AAV vectors encoding diamine oxidase and uses thereof

Abstract

The present invention provides recombinant adeno-associated virus (rAAV) vectors, methods of treating conditions (e.g., ocular conditions), pharmaceutical compositions, and other compositions and methods, wherein the rAAV vectors comprise a polynucleotide encoding a diamine oxidase (DAO1). Methods of treatment may include administration to the lacrimal gland.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 383,026, filed on November 9, 2022, and U.S. Provisional Patent Application No. 63 / 348,856, filed on June 3, 2022, the disclosures of which are incorporated herein by reference in their entirety.

[0003] Sequence Listing

[0004] This application incorporates by reference in its entirety a sequence listing XML file entitled “037525-00558.xml” (71 KB), which was created on May 24, 2023 and is submitted electronically with this document. Background Art

[0005] Medical treatments are known for eye conditions that affect the ocular surface, such as allergic conjunctivitis, including perennial and / or seasonal rhinoconjunctivitis, atopic keratoconjunctivitis, giant papillary conjunctivitis, and vernal (VKC) or allergic keratoconjunctivitis. These medical treatments include topical eye drops, oral medications, and the like.

[0006] Despite available treatments, conjunctivitis remains a challenge for eye care practitioners (ophthalmologists and optometrists), and treatment strategies focus on the severity of the disease. Treatment for less severe cases may include antihistamines, mast cell stabilizers, anti-inflammatory agents, and / or steroid topical eye drops. For more severe cases, oral antihistamines or oral leukotriene inhibitor medications may be used.

[0007] The delivery of antihistamines, mast cell stabilizers, calcineurin inhibitors, anti-inflammatory agents and / or steroid topical eye drops for the treatment of conjunctivitis currently relies on repeated administration of eye drops, which does not provide adequate therapeutic results in many severe cases, such as in vernal or atopic keratoconjunctivitis, where corneal ulcers, cataract formation, or glaucoma may result even with treatment. Summary of the invention

[0008] In view of the shortcomings of current medical treatment options for ocular surface disorders, there is a need for improved methods for delivering antihistamine therapy to the ocular surface, such as without repeated topical administration, which may be challenging or impossible for some patients and may lead to poor patient compliance. In addition, pulse administration of antihistamine therapy may only provide temporary or "occasional" relief and may not be suitable for chronic conditions. In addition to providing various other benefits as described herein and shown in the accompanying drawings, the present disclosure provides recombinant constructs and methods that address these and other needs in some aspects.

[0009] In some aspects, the present disclosure provides adeno-associated viral (AAV) vectors for expressing diamine oxidase ("DAO1"; alternatively abbreviated herein as "DAO") in the eye following in vivo administration.

[0010] In one aspect, the present disclosure provides a recombinant adeno-associated virus (rAAV) vector comprising an AAV capsid and an expression cassette comprising a polynucleotide encoding diamine oxidase (also known as DAO1, DAO, or histaminase) operably linked to a promoter. In some embodiments, the polynucleotide encodes a protein at least 95% identical to SEQ ID NO: 1 or 2. In some embodiments, the polynucleotide comprises a nucleotide sequence at least 95% identical to a nucleotide sequence selected from SEQ ID NO: 29-31. In some embodiments, the polynucleotide comprises a nucleotide sequence selected from SEQ ID NO: 29-31.

[0011] In another aspect, the present disclosure provides a composition comprising a rAAV vector, wherein the rAAV vector comprises: (a) an AAV capsid, and (b) an expression cassette, wherein the expression cassette comprises a polynucleotide comprising a nucleotide sequence having at least 95% identity to a nucleotide sequence selected from SEQ ID NOs: 29-31, and wherein the polynucleotide is linked to a promoter.

[0012] In some embodiments, the promoter is a CMV promoter. In some embodiments, the expression cassette comprises a CMV enhancer. In some embodiments, the promoter is a CAG promoter. In some embodiments, the expression cassette comprises a polyadenylation (polyA) sequence. In some embodiments, the polyA sequence is a BGH polyA sequence. 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. In some embodiments, the expression cassette is flanked by two inverted terminal repeats (ITRs). In some embodiments, the ITRs are AAV2 ITRs.

[0013] In some embodiments, the expression cassette comprises a nucleotide sequence that is at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:23.

[0014] In some embodiments, the AAV capsid comprises a VP3 that shares at least 95%, 98%, or 100% identity with AAV2 VP3 (SEQ ID NO: 8), AAV5 VP3 (SEQ ID NO: 10), AAV8 VP3 (SEQ ID NO: 12), or AAV9 VP3 (SEQ ID NO: 14). In some embodiments, the AAV capsid comprises a VP3 that shares at least 95%, 98%, or 100% identity with AAV9 VP3 (SEQ ID NO: 14).

[0015] In yet another aspect, the present disclosure provides 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 polynucleotide comprising a nucleotide sequence having at least 95% identity to a nucleotide sequence selected from SEQ ID NOs: 29-31, and wherein the polynucleotide is linked to a promoter.

[0016] In some embodiments, the expression cassette comprises a polynucleotide sequence that shares at least 95% identity with SEQ ID NO:23.

[0017] In some embodiments, the AAV capsid is AAV2. In some embodiments, the AAV capsid is AAV5. In some embodiments, the AAV capsid is AAV9.

[0018] In one aspect, the present disclosure provides a pharmaceutical composition comprising a rAAV vector described herein or a composition described herein (eg, a polypeptide comprising DAO1 or a fragment thereof) and a pharmaceutically acceptable carrier.

[0019] In some embodiments, the pharmaceutical composition comprises about 1×10 5 About 1×10 16 In some embodiments, the pharmaceutical composition comprises about 1×10 12 About 6.2×10 12 genome copies.

[0020] In some embodiments, the pharmaceutical composition may include an rAAV vector (or any other vector described herein, such as a plasmid configured to express DAO1) in an amount or concentration sufficient to cause expression of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 μg / mL of DAO1 in the tear film of a subject, or a concentration within a range defined by any pair of the foregoing values. Expression can be measured, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 hours after administration, or after a longer duration, for example after 1, 2, 3, 4, or 5 days.

[0021] In some embodiments, the pharmaceutical composition may comprise 0.25, 0.50, 0.75, 1.00, 1.25, 1.50, 1.75, 2.00, 2.25, 2.50, 2.75, 3.00, 3.25, 3.50, 3.75, 4.00, 4.25, 4.50, 4.75, 5.00, 5.25, 5.50, 5.75, or 6.00 μg of a polypeptide comprising DAO1 (or a fragment thereof), or an amount within a range defined by any pair of the foregoing values. In some embodiments, the pharmaceutical composition may further comprise one or more excipients, carriers, and / or diluents suitable for administration to the eye of a human or animal subject.

[0022] In some embodiments, the pharmaceutical composition is formulated for administration into the lacrimal gland. In some embodiments, the pharmaceutical composition is formulated for administration onto the surface of the eye. In some embodiments, the pharmaceutical composition is formulated for or suitable for use in treating an ocular disease, disorder, or condition.

[0023] In some aspects, the present disclosure provides a method for treating a condition in an individual in need thereof, the method comprising administering to an eye of the individual an effective amount of a pharmaceutical composition described herein.

[0024] In some embodiments, the pharmaceutical composition is delivered to an ocular secretory gland of an individual. In some embodiments, the pharmaceutical composition is delivered to a lacrimal gland. In some embodiments, the cells in the lacrimal gland are acinar cells. In some embodiments, the cells in the lacrimal gland are ductile epithelial cells. In some embodiments, the cells in the lacrimal gland are transduced by a rAAV vector. In some embodiments, the transduced cells in the lacrimal gland express an effective amount of DAO1 into the tear film and optionally onto the ocular surface of the individual. In some embodiments, the pharmaceutical composition is delivered to the accessory lacrimal gland (e.g., to Krause's glands, Wolfring's glands and / or Popov's gland).

[0025] In some embodiments, about 1×10 of the rAAV vector is administered. 9 About 1×10 10 , about 1×10 10 About 1×10 11 , about 1×10 11 About 1×10 12 , about 1×10 12 About 1×10 13 or about 1×10 13 About 1×10 15 genome copies.

[0026] In some embodiments, the condition is an eye condition. In some embodiments, the condition or eye condition is associated with increased histamine production and / or increased histamine signaling. In some embodiments, the condition or eye condition is an inflammatory condition. In some embodiments, the condition or eye condition is an autoimmune condition. In some embodiments, the condition or eye condition is an allergic condition. In some embodiments, the condition or eye condition is an allergic reaction to a therapeutic agent. In some embodiments, the condition or eye condition is an allergic reaction to microbial infection. In some embodiments, the condition or eye condition is vernal keratoconjunctivitis. In some embodiments, the condition or eye condition is atopic keratoconjunctivitis. In some embodiments, the condition or eye condition is seasonal or perennial allergic conjunctivitis.

