc., are to be construed as covering both the singular and the plural unless the context indicates otherwise or unless expressly indicated otherwise. Furthermore, ordinal designations of distinguishing elements (e.g., "first," "second," "third," etc.) are used to indicate a distinction between the elements and do not necessarily indicate the position or order of the respective elements.

[0433] The description of specific examples is intended to illuminate the invention and not to limit its scope. Not all elements of the invention described in this specification should be construed as necessarily claimed elements unless expressly indicated as such.

[0434] All patents, patent publications, and other publications are individually and expressly incorporated by reference into this specification for the purpose of describing structures and methodologies therein. These publications are provided solely for their disclosure prior to the date of this patent application. No admission is made that such disclosures constitute prior invention.

[0435] Finally, 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 as defined by the claims. Therefore, the present invention is not limited to that precisely as shown and described.

[0436] [Table 16-01] [Table 16-02] [Table 16-03] [Table 16-04] [Table 16-05]

Table 16-06

Table 16-07

Table 16-08

Table 16-09

Table 16-10

Table 16-11

Table 16-12

Table 16-13

Claims

1. A recombinant adeno-associated virus (rAAV) vector having an expression cassette containing a polynucleotide encoding an oxidoreductase 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 polynucleotide has a sequence at least 95% identical to SEQ ID NO: 2 or SEQ ID NO:

28. The rAAV vector of claim 2 or 5, comprising a sequence.

7. Claim 3, wherein the polynucleotide comprises a sequence at least 95% identical to SEQ ID NO:

30. Or the rAAV vector described in 4.

8. The promoter is a CMV promoter comprising the nucleotide sequence shown in SEQ ID NO:

17. Item 8. The rAAV vector according to any one of items 1 to 7.

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

10. 10. The method according to claim 1, wherein the expression cassette comprises a polyadenylation (polyA) sequence. rAAV vectors as described.

11. The rAAV vector of claim 10, wherein the polyA sequence is a BGH polyA 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 an rAAV vector comprising an expression cassette linked to a promoter, the expression cassette comprising a polynucleotide sharing at least 95% sequence identity with the AAV capsid. 。

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

16. The rAAV vector of claim 15, wherein the ITRs are AAV2 ITRs.

17. rA according to any one of claims 1 to 2 and 5 to 13, wherein the expression cassette comprises nucleotide sequences that share at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. An AV vector or a composition according to any one of claims 14 to 16.

18. 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). Finished product.

19. The AAV capsid shares at least 95%, 98%, or 100% identity with AAV9 (SEQ ID NO: 14).

18. The rAAV vector or composition of any one of claims 1 to 17, comprising a VP3 having the following structure:

20. The expression cassette comprises a polynucleotide and a promoter that share at least 95% sequence identity. A composition comprising an rAAV vector comprising an expression cassette linked to a target and an AAV2, AAV5, AAV8, or AAV9 capsid.

21. A composition comprising an rAAV vector comprising an expression cassette and an AAV2, AAV5, AAV8, or AAV9 capsid, with which the expression cassette shares at least 95% sequence identity.

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. 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 an rAAV vector or composition described in any one of claims 1 to 25 and a pharmaceutically acceptable carrier.

27. The composition is approximately 1 x 10 7 From approximately 1 x 10 14 27. The pharmaceutical composition of claim 26, comprising genome copies / mL of an rAAV vector.

28. The composition is approximately 1 x 10 12 From approximately 6.2 x 10 12 rAAV vectors containing 100 genome copies / mL of the vector.

27. The pharmaceutical composition described in 26.

29. A method for treating an eye disease by administering the pharmaceutical composition according to any one of claims 26 to 28 to the eye of a patient.

30. 30. The method of claim 29, wherein the pharmaceutical composition is administered to the ocular glands of the patient.

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

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

33. 33. The method of claim 32, wherein the accessory lacrimal gland is a meibomian gland.

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

35. Approximately 1 x 10 9 From approximately 1 x 10 10 , about 1 x 10 10 From approximately 1 x 10 11 , about 1 x 10 11 From approximately 1 x 10 12 , Approximately 1 x 10 12 From approximately 1 x 10 13 , or approximately 1 x 10 13 From approximately 1 x 10 15 Genome copy rAAV vector The method of any one of claims 29 to 34, wherein

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

37. Claims 29 to 35, wherein the eye disease is associated with loss of expression and / or function of oxidoreductases.

10. The method according to any one of the preceding claims.

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

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

40. 36. The method of any one of claims 39 to 35, wherein the eye disease is characterized by loss of myopia.