[0027] In some embodiments, administration causes expression of DAO1 in cells of the lacrimal gland and / or accessory lacrimal gland. In some embodiments, administration causes secretion of DAO1 into the tear film. In some embodiments, secretion of DAO1 into the tear film is stimulated by administration of electrical stimulation, mechanical stimulation, ultrasound stimulation, and / or a drug. An example of electrical stimulation is an intranasal stimulator, such as Intranasal tear nerve stimulator. An example of mechanical stimulation is provided by e.g. 100. An example of ultrasonic stimulation is the use of a neuromodulatory device, such as the iTear system (Olympic Ophthalmics) developed for the treatment of dry eye disease. In some embodiments, the drug that stimulates the secretion of DAO1 into the tear film is a cholinergic agonist (e.g., pilocarpine or cevimeline). In some embodiments, the drug is a nicotinic acetylcholine receptor (nAChR) agonist (e.g., varenicline). In some embodiments, the drug is a secretagogue or a mucosal protectant (e.g., diquafosol, rebamipide, or ecabet). In some embodiments, a drug that stimulates the secretion of DAO1 into the tear film is administered to the eye. In some embodiments, a drug, such as a cholinergic agonist, is administered orally. In some embodiments, the secretion of DAO1 into the tear film is stimulated by a drug (e.g., a cholinergic agonist or a nAChR agonist) administered to the nasal cavity. In some aspects, DAO1 or rAAV vectors, plasmids, or any other constructs configured to express DAO1 described herein can be administered to an individual as a combination therapy in combination with tear-increasing stimulants and / or drugs, such as those described in this paragraph. In some aspects, such a combination therapy can follow any of the protocols described in International Patent Application Publication WO 2022 / 235786, or use any components or parameters, the entire contents of which are incorporated herein.

[0028] In some embodiments, administration causes improvement of one or more symptoms of ocular conditions. In some embodiments, symptoms are selected from the group consisting of itching, swelling, tearing and redness. In some embodiments, administration causes improvement of 0.5 points, 1 point, 1.5 points, 2 points, 2.5 points, 3 points, 3.5 points or 4 points on the conjunctival itching grading scale. In some embodiments, administration causes improvement of 1 point, 2 points, 3 points or 4 points on the conjunctival redness assessment grading scale. In some embodiments, administration causes improvement of 1 point, 2 points, 3 points or 4 points on the eye tearing scoring grading scale. In some embodiments, administration causes improvement of about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about 50%, about 50% to about 60%, about 60% to about 70%, about 70% to about 80%, about 80% to about 90% or more than about 95% of at least one area on the corneal staining grading scale.

[0029] In some embodiments, the improvement is measured at about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 9 months, or about 12 months after administration. In some embodiments, the improvement persists for at least 3 months, at least 6 months, at least 9 months, at least 12 months, at least 15 months, at least 18 months, at least 24 months, at least 3 years, at least 4 years, or at least 5 years after administration.

[0030] In some embodiments, the method further comprises administering 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 is an agent that increases tear production that is a cholinergic agonist. In some embodiments, the individual is a human.

[0031] In some embodiments, the present disclosure provides DAO1, rAAV or other vector configured to express DAO1, a composition, or a pharmaceutical composition described herein for use in a method of treating a condition in an individual in need thereof, the method comprising administering an effective amount of the pharmaceutical composition to an eye of the individual.

[0032] In some embodiments, the present disclosure provides DAO1, rAAV or other vectors configured to express DAO1, compositions, or pharmaceutical compositions described herein for use in the manufacture of a medicament for treating a condition in an individual in need thereof.

[0033] In some embodiments, the disclosure provides a kit comprising DAO1, rAAV or other vector configured to express DAO1 or a composition described herein, and a pharmaceutically acceptable carrier, and instructions for treating a condition in a subject. In some embodiments, the instructions comprise administering the pharmaceutical composition to the eye of the subject.

[0034] In some embodiments, the present disclosure provides a kit comprising DAO1, rAAV or other vector configured to express DAO1 or a composition described herein, and a pharmaceutically acceptable carrier, and instructions for treating a condition associated with increased histamine production and / or histamine signaling in an individual. In some embodiments, the instructions comprise administering the pharmaceutical composition to the eye of the individual.

[0035] In some embodiments, the disclosure provides a kit comprising DAO1, rAAV or other vector configured to express DAO1 or a composition described herein, and a pharmaceutically acceptable carrier, and instructions for treating an autoimmune condition in a subject. In some embodiments, the instructions comprise administering the pharmaceutical composition to the eye of the subject.

[0036] In some embodiments, the disclosure provides a kit comprising DAO1, rAAV or other vector configured to express DAO1 or a composition described herein, and a pharmaceutically acceptable carrier, and instructions for treating an allergic condition in a subject. In some embodiments, the instructions comprise administering the pharmaceutical composition to the eye of the subject. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1A A vector map of the rAAV expression cassette with inverted terminal repeats ("ITRs"), promoter, and DAO1 polynucleotide elements is shown.

[0038] Figure 1B A schematic diagram is provided depicting the elements located between the ITRs of the rAAV expression cassette, including from 5' to 3': the cytomegalovirus ("CMV") enhancer / promoter, the N-terminal secretion signal, the nucleotide sequence encoding DAO1, the woodchuck hepatitis virus post-translational regulatory element ("WPRE"), and the polyadenylation ("pA") signal.

[0039] Figure 2A and 2B An example of delivery of a viral vector to the lacrimal gland of a human subject is shown.

[0040] Figure 3 Shown is a graphic representation of the corneal staining grading scale, which is a grading diagram of the corneal surface divisions based on the NEI / Industry Workshop scale.

[0041] Figure 4 A provides images obtained by confocal fluorescence microscopy at 20× magnification (top row) or 63× magnification (middle row) of 293T cells transfected with AAV plasmid encoding DAO1 compared to untransfected control 293T cells at 20× magnification (bottom row). The leftmost column shows the detection of the fluorescence signal of the secondary antibody labeled with AF488 for detection of the primary anti-DAO1 antibody, the middle column shows the fluorescence signal of nuclear staining using DAPI, and the rightmost column shows the overlap of the AF488 and DAPI fluorescence signals.

[0042] Figure 4 B provides images obtained by confocal fluorescence microscopy at 20× magnification (middle row) or 63× magnification (bottom row) of 293T cells transfected with AAV plasmid encoding DAO1 compared to untransfected control 293T cells at 20× magnification (top row). The rightmost column shows the fluorescence signal of nuclear staining using DAPI, the middle column shows the fluorescence signal of the secondary antibody labeled with AF488 used to detect the primary anti-DAO1 antibody, and the rightmost column shows the overlap of the AF488 and DAPI fluorescence signals.

[0043] Figure 4 C provides images obtained by confocal fluorescence microscopy at 20× magnification (bottom row) of PANC-1 cells transfected with an AAV plasmid encoding DAO1 compared to untransfected control cells at 40× magnification and labeled with secondary antibodies (top row) or unlabeled at 20× magnification (middle row). Figure 4 As described in B.

[0044] Figure 4 D provides images obtained by confocal fluorescence microscopy at 40× magnification (top row) of PANC-1 cells transfected with an AAV plasmid encoding DAO1 compared to untransfected control cells at 20× magnification (bottom row). Figure 4 As described in B.

[0045] Figure 5A Shown are images of Western blots of whole cell lysates collected from 293T cells 48 hours after transient transfection with AAV.DAO1 (lane labeled "AAV-DAO1") compared to control untransfected 293T cells (lane labeled "293T"), using rabbit anti-DAO1 antibody to detect DAO1. Arrows indicate protein bands corresponding to DAO1 polypeptides.

[0046] Figure 5B An image of a Western blot of recombinant human DAO1 polypeptide is shown, where DAO1 was detected using a mouse anti-his antibody.

[0047] Figures 5C-5D show images of Western blots for detection of DAO1 from whole cell lysates collected from 293T cells 24 hours after transient transfection with AAV.DAO1 (lanes labeled "AAV-DAO") compared to control untransfected 293T cells (lanes labeled "293T") and recombinant human DAO1 polypeptide (lanes labeled "recombinant DAO"). Arrows indicate protein bands corresponding to DAO1 polypeptide (Figure 5C). GAPDH Western blot (15 μg / well) was used as an internal reference (Figure 5D).

[0048] Figure 5E shows images of a Western blot for detection of DAO1 in conditioned medium obtained from 293T cells transiently transfected with AAV.DAO1 ("AAV.DAO") or not transfected ("293T") 2, 4 and 6 hours after fresh medium replacement. Arrows indicate protein bands corresponding to DAO1 polypeptides.

[0049] Figure 6A-6BShown are data generated using a colorimetric functional activity assay to measure DAO1 substrate cleavage.DAO1 cleaves the first substrate to produce H2O2 (hydrogen peroxide), which reacts with the second substrate to produce a signal detected using fluorescence spectroscopy (excitation wavelength of 535 nm and emission wavelength of 587 nm). Fig. 6A Shown is a calibration curve generated by measuring the fluorescence signal of the second substrate as a function of the concentration of H2O2. Figure 6B Shown are graphs quantifying H2O2 produced by the activity of DAO1 present in the culture medium or cell lysates of 293T cells transiently transfected with an AAV plasmid encoding DAO1, compared to the activity in the culture medium of untransfected control cells.

[0050] Figure 6C provides a graph quantifying H2O2 produced by the activity of DAO1 present in combined conditioned media and whole cell extracts obtained from 293T cells (transiently transfected with an AAV plasmid encoding DAO1) or untransfected control cells at 30, 60 and 90 minutes. Recombinant DAO1 was used as a positive control.

[0051] Figure 7 A schematic diagram depicting the elements between the ITRs of the AAV plasmid is shown. The plasmid encodes EGFP ("secEGFP") linked to its secretion signal at its N-terminus under the control of the CMV promoter. The woodchuck hepatitis virus post-translational regulatory element (WPRE) is used to increase transgene expression and is close to the bovine growth hormone polyadenylation (pA) signal.

[0052] Figures 8A-8K is an image of lacrimal gland tissue stained with an anti-eGFP antibody. The lacrimal gland was administered with an rAAV vector containing an expression cassette with an eGFP transgene by intra-lacrimal injection. The lacrimal gland tissue was stained with an anti-eGFP antibody to assess eGFP expression. The black arrow indicates staining showing eGFP expression.

[0053] Fig. 9 Images of porcine lacrimal glands injected with AAV-secEGFP (AAV2 or AAV9 serotypes) and collected and fixed in paraffin at day 103 are provided. IHC was performed on 5 μM sections using anti-GFP antibody and DAPI (nuclear) contrast stain. Images were captured using a confocal microscope at 100× magnification. Negative control animals did not receive injections.

[0054] Fig.10 Images of porcine lacrimal glands injected with AAV9-secEGFP and collected and fixed in paraffin at day 103 are provided. 5 μM sections were subjected to IHC using anti-GFP antibody and DAPI (nuclear) counter stain. In addition to lacrimal gland acinar cells, ductile epithelial cells appear to be transduced by AAV9 (white arrows).

[0055] Fig.11 Images are provided of porcine lacrimal glands that received an AAV injection and were collected and fixed in paraffin on day 103. H&E staining of 5 μM paraffin sections at 100× showed no inflammatory infiltrates, macroscopic or microscopic abnormalities.

[0056] Fig.12 A schematic diagram of the treatment regimen for administering AAV encoding a model protein via injection into the lacrimal glands of pigs in combination with OC-01 (varenicline) administered by nasal spray is provided. Time points for tear collection and study termination are indicated.

[0057] Fig.13 The calibration curve generated by measuring the fluorescence signal of the second substrate as a function of the concentration of histamine is shown. The calibration curve was used to evaluate the histamine degradation kinetics of recombinant porcine diamine oxidase ( Figure 14-15 ) and histamine degradation in conditioned medium ( Figure 16-17 ), the conditioned medium was obtained from untransfected control 293T cells and 293T cells transfected with a plasmid engineered to express DAO1.

[0058] Figure 14-15 Provided is a graph showing that recombinant porcine diamine oxidase Fig.14 ) and 24 hours ( Fig.15 ) is a graph showing the histamine degradation kinetics when .

[0059] Figure 16-17 Conditioned media obtained from untransfected control 293T cells and 293T cells transfected with a plasmid engineered to express DAO1 are provided showing that the expression of DAO1 in the 4 h Fig.16 ) and 24 hours ( Fig.17 ) diagram of histamine degradation. DETAILED DESCRIPTION

[0060] The present disclosure provides a method for treating an ocular condition in an individual in need thereof. Such a method comprises, for example, the expression and subsequent secretion of diamine oxidase (DAO1) from the lacrimal gland, which can then be transferred to other components of the tear film and combined with other components of the tear film onto the surface of the eye. In healthy individuals, the main function of DAO1 is to decompose excess histamine in the body. DAO1 is almost absent from human blood circulation, although it has been shown to be significantly increased in pregnant women, which indicates a protective mechanism against bad histamine. It has also been shown that in individuals with histamine intolerance and many ocular conjunctivitis, there is an imbalance between accumulated histamine and histamine degradation. After various noxious stimuli, histamine can be released excessively from basophils and mast cells, thereby producing tissue edema and starting a chain of events that lead to inflammation and allergic reactions. Elevated histamine levels in the tear film are a sign of perennial and / or seasonal rhinoconjunctivitis, atopic keratoconjunctivitis, giant papillary conjunctivitis, and spring or allergic keratoconjunctivitis. Histamine induces itching, redness, and tearing by activating H1 receptors on blood vessels and nociceptive nerves. In severe cases, patients may experience vision impairment due to corneal damage, cataracts, or glaucoma.

[0061] As disclosed herein, AAV-based delivery of transgenes to the eye provides a method for treating ocular conditions. rAAV vectors have unique advantages for transgene delivery. Transgenes delivered by rAAV vectors may be incorporated into the genome of transduced cells, allowing potential long-term expression of transgenic products. In addition, compared with other viral delivery vectors (such as adenovirus), rAAV vectors may have lower immunogenicity. AAV-based delivery vectors for transducing cells in the posterior segment of the eye using subretinal and intravitreal injections are described, and in vivo efficacy is shown (U.S. Patent No. 10,308,957; Petrs-Silva et al., Mol Ther. 19: 293-301 (2011); Rodriques et al., Pharm Res. 36: 29 (2019)).

[0062] AAV serotypes used for AAV-based delivery of transgenes to the eye include AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, and AAV9 for delivering transgenes (Lebherz et al. J Gene Med.; 10(4):375-382 (2008)). AAV serotypes used for AAV-based delivery of transgenes to the lacrimal gland include AAV2, AAV4, AAV5, AAV5w8, AAV x5, AAV 9, AAV 12, and BAAV (Rocha et al. Invest Ophthalmol Vis Sci. 52:9567-9572 (2011)).

[0063] In some embodiments, rAAV vectors are provided for expressing DAO1. In some embodiments, cells of at least one eye and / or lacrimal gland are transduced with the rAAV vectors disclosed herein. The lacrimal gland is the main source of tears and is responsible for promoting a healthy ocular surface and maintaining normal visual function. The main lacrimal gland contains the eyelid and orbital lobes, which are continuous with each other at the lateral edge of the levator palpebrae superioris aponeurosis. The lacrimal lobule has many alveoli and intralobular ducts, which form excretory ducts leading to the conjunctival fornix. The main lacrimal gland contains acinar cells, duct cells and / or myoepithelial cells (Obata Cornea.; 25 (10 Supplement 1): S82-9 (2006)). The main lacrimal gland secretes the aqueous layer of the tear film and mucin onto the ocular surface of the individual eye (see, for example, Paulsen, F. et al., (2004) Cell and tissue research, 316 (2), 167-177). As used herein, the term "tear gland" refers to the main tear gland of an individual, as well as the Wolfflin gland and the Kraus gland. The accessory glands known as the Wolfflin gland and the Kraus gland are located in the eyelids. There are about 2 to 5 Wolfflin glands and about 40 Kraus glands in the upper eyelid. There are about 6 to 8 Kraus glands in the lower eyelid. The specific location and anatomical structure of the lacrimal functional unit are well known (Conrady et al., Journal of Ophthalmology (JOphthalmol.) Article No. 7542929 (2016)).

[0064] Expression cassette

[0065] The rAAV vector of the present disclosure comprises an expression cassette. As used herein, the term "expression cassette" refers to a polynucleotide comprising at least one polynucleotide sequence encoding a protein of interest (e.g., DAO1) flanked by two inverted terminal repeat sequences. In some embodiments, the expression cassette further comprises other polynucleotide sequences, such as a promoter, a regulatory element (e.g., one or more promoters), a translation initiation sequence, a coding sequence, and a termination sequence ( Figure 1A and 1B ).