41. The method according to any one of claims 29 to 40, wherein the eye disease is presbyopia.

42. 41. The method according to any one of claims 29 to 40, wherein the eye disease is cataract formation.

43. 41. The method of any one of claims 29 to 40, wherein the eye disease is ocular hypertension.

44. The method according to any one of claims 29 to 40, wherein the eye disease is meibomian gland dysfunction (MDI). How to post.

45. The method according to any one of claims 29 to 40, wherein the eye disease is glaucoma.

46. The method involves the expression of oxidoreductases in cells of the lacrimal gland and / or accessory lacrimal gland, or the plexus trabecularis. The method according to any one of claims 29 to 45.

47. 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, or the meshwork of the trabecularis.

48. 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 ocular surface of the subject.

49. 49. The method of any one of claims 29 to 48, wherein the method results in amelioration of one or more symptoms of the eye disease.

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

51. A method according to any one of claims 29 to 50, wherein the method reduces the need for corrective lenses.

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

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

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

55. 55. The method of claim 53 or 54, wherein the control subject is an age-matched subject not treated with an rAAV vector containing the expression cassette.

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

57. The subject's visual acuity is at least approximately 6 to 9 months, approximately 9 to 12 months, or approximately 12 to 15 months. , about 15 to 18 months, about 18 to 21 months, about 21 to 24 months, about 2 to 3 years, about 3 1 to 4 years, about 4 to 5 years, about 5 to 6 years, about 6 to 7 years, about 7 to 8 years, about 8 to 9 years , approximately 9 to 10 years, approximately 10 to 15 years, approximately 15 to 20 years, or unchanged for more than 20 years, claim Item 56. The method according to any one of Items 29 to 55.

58. The method of any one of claims 29 to 57 further comprising administering one or more additional therapeutic agents. The method according to any one of claims 1 to 5.

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

60. 29. The pharmaceutical composition of any one of claims 26 to 28 for use in a method for treating an ocular disease comprising administering an effective amount of the pharmaceutical composition to the eye of a 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 eye disease.

62. A compound for use in treating an eye disease in a subject, comprising administering an rAAV vector or composition described in any one of claims 1 to 25 or a pharmaceutical composition of any one of claims 26 to 28.

63. A kit comprising an rAAV vector or composition described in any one of claims 1 to 25 or a pharmaceutical composition described in any one of claims 26 to 28, and instructions for use to treat an eye disease in a subject, comprising administering the pharmaceutical composition to the eye of a subject.

64. A kit comprising an rAAV vector or composition described in any one of claims 1 to 25 or a pharmaceutical composition described in any one of claims 26 to 28, and instructions on how to use the pharmaceutical composition to treat presbyopia in a subject, comprising administering the pharmaceutical composition to the eye of the subject.

65. A kit comprising an rAAV vector or composition described in any one of claims 1 to 25 or a pharmaceutical composition described in any one of claims 26 to 28, and instructions on how to use the pharmaceutical composition to treat cataract formation in a subject, comprising administering the pharmaceutical composition to the eye of the subject.

66. rAAV vector or composition according to any one of claims 1 to 25 or claim 26 29. A kit comprising a pharmaceutical composition according to any one of claims 1 to 28 and instructions on how to use the pharmaceutical composition to treat loss of accommodation in a subject, comprising administering the pharmaceutical composition to the eye of the subject.

67. A kit comprising an rAAV vector or composition described in any one of claims 1 to 25 or a pharmaceutical composition described in any one of claims 26 to 28, and instructions on how to use the pharmaceutical composition to treat ocular hypertension in a subject, comprising administering the pharmaceutical composition to the eye of the subject.

68. 20. 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 instructions for use in treating meibomian gland dysfunction (MGD) in a subject, comprising administering the pharmaceutical composition to the eye of the subject. Kit including:

69. a) a polypeptide comprising an oxidoreductase or a fragment thereof, optionally the polypeptide comprising 90% or more a polypeptide having an amino acid sequence having identity to the above (SEQ ID NOs: 1 and 26), and b) a pharmaceutically acceptable carrier suitable for administration to the human eye; A pharmaceutical composition comprising:

70. a) a polynucleotide encoding a polypeptide comprising an oxidoreductase or a fragment thereof a vector comprising the polypeptide, optionally a polypeptide having an amino acid sequence with 90% or more identity (SEQ ID NOs: 1 and 26); b) a pharmaceutically acceptable carrier suitable for administration to the human eye; A pharmaceutical composition comprising:

71. Pharmaceutically acceptable carriers include water, sterile water, aseptic water, phosphate buffered saline, and HEPES buffered saline. , an isotonic saline solution, a balanced salt solution, a wetting agent, a surfactant, an isotonic agent, a pH adjuster, a viscosity adjuster, a buffer, a disaccharide (e.g., sucrose or trehalose), cellulose or a derivative thereof, an amino acid (e.g., histidine), or any combination thereof.