[0066] In mammals, histamine is metabolized by oxidative deamination via DAO1 (also known as histaminase, amiloride-binding protein, copper-containing amine oxidase 1, AOC1) (see, e.g., Elmore et al., (2002) J Biol Inorg Chem 7:565; Schwelberger et al., (2018) Inflamm Res 67:245; Finney et al. (2014) Arch Biochem Biophys 546:19). The enzyme is a homodimer that catalyzes the oxidative deamination of the primary amine group of histamine.

[0067] Histamine intolerance is excessive levels of histamine that accumulate due to reduced catabolism and / or increased production. Vakal et al., Molecules 2020, 25, 1293. Histamine is released during allergic reactions and mast cell degranulation, and mast cell degranulation has been shown to increase DAO1 release, and DAO1 is increased in severe allergic reactions in patients with mastocytosis (Boehm et al., Allergy 2019, 74: 58). In addition, several therapeutic agents have been shown to inhibit DAO1, including, for example, berenil, pentamidine, aminoguanidine, metformin (Vakal 2020).

[0068] Excess histamine causes a range of symptoms (see, e.g., Schnedl et al., Food Sci Biotechnol (2019) 28(6): 1779-1784). Histamine is a proinflammatory mediator that contributes to allergic reactions (see, e.g., White MV (1990), J Allergy Clin Immunol 86: 599-605) and can contribute to vernal keratoconjunctivitis (VKC) (see, e.g., Abelson et al., Opthamology 1995, 102(12): 1958-1963; Bonini et al., (1992), J Allergy Clin Immunol, 89(1), 103-107).

[0069] Thus, the present disclosure provides expression cassettes comprising a polynucleotide sequence encoding DAO1 for use in therapies that induce the breakdown of histamine (e.g., histamine present in tears). In some embodiments, the expression cassettes are used in methods of treating conditions associated with increased histamine production (e.g., ocular conditions).

[0070] DAO1 enzyme

[0071] In some embodiments, the expression cassette of the present disclosure comprises a polynucleotide sequence encoding a DAO1 enzyme or a functional variant thereof. As used herein, the term "DAO1 enzyme" refers to a DAO1 enzyme from any species. The term "functional variant" refers to a variant having a sequence substitution, insertion, deletion, and / or N-terminal or C-terminal truncation, wherein the functional variant retains one or more functions of a reference protein (e.g., a native DAO1 enzyme). DAO1 is a 200-kDa homopolyglycoprotein that catalyzes the oxidative deamination of the primary amino group of histamine.

[0072] In some embodiments, the DAO1 enzyme is a human DAO1 enzyme. In some embodiments, the human DAO1 enzyme is identified in a public database. It is known in the art that human DAO1 isoforms are generally identified by public databases. For example, the National Center for Biotechnology Information (NCBI) gene database of the National Library of Medicine of the United States (accessible via the World Wide Web: ncbi.nlm.nih.gov / ) is a searchable gene database that provides nomenclature, chromosome location, gene product, gene attributes, related markers, phenotypes, interactions, reference links, sequence information, information about sequence variants, gene maps, expression reports, homologs, protein domain content, and access to external databases. As understood by those skilled in the art, sequence information of human DAO1 isoforms known in the art can be identified by entering the appropriate accession number into the NCBI gene database and selecting the sequence information of the desired computer readable format (e.g., FASTA). Such sequence information may include, but is not limited to, the nucleotide sequence of the full-length gene encoding DAO, the nucleotide sequence of the pre-mRNA transcript encoding DAO, the nucleotide sequence of the mRNA encoding DAO, the nucleotide sequence of the open reading frame (ORF) encoding DAO, and the amino acid sequence of DAO. For example, one isoform of human DAO1 can be identified in the NCBI gene database by accession number NM_001272072.2 and has the amino acid sequence shown in SEQ ID NO: 2. In another example, one isoform of human DAO1 can be identified in the NCBI gene database by accession number NM_001091 and consists of the amino acid sequence shown in SEQ ID NO: 2.

[0073] Exemplary amino acid sequences of human DAO1 and exemplary polynucleotide sequences (mRNA and ORF) encoding human DAO1 are listed in Table 1. In some embodiments, the DAO1 enzyme comprises or consists of the amino acid sequence shown in Table 1. In some embodiments, the DAO1 enzyme comprises or consists of the amino acid sequence encoded by the mRNA sequence shown in Table 1. In some embodiments, the DAO1 enzyme comprises or consists of the amino acid sequence encoded by the ORF shown in Table 1.

[0074] In some embodiments, the DAO1 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 DAO1 enzyme comprises or consists of SEQ ID NO: 1. In some embodiments, the DAO1 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: 2. In some embodiments, the DAO1 enzyme comprises or consists of SEQ ID NO: 2.

[0075] In some embodiments, the DAO1 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 one or more segments of SEQ ID NO: 1, including but not limited to a segment spanning positions 39-125, 141-241, and / or 300-727 of SEQ ID NO: 1. In some embodiments, the DAO1 enzyme is encoded by an mRNA comprising or consisting of a nucleotide sequence encoding one or more segments of SEQ ID NO: 1 (e.g., a segment spanning positions 39-125, 141-241, and / or 300-727 of SEQ ID NO: 1). In some embodiments, the DAO1 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 one or more segments of SEQ ID NO: 2, including but not limited to a segment spanning positions 39-125, 141-241, and / or 300-708 of SEQ ID NO: 2. In some embodiments, the DAO1 enzyme is encoded by an mRNA comprising or consisting of a nucleotide sequence encoding one or more segments of SEQ ID NO: 2 (e.g., a segment spanning positions 39-125, 141-241, and / or 300-708 of SEQ ID NO: 2).

[0076] In some embodiments, the DAO1 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 one or more segments of SEQ ID NO: 1 or SEQ ID NO: 2, wherein each segment 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 contiguous amino acids of SEQ ID NO: 1 or SEQ ID NO: 2, including segments of length within the range defined by any pair of the aforesaid values. For example, a functional DAO1 enzyme may share high sequence identity (e.g., 95%) with one or more predicted functional domains (e.g., positions 39-125, 141-241, and / or 300-727 of SEQ ID NO: 1; or positions 39-125, 141-241, and / or 300-708 of SEQ ID NO: 2), while having greater sequence divergence in other portions of the DAO1 enzyme. Similarly, in some embodiments, a functional DAO1 enzyme may have 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% sequence identity with a segment spanning position 20 to the end of SEQ ID NO: 1 or SEQ ID NO: 2 (e.g., omitting the predicted N-terminal signal peptide common to both sequences).

[0077] In some embodiments, the DAO1 enzyme is encoded by an mRNA comprising or consisting of a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO:3. In some embodiments, the DAO1 enzyme is encoded by an mRNA comprising or consisting of a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO:4. In some embodiments, the DAO1 enzyme is encoded by an mRNA comprising or consisting of SEQ ID NO:4.

[0078] In some embodiments, the DAO1 enzyme is encoded by an ORF comprising or consisting of a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 29. In some embodiments, the DAO1 enzyme is encoded by an ORF comprising or consisting of SEQ ID NO: 29. In some embodiments, the DAO1 enzyme is encoded by an ORF comprising or consisting of a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 30. In some embodiments, the DAO1 enzyme is encoded by an ORF comprising or consisting of SEQ ID NO: 30. In some embodiments, the DAO1 enzyme is encoded by an ORF comprising or consisting of a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 31. In some embodiments, the DAO1 enzyme is encoded by an ORF comprising or consisting of SEQ ID NO: 31.

[0079] Polynucleotide encoding DAO1 enzyme

[0080] In some embodiments, the expression cassette comprises a polynucleotide encoding a DAO1 enzyme described herein (eg, a human DAO1 enzyme). In some embodiments, the polynucleotide comprises an ORF encoding a DAO1 enzyme described herein (eg, a human DAO1 enzyme).

[0081] In some embodiments, the expression cassette provides increased DAO1 expression in at least one eye and / or lacrimal gland. In some embodiments, the expression of DAO1 may be increased by 5%, 10%, 15%, 20%, or 25% compared to the expression of DAO1 in an untreated individual or in the contralateral eye of a treated individual. As used herein, "individual" refers to any mammal, including mice, rabbits, non-human primates (NHPs), and humans. In some embodiments, the individual is a human or NHP. In addition, "individual" or "patient" may be used interchangeably with "subject". In some embodiments, the expression of DAO1 may be increased by at least 1.5 times, 2 times, 3 times, 4 times, or 5 times compared to the expression of DAO1 in an untreated individual or in the contralateral eye of a treated individual. In some embodiments, DAO1 may be expressed at any detectable level in the treated eye, while DAO1 may not be expressed or expressed at an undetectable level in an untreated individual or in the contralateral eye of a treated individual. In other words, an eye or lacrimal gland administered with a rAAV vector described herein can have a higher abundance of DAO1 expression than an eye or lacrimal gland having only endogenous (ie, native) DAO1 expression or an eye having low or impaired endogenous (ie, native) DAO1 secretion.

[0082] The term "functional variant" refers to a variant having sequence substitutions, insertions, deletions and / or N-terminal or C-terminal truncations, wherein the functional variant retains one or more functions of a reference protein (eg, native DAO1).

[0083] In some embodiments, the expression cassette comprises a polynucleotide encoding a protein, the protein being human DAO1 or a functional variant thereof. In some embodiments, the expression cassette comprises a polynucleotide comprising a nucleotide sequence encoding an amino acid sequence identified in Table 1. In some embodiments, the expression cassette comprises a polynucleotide comprising a nucleotide sequence identified in Table 1.

[0084] In some embodiments, the expression cassette comprises a polynucleotide comprising a nucleotide sequence encoding a protein having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence shown in SEQ ID NO: 1. In some embodiments, the expression cassette comprises a polynucleotide comprising a nucleotide sequence encoding the amino acid sequence shown 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: 2. In some embodiments, the expression cassette comprises a polynucleotide comprising a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 2.

[0085] In some embodiments, the expression cassette comprises a polynucleotide comprising a nucleotide sequence encoding a DAO1 enzyme, wherein the DAO1 enzyme comprises or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 1. In some embodiments, the DAO1 enzyme comprises or consists of SEQ ID NO: 1. In some embodiments, the expression cassette comprises a polynucleotide comprising a nucleotide sequence encoding a DAO1 enzyme, wherein the DAO1 enzyme comprises or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 2. In some embodiments, the DAO1 enzyme comprises or consists of SEQ ID NO: 2.

[0086] In some embodiments, the expression cassette comprises a polynucleotide encoding DAO1 comprising a nucleotide sequence that shares 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 with SEQ ID NO:30. In some embodiments, the expression cassette comprises a polynucleotide encoding DAO1 comprising or consisting of the nucleotide sequence shown in SEQ ID NO:30. In some embodiments, the expression cassette comprises a polynucleotide encoding DAO1 comprising or consisting of a nucleotide sequence that shares 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 with SEQ ID NO:31. In some embodiments, the expression cassette comprises a polynucleotide comprising or consisting of the nucleotide sequence shown in SEQ ID NO:31. In some embodiments, the protein is human DAO1 or a functional variant thereof.

[0087] Codon optimization

[0088] In some embodiments, the expression cassette comprises a polynucleotide encoding a DAO1 enzyme, wherein the polynucleotide comprises or consists of a nucleotide sequence that is codon-optimized for expression in a target cell. In some embodiments, the target cell is a mammalian cell. In some embodiments, the target cell is a human cell, a murine cell, or a non-human primate (NHP) cell.

[0089] A codon-optimized nucleotide sequence, such as a codon-optimized nucleotide sequence encoding a DAO1 enzyme, is typically a sequence comprising at least one synonymous nucleobase substitution relative to a reference sequence (e.g., a wild-type ORF encoding a DAO1 enzyme). A codon-optimized nucleotide sequence may be partially or completely different in sequence from a reference sequence. For example, a reference sequence encoding a polyserine uniformly encoded by a TCT codon may be sequence optimized by replacing 100% of its nucleobases (for each codon, T in position 1 is replaced by A, C in position 2 is replaced by G, and T in position 3 is replaced by C) to generate a sequence encoding a polyserine uniformly encoded by an AGC codon. The sequence identity percentage obtained from the global pairwise alignment between the reference polyserine nucleic acid sequence and the sequence-optimized polyserine nucleic acid sequence is 0%. However, the protein products from the two sequences will be 100% consistent.

[0090] Codon optimization methods are known in the art and can be used to achieve one or more desired results, such as increasing expression of a synthetic gene in a target cell. In some embodiments, the expression cassette comprises a nucleotide sequence that has been sequence optimized relative to a reference sequence using a sequence optimization method. Methods for sequence optimization are known in the art and include known sequence optimization tools, algorithms, and services. Non-limiting examples include those from GeneArt (Life Technologies), DNA2.0 (Menlo Park CA), and (Atum, Newark, CA) services.

[0091] In some embodiments, the expression cassette of the present disclosure comprises a polynucleotide encoding a DAO1 enzyme, which is generated using sequence optimization methods (e.g. For example ) is performed relative to a reference sequence. In some embodiments, methods for sequence optimization include US 7,561,972; US 7,561,973; US 8,126,653; and US 8,401,798, each of which is incorporated herein by reference. In some embodiments, sequence optimization methods known in the art (e.g., For example ), the polynucleotide sequence is sequence optimized based on the codon usage bias of the host cell (e.g., a mammalian cell, such as a human cell, a murine cell, a non-human primate cell) relative to the reference sequence. In some embodiments, the polynucleotide sequence comprises or consists of a nucleotide sequence codon-optimized relative to a reference sequence using a sequence optimization method, wherein the reference sequence is selected from SEQ ID NOs: 30 and 31. In some embodiments, the polynucleotide sequence comprises or consists of a nucleotide sequence codon-optimized relative to a reference sequence for expression in a human host cell, wherein the reference sequence is selected from SEQ ID NOs: 30 and 31. In some embodiments, the polynucleotide sequence comprises or consists of SEQ ID NO: 29.

[0092] In some embodiments, the disclosure provides an expression cassette comprising a polynucleotide comprising or consisting of a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 29. In some embodiments, the polynucleotide comprises or consists of SEQ ID NO: 29.

[0093] Table 1: Human DAO1 amino acid and polynucleotide sequences

[0094]

[0095] Expression cassette elements

[0096] In some embodiments, the expression cassette of the present disclosure comprises a promoter. As used herein, "promoter" refers to a DNA sequence that guides the binding of RNA polymerase and thereby promotes RNA synthesis, i.e., the minimum sequence sufficient to guide transcription. Promoters and corresponding protein or polypeptide expression can be ubiquitous, meaning strong activity or cell type specificity, tissue specificity or species specificity in a wide range of cells, tissues and species. The promoter can be "constitutive", meaning continuous activity, or "inducible", meaning that the promoter can be activated or deactivated by the presence or absence of biological or non-biological factors. The nucleic acid construct or vector of the present invention also includes an enhancer sequence, which may or may not be adjacent to the promoter sequence. The enhancer sequence affects promoter-dependent gene expression and can be located in the 5' or 3' region of the natural gene.

[0097] Any suitable promoter region or promoter sequence therein can be used in an individual polynucleotide cassette, so long as the promoter region promotes expression of a polynucleotide sequence encoding a DAO1 enzyme in at least one eye and / or lacrimal gland. In some embodiments, the promoter promotes expression of a gene in a mammalian eye and / or lacrimal gland. In some embodiments, the expression cassette comprises a cell type specific promoter. The promoter can specifically promote transcription in ocular cells and / or lacrimal gland cells.

[0098] In some embodiments, the promoter is a CAG promoter. In some embodiments, the promoter comprises a nucleotide sequence having at least 90%, 95%, 96%, 97%, 98% or 99% identity to the nucleotide sequence shown in SEQ ID NO: 5. In some embodiments, the promoter comprises SEQ ID NO: 5.

[0099] In some embodiments, the promoter is a CMV promoter. In some embodiments, the promoter comprises a nucleotide sequence having at least 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 21. In some embodiments, the promoter comprises or consists of SEQ ID NO: 21.