72. 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 polypeptide is 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 73. The pharmaceutical composition of any one of claims 69 to 72, comprising:

74. 74. The pharmaceutical composition of any one of claims 69 to 73, which is a liquid formulation prepared for application to or penetration into the ocular surface, or for intralacrimal injection.

75. 75. The pharmaceutical composition of any one of claims 70 to 74, comprising a vector, wherein the composition has the effect of expressing the polypeptide in the tear film in the range of 100 pg / mL to 50 μg / mL.

76. 76. The pharmaceutical composition of any one of claims 70 to 75, wherein the polynucleotide is substantially linked to a promoter.

77. The vector is designed to constitutively express polypeptides comprising the sequences of SEQ ID NOs: 1 and 26. The pharmaceutical composition according to any one of claims 70 to 76, which is designed.

78. 78. The pharmaceutical composition of any one of claims 70 to 77, wherein the vector comprises an adenoviral or lentiviral vector, a plasmid, an episome, or an artificial chromosome, and further comprises one or more lipids, polyvalent cations, DNA carrier proteins, histones, pseudocapsids, chimeric proteins, or endocytic receptor proteins.

79. 75. The pharmaceutical composition of any one of claims 69 or 71 to 74, wherein the polypeptide is contained in an amount ranging from 100 pg / mL to 50 μg / mL.

80. The polypeptide is contained in an amount ranging from 500 ng to 5 μg according to claims 69, 71 to 74, or 79. A pharmaceutical composition according to any one of claims 79 to 79.

81. 81. The pharmaceutical composition of any one of claims 69, 71-74, 79 or 80, wherein the polypeptide is contained in a unit dosage.

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

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

84. 84. The method of claim 83, wherein the accessory lacrimal gland is a meibomian gland.

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

86. The method of any one of claims 82 to 85, wherein the eye disease 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 TRX expression and / or function; and / or d) associated with loss of PDI expression and / or function.

87. 87. The method of any one of claims 82 to 86, wherein the eye disease is characterized by loss of near vision.

88. The method of any one of claims 82 to 87, wherein the eye disease is: a) presbyopia; b) cataract formation; c) ocular hypertension; d) meibomian gland dysfunction (MGD); and / or e) Glaucoma.

89. The method of any one of claims 82 to 88, wherein the method results in: a) Expression of oxidoreductase enzymes in cells of the lacrimal gland and / or accessory lacrimal gland, and / or the rete trabecularis; b) expression of TRX in cells of the lacrimal gland and / or accessory lacrimal gland, and / or the trabecular meshwork; and / or c) Secretion of TRX into the tear film and / or ocular surface of the subject.

90. The method of any one of claims 82 to 88, wherein the method results in: a) Improvement of one or more symptoms of eye disease; b) improved vision; c) a reduction in the need for corrective lenses; and / or d) Slowing the progression of eye disease.

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

92. The administration induces the expression of functional oxidoreductases in one or more cells of the lacrimal gland and / or accessory lacrimal gland of the subject.

93. The method of any one of claims 82 to 92, resulting in

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

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

95. 95. The method of any one of claims 82 to 94, wherein administration is to the ocular surface of the subject, or to the ocular surface or to the lacrimal gland.

96. 96. The method of any one of claims 82 to 95, wherein the symptoms are selected from itching, swelling, watery eyes, and redness.

97. 97. The method of any one of claims 82-96, wherein administration results in a 0.5 point, 1 point, 1.5 points, 2 points, 2.5 points, 3 points, 3.5 points, or 4 point improvement on a conjunctival itching rating scale.

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

99. 82. A kit comprising the pharmaceutical composition of any one of claims 69 to 81 and instructions on how to use the pharmaceutical composition to treat a condition in a human subject, comprising administering the pharmaceutical composition to the eye of the human.

100. 82. The pharmaceutical composition of 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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