[0100] The expression cassette may contain a polyadenylation (polyA) sequence. In some embodiments, the expression cassette described herein comprises a transcription termination signal. Elements that guide efficient termination and polyadenylation of heterologous nucleic acid transcripts will increase heterologous gene expression. Transcription termination signals are generally found downstream of the polyadenylation signal. In some embodiments, the vector comprises a polyadenylation sequence at the 3′ position of the polynucleotide encoding the polypeptide to be expressed. As used herein, the term “polyadenylic acid site (polyA site)” or “polyadenylic acid sequence (polyA sequence)” represents a DNA sequence that guides the termination and polyadenylation of nascent RNA transcripts caused by RNA polymerase II. The polyadenylation sequence can enhance mRNA stability by adding a polyA tail to the 3′ end of the coding sequence, and thus contributes to increased translation efficiency. Cleavage and polyadenylation are guided by the poly (A) sequence in the RNA. The core polyadenylic acid sequence of mammalian pre-mRNA has two recognition elements flanking the cleavage-polyadenylation site. Typically, the nearly invariant AAUAAA hexamer is located 20 to 50 nucleotides upstream of the more variable element rich in U or GU residues. Cleavage of the nascent transcript occurs between these two elements and is coupled to add up to 250 adenosines to the 5' cleavage product. In specific embodiments, the core poly (A) sequence is an ideal poly A sequence (e.g., AATAAA, ATTAAA, AGTAAA). In specific embodiments, the poly (A) sequence is an SV40 poly A sequence, a bovine growth hormone poly A sequence (BGH poly A), a rabbit β-globulin poly A sequence (rβgpA), a variant thereof, or another suitable heterologous or endogenous poly A sequence known in the art. In some embodiments, the expression cassette described herein comprises a poly A sequence. In some embodiments, the poly A sequence is a BGH poly A sequence. In some embodiments, the BGH poly A sequence comprises a nucleotide sequence or a composition thereof having at least 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 18. In some embodiments, the BGH poly A sequence comprises or consists of SEQ ID NO: 18.

[0101] Optionally, 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:22.

[0102] Recombinant AAV vector

[0103] In some embodiments of the present disclosure, the expression cassette of the present disclosure is used to deliver DAO1 or a functional variant thereof to at least one eye and / or lacrimal gland of an individual, for example, to treat an eye disorder. Therefore, in some embodiments of the present disclosure, the composition for providing expression of DAO1 or a functional variant thereof in at least one eye and / or lacrimal gland of an individual is a gene delivery vector, wherein the gene delivery vector comprises the expression cassette described herein.

[0104] In some embodiments, the gene delivery vector is an rAAV vector. In such embodiments, the expression cassette is flanked by functional AAV reverse terminal repeat (ITR) sequences at the 5' and 3' ends. "Functional AAV ITR sequence" means that the ITR sequence is used to rescue, replicate and package the AAV vector. Therefore, the AAV ITR for the gene delivery vector of the present disclosure does not need to have a wild-type nucleotide sequence and can be changed by insertion, deletion or substitution of nucleotides, or the AAV ITR can be derived from any AAV serotype in several AAV serotypes, such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10. In some embodiments, AAV ITR is derived from AAV1. In some embodiments, AAV ITR is derived from AAV2. In some embodiments, AAV ITR is derived from AAV3. In some embodiments, AAV ITR is derived from AAV4. In some embodiments, AAV ITR is derived from AAV5. In some embodiments, AAV ITR is derived from AAV6. In some embodiments, AAVITR is derived from AAV7. In some embodiments, the AAV ITR is derived from AAV9. In some embodiments, the AAV ITR is derived from AAV1. In some embodiments, the AAV ITR is derived from AAV10. Certain rAAV vectors have all or part of the wild-type REP gene and CAP gene deleted, but retain functional flanking ITR sequences. In some embodiments, the AAV ITR is an AAV ITR identified in Table 2.

[0105] Table 2. Exemplary ITR sequences

[0106]

[0107] In such embodiments, the rAAV vector comprises an AAV capsid derived from any adeno-associated virus serotype known or expected to be found in the art, including but not limited to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, etc. For example, the AAV capsid can be a wild-type (or "natural") capsid. In some embodiments, the rAAV vector comprises an AAV capsid derived from AAV1. 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 some embodiments, the rAAV vector comprises an AAV capsid derived from AAV5. In some embodiments, the rAAV vector comprises an AAV capsid derived from AAV6. In some embodiments, the rAAV vector comprises an AAV capsid derived from AAV7. In some embodiments, the rAAV vector comprises an AAV capsid derived from AAV8. In some embodiments, the rAAV vector comprises an AAV capsid derived from AAV9. In some embodiments, the rAAV vector comprises an AAV capsid derived from AAV10. AAV capsids of particular interest include AAV2, AAV5, AAV8, and AAV9. However, like ITR, the capsid does not need to have a wild-type nucleotide sequence, but as long as the capsid is capable of transducing cells of the eye and / or lacrimal gland, it can be changed by insertion, deletion, or substitution of nucleotides in the VP1, VP2, or VP3 sequence. In other words, the AAV capsid can be a variant AAV capsid. In some embodiments, the rAAV vector is a "pseudotyped" AAV produced by using a capsid (cap) gene of an AAV and rep genes and ITRs from different AAVs, such as pseudotyped AAV2 created by using rep from AAV2 and caps from AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, or AAV9 and 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.

[0108] In some embodiments, rAAV is replication-defective because the rAAV vector cannot independently replicate and package its genome. For example, when the eye and / or lacrimal gland are transduced with a rAAV vector, the gene is expressed in the transduced eye and / or lacrimal gland, however, due to the fact that the transduced eye and / or lacrimal gland lack the AAV rep gene and cap gene and auxiliary function genes, the rAAV cannot replicate.

[0109] The rAAV vector of the present disclosure encapsulating the expression cassette as described herein can be produced using non-assisted production. rAAV is a replication-deficient virus, and components from live helper viruses such as adenovirus are generally required in host cells to package infectious rAAV vectors. The rAAV non-assisted production system allows the production of infectious rAAV vectors without the use of live helper viruses. In the non-assisted system, the host packaging cell line is co-transfected with three plasmids. The first plasmid can contain adenoviral gene products (e.g., E2A, E4, and VA RNA genes) required for packaging rAAV vectors. The second plasmid contains the required AAV genes (i.e., REP and CAP genes). The third plasmid contains a polynucleotide sequence encoding DAO1 or a functional variant thereof and a promoter flanked by ITRs. The host packaging cell line can be, for example, an AAV-293 host cell. Suitable host cells contain additional components required for packaging infectious rAAV vectors that are not provided by plasmids. In some embodiments, the CAP gene can encode, for example, an AAV capsid protein as described herein. In some embodiments, the promoter is a promoter sequence as described herein. In some embodiments, the promoter sequence is a CAG sequence. In some embodiments, the encoded enzyme is DAO1.

[0110] AAV serotypes shown to infect the eye and / or lacrimal gland include AAV2, AAV5, AAV 5w8, and AAV9 (Rocha et al., supra). Exemplary amino acid and nucleotide sequences of AAV capsid proteins are identified in Table 3.

[0111] In some embodiments, the AAV serotype used to infect the eye and / or lacrimal gland is AAV2. In some embodiments, the AAV capsid protein comprises SEQ ID NO: 6. In some embodiments, the AAV capsid protein comprises a sequence that is at least 95%, 96%, 97%, 98%, 99% or 100% similar to SEQ ID NO: 6. In some embodiments, the AAV capsid protein shares at least 95%, 98% or 100% identity with the AAV2 VP1 protein (SEQ ID NO: 6).

[0112] In some embodiments, the polynucleotide sequence encoding the AAV2 VP1 protein comprises SEQ ID NO: 7. In some embodiments, the polynucleotide sequence encoding the AAV2 VP1 protein comprises a sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% similar to SEQ ID NO: 7. In some embodiments, the polynucleotide sequence encoding the AAV2 VP1 protein shares at least 95%, 98%, or 100% identity with SEQ ID NO: 7.

[0113] In some embodiments, the AAV capsid protein comprises SEQ ID NO: 8. In some embodiments, the AAV capsid protein comprises a sequence at least 95%, 96%, 97%, 98%, 99%, or 100% similar to SEQ ID NO: 8. In some embodiments, the AAV capsid protein shares at least 95%, 98%, or 100% identity with the AAV2 VP3 protein (SEQ ID NO: 8).

[0114] In some embodiments, the polynucleotide sequence encoding the AAV2 VP3 protein comprises SEQ ID NO: 9. In some embodiments, the polynucleotide sequence encoding the AAV capsid protein comprises a sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% similar to SEQ ID NO: 9. In some embodiments, the polynucleotide sequence encoding the AAV2 VP3 protein shares at least 95%, 98%, or 100% identity with SEQ ID NO: 9.

[0115] In some embodiments, the AAV serotype used to infect the eye and / or lacrimal gland is AAV5. In some embodiments, the AAV capsid protein comprises SEQ ID NO: 10. In some embodiments, the AAV capsid protein comprises a sequence that is at least 95%, 96%, 97%, 98%, 99% or 100% similar to SEQ ID NO: 10. In some embodiments, the AAV capsid protein shares at least 95%, 98% or 100% identity with the AAV5 capsid protein (SEQ ID NO: 10).

[0116] In some embodiments, the polynucleotide sequence encoding the AAV5 capsid protein comprises SEQ ID NO: 11. In some embodiments, the polynucleotide sequence encoding the AAV capsid protein comprises a sequence at least 95%, 96%, 97%, 98%, 99% or 100% similar to SEQ ID NO: 11. In some embodiments, the polynucleotide sequence encoding the AAV5 capsid protein shares at least 95%, 98% or 100% identity with SEQ ID NO: 11.

[0117] In some embodiments, the AAV serotype used to infect the eye and / or lacrimal gland is AAV8. In some embodiments, the capsid protein comprises SEQ ID NO: 12. In some embodiments, the AAV capsid protein comprises a sequence that is at least 95%, 96%, 97%, 98%, 99% or 100% similar to SEQ ID NO: 12. In some embodiments, the AAV capsid protein shares at least 95%, 98% or 100% identity with the AAV8 capsid protein (SEQ ID NO: 12).

[0118] In some embodiments, the polynucleotide encoding the AAV8 capsid protein comprises SEQ ID NO: 13. In some embodiments, the polynucleotide sequence encoding the AAV capsid protein comprises a sequence at least 95%, 96%, 97%, 98%, 99% or 100% similar to SEQ ID NO: 13. In some embodiments, the polynucleotide sequence encoding the AAV8 capsid protein shares at least 95%, 98% or 100% identity with SEQ ID NO: 13.

[0119] In some embodiments, the AAV serotype used to infect the eye and / or lacrimal gland is AAV9. In some embodiments, the AAV capsid protein comprises SEQ ID NO: 14. In some embodiments, the AAV capsid protein comprises a sequence that is at least 95%, 96%, 97%, 98%, 99% or 100% similar to SEQ ID NO: 14. In some embodiments, the AAV capsid protein shares at least 95%, 98% or 100% identity with the AAV9 capsid protein (SEQ ID NO: 14).

[0120] In some embodiments, the polynucleotide sequence encoding the AAV9 capsid protein comprises SEQ ID NO: 15. In some embodiments, the polynucleotide sequence encoding the AAV capsid protein comprises a sequence at least 95%, 96%, 97%, 98%, 99% or 100% similar to SEQ ID NO: 15. In some embodiments, the polynucleotide sequence encoding the AAV9 capsid protein shares at least 95%, 98% or 100% identity with SEQ ID NO: 15.

[0121] Table 3. AAV capsid sequences

[0122] protein Nucleotide (open reading frame) AAV2 VP1 SEQ ID NO:6 SEQ ID NO:7 AAV2 VP3 SEQ ID NO:8 SEQ ID NO:9 AAV5 SEQ ID NO:10 SEQ ID NO:11 AAV8 SEQ ID NO:12 SEQ ID NO:13 AAV9 SEQ ID NO:14 SEQ ID NO:15

[0123] Exemplary rAAV

[0124] In some embodiments, rAAV comprises an AAV capsid. In some embodiments, the rAAV described herein comprises an expression cassette comprising a polynucleotide comprising a nucleotide sequence encoding DAO1. In some embodiments, the polynucleotide is operably linked to a promoter. In some embodiments, the rAAV comprises an AAV capsid and an expression cassette, wherein the expression cassette comprises a polynucleotide operably linked to a promoter, wherein the polynucleotide comprises a nucleotide sequence encoding DAO1. In some embodiments, the promoter is any promoter described herein or known in the art. In some embodiments, the promoter is a CAG promoter. In some embodiments, the promoter is a CMV promoter. In some embodiments, the expression cassette further comprises a 5′ITR and / or a 3′ITR. In some embodiments, the 5′ITR is an AAV2 5′ITR. In some embodiments, the 3′ITR is an AAV2 3′ITR. In some embodiments, the expression cassette comprises a nucleotide sequence comprising, from 5′ to 3′: a 5′ ITR (e.g., AAV2 5′ ITR), a promoter (e.g., a CMV promoter), a 5′ untranslated region (5′ UTR), a polynucleotide sequence comprising a nucleotide sequence encoding a DAO1 enzyme, a polyA sequence, and a 3′ ITR (e.g., AAV2 3′ ITR). In some embodiments, the expression cassette comprises a nucleotide sequence comprising, from 5′ to 3′: a 5′ ITR (e.g., AAV2 5′ ITR), an enhancer (e.g., a CMV enhancer), a promoter (e.g., a CMV promoter), a 5′ UTR, a polynucleotide sequence comprising a nucleotide sequence encoding a DAO1 enzyme, a WPRE sequence, a polyA sequence, and a 3′ ITR (e.g., AAV2 3′ ITR). In some embodiments, the 5′ UTR comprises a Kozak sequence located immediately upstream and adjacent to the start codon in the nucleotide sequence encoding the DAO1 enzyme.

[0125] In some embodiments, the rAAV comprises an AAV capsid and an expression cassette, wherein the expression cassette comprises a polynucleotide operably linked to a promoter, wherein the polynucleotide comprises a nucleotide sequence encoding a human DAO1 enzyme described herein. In some embodiments, the rAAV comprises an AAV capsid and an expression cassette, wherein the expression cassette comprises a polynucleotide operably linked to a CAG promoter or a CMV promoter, wherein the polynucleotide comprises a nucleotide sequence encoding a human DAO1 enzyme described herein.

[0126] In some embodiments, the rAAV comprises an AAV capsid and an expression cassette comprising a polynucleotide operably linked to a promoter (e.g., a CAG promoter or a CMV promoter), wherein the polynucleotide comprises a nucleotide sequence encoding a DAO1 enzyme, wherein the DAO1 enzyme comprises or consists of an amino acid sequence that shares at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 1. In some embodiments, the DAO1 enzyme comprises or consists of the amino acid sequence shown in SEQ ID NO: 1. In some embodiments, the rAAV comprises an AAV capsid and an expression cassette comprising a polynucleotide operably linked to a promoter (e.g., a CAG promoter), wherein the polynucleotide comprises a nucleotide sequence encoding a DAO1 enzyme, wherein the nucleotide sequence comprises a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 30. In some embodiments, the nucleotide sequence comprises SEQ ID NO: 30.

[0127] In some embodiments, the rAAV comprises an AAV capsid and an expression cassette comprising a polynucleotide operably linked to a promoter (e.g., a CAG promoter or a CMV promoter), wherein the polynucleotide comprises a nucleotide sequence encoding a DAO1 enzyme, wherein the DAO1 enzyme comprises or consists of an amino acid sequence that shares at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 2. In some embodiments, the DAO1 enzyme comprises or consists of the amino acid sequence shown in SEQ ID NO: 2. In some embodiments, the rAAV comprises an AAV capsid and an expression cassette comprising a polynucleotide operably linked to a promoter (e.g., a CAG promoter or a CMV promoter), wherein the polynucleotide comprises a nucleotide encoding a DAO1 enzyme, wherein the nucleotide sequence comprises a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 31. In some embodiments, the nucleotide sequence comprises SEQ ID NO: 31. In some embodiments, the rAAV comprises an AAV capsid and an expression cassette comprising a polynucleotide operably linked to a promoter (e.g., a CAG promoter or a CMV promoter), wherein the polynucleotide comprises a nucleotide encoding a DAO1 enzyme, wherein the nucleotide sequence comprises a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 29. In some embodiments, the nucleotide sequence comprises SEQ ID NO: 29.

[0128] In some embodiments, the rAAV comprises an AAV capsid and an expression cassette comprising or consisting of a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 23. In some embodiments, the expression cassette comprises or consists of SEQ ID NO: 23.

[0129] In some embodiments, the rAAV comprises an AAV2 capsid and an expression cassette, wherein the expression cassette comprises a polynucleotide operably linked to a promoter, wherein the polynucleotide comprises a nucleotide sequence encoding a human DAO1 enzyme described herein. In some embodiments, the rAAV comprises an AAV2 capsid and an expression cassette, wherein the expression cassette comprises a polynucleotide operably linked to a CAG promoter or a CMV promoter, wherein the polynucleotide comprises a nucleotide sequence encoding a human DAO1 enzyme described herein.

[0130] In some embodiments, the rAAV comprises an AAV2 capsid and an expression cassette comprising a polynucleotide operably linked to a promoter (e.g., a CAG promoter or a CMV promoter), wherein the polynucleotide comprises a nucleotide sequence encoding a DAO1 enzyme, wherein the DAO1 enzyme comprises or consists of an amino acid sequence that shares at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 1. In some embodiments, the DAO1 enzyme comprises or consists of the amino acid sequence shown in SEQ ID NO: 1. In some embodiments, the rAAV comprises an AAV2 capsid and an expression cassette comprising a polynucleotide operably linked to a promoter (e.g., a CAG promoter or a CMV promoter), wherein the polynucleotide comprises a nucleotide encoding a DAO1 enzyme, wherein the nucleotide sequence comprises a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 30. In some embodiments, the nucleotide sequence comprises SEQ ID NO: 30.

[0131] In some embodiments, the rAAV comprises an AAV2 capsid and an expression cassette comprising a polynucleotide operably linked to a promoter (e.g., a CAG promoter or a CMV promoter), wherein the polynucleotide comprises a nucleotide sequence encoding a DAO1 enzyme, wherein the DAO1 enzyme comprises or consists of an amino acid sequence that shares at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 2. In some embodiments, the DAO1 enzyme comprises the amino acid sequence shown in SEQ ID NO: 2. In some embodiments, the rAAV comprises an AAV2 capsid and an expression cassette comprising a polynucleotide operably linked to a promoter (e.g., a CAG promoter or a CMV promoter), wherein the polynucleotide comprises a nucleotide encoding a DAO1 enzyme, wherein the nucleotide sequence comprises a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 31. In some embodiments, the nucleotide sequence comprises SEQ ID NO: 31. In some embodiments, the rAAV comprises an AAV2 capsid and an expression cassette comprising a polynucleotide operably linked to a promoter (e.g., a CAG promoter or a CMV promoter), wherein the polynucleotide comprises nucleotides encoding a DAO1 enzyme, wherein the nucleotide sequence comprises a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 29. In some embodiments, the nucleotide sequence comprises SEQ ID NO: 29.

[0132] In some embodiments, the rAAV comprises an AAV2 capsid and an expression cassette comprising or consisting of a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 23. In some embodiments, the rAAV comprises an AAV2 capsid and an expression cassette comprising or consisting of SEQ ID NO: 23.

[0133] In some embodiments, the rAAV comprises an AAV5 capsid and an expression cassette, wherein the expression cassette comprises a polynucleotide operably linked to a promoter, wherein the polynucleotide comprises a nucleotide sequence encoding a human DAO1 enzyme described herein. In some embodiments, the rAAV comprises an AAV5 capsid and an expression cassette, wherein the expression cassette comprises a polynucleotide operably linked to a CAG promoter, wherein the polynucleotide comprises a nucleotide sequence encoding a DAO1 enzyme described herein.

[0134] In some embodiments, the rAAV comprises an AAV5 capsid and an expression cassette comprising a polynucleotide operably linked to a promoter (e.g., a CAG promoter or a CMV promoter), wherein the polynucleotide comprises a nucleotide sequence encoding a DAO1 enzyme, wherein the DAO1 enzyme comprises or consists of an amino acid sequence that shares at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 1. In some embodiments, the DAO1 enzyme comprises the amino acid sequence shown in SEQ ID NO: 1. In some embodiments, the rAAV comprises an AAV5 capsid and an expression cassette comprising a polynucleotide operably linked to a promoter (e.g., a CAG promoter or a CMV promoter), wherein the polynucleotide comprises a nucleotide encoding a DAO1 enzyme, wherein the nucleotide sequence comprises a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 30. In some embodiments, the nucleotide sequence comprises SEQ ID NO: 30.

[0135] In some embodiments, the rAAV comprises an AAV5 capsid and an expression cassette comprising a polynucleotide operably linked to a promoter (e.g., a CAG promoter or a CMV promoter), wherein the polynucleotide comprises a nucleotide sequence encoding a DAO1 enzyme, wherein the DAO1 enzyme comprises an amino acid sequence that shares at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 2. In some embodiments, the DAO1 enzyme comprises the amino acid sequence shown in SEQ ID NO: 2. In some embodiments, the rAAV comprises an AAV5 capsid and an expression cassette comprising a polynucleotide operably linked to a promoter (e.g., a CAG promoter or a CMV promoter), wherein the polynucleotide comprises a nucleotide encoding a DAO1 enzyme, wherein the nucleotide sequence comprises a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 31. In some embodiments, the nucleotide sequence comprises SEQ ID NO: 31. In some embodiments, the rAAV comprises an AAV5 capsid and an expression cassette comprising a polynucleotide operably linked to a promoter (e.g., a CAG promoter or a CMV promoter), wherein the polynucleotide comprises nucleotides encoding a DAO1 enzyme, wherein the nucleotide sequence comprises a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 29. In some embodiments, the nucleotide sequence comprises SEQ ID NO: 29.

[0136] In some embodiments, the rAAV comprises an AAV5 capsid and an expression cassette comprising or consisting of a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 23. In some embodiments, the rAAV comprises an AAV5 capsid and an expression cassette comprising or consisting of SEQ ID NO: 23.

[0137] In some embodiments, the rAAV comprises an AAV9 capsid and an expression cassette, wherein the expression cassette comprises a polynucleotide operably linked to a promoter, wherein the polynucleotide comprises a nucleotide sequence encoding a human DAO1 enzyme described herein. In some embodiments, the rAAV comprises an AAV9 capsid and an expression cassette, wherein the expression cassette comprises a polynucleotide operably linked to a CAG promoter, wherein the polynucleotide comprises a nucleotide sequence encoding a DAO1 enzyme described herein.

[0138] In some embodiments, the rAAV comprises an AAV9 capsid and an expression cassette comprising a polynucleotide operably linked to a promoter (e.g., a CAG promoter or a CMV promoter), wherein the polynucleotide comprises a nucleotide sequence encoding a DAO1 enzyme, wherein the DAO1 enzyme comprises an amino acid sequence that shares at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 1. In some embodiments, the DAO1 enzyme comprises the amino acid sequence shown in SEQ ID NO: 1. In some embodiments, the rAAV comprises an AAV9 capsid and an expression cassette comprising a polynucleotide operably linked to a promoter (e.g., a CAG promoter or a CMV promoter), wherein the polynucleotide comprises a nucleotide encoding a DAO1 enzyme, wherein the nucleotide sequence comprises a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 30. In some embodiments, the nucleotide sequence comprises SEQ ID NO: 30.

[0139] In some embodiments, the rAAV comprises an AAV9 capsid and an expression cassette comprising a polynucleotide operably linked to a promoter (e.g., a CAG promoter or a CMV promoter), wherein the polynucleotide comprises a nucleotide sequence encoding a DAO1 enzyme, wherein the DAO1 enzyme comprises or consists of an amino acid sequence that shares at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 2. In some embodiments, the DAO1 enzyme comprises the amino acid sequence shown in SEQ ID NO: 2. In some embodiments, the rAAV comprises an AAV9 capsid and an expression cassette comprising a polynucleotide operably linked to a promoter (e.g., a CAG promoter or a CMV promoter), wherein the polynucleotide comprises a nucleotide encoding a DAO1 enzyme, wherein the nucleotide sequence comprises a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 31. In some embodiments, the nucleotide sequence comprises SEQ ID NO: 31. In some embodiments, the rAAV comprises an AAV9 capsid and an expression cassette comprising a polynucleotide operably linked to a promoter (e.g., a CAG promoter or a CMV promoter), wherein the polynucleotide comprises a nucleotide encoding a DAO1 enzyme, wherein the nucleotide sequence comprises a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 29. In some embodiments, the nucleotide sequence comprises SEQ ID NO: 29.

[0140] In some embodiments, the rAAV comprises an AAV9 capsid and an expression cassette comprising or consisting of a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 23. In some embodiments, the rAAV comprises an AAV9 capsid and an expression cassette comprising or consisting of SEQ ID NO: 23.

[0141] How to use

[0142] The methods and compositions described herein can be used to treat ocular conditions and reduce symptoms associated with ocular conditions. The terms "treatment", "treating", etc. are generally used herein to mean obtaining a desired pharmacological and / or physiological effect. The effect can be preventive in terms of completely or partially preventing a disease or its symptoms, such as reducing the likelihood of a disease or its symptoms occurring in an individual, and / or can be therapeutic in terms of partially or completely curing a disease and / or side effects caused by the disease. "Treatment" as used herein encompasses any treatment of a mammalian disease, and includes, but is not limited to: (a) inhibiting the progression of the disease; (b) alleviating, reducing one or more symptoms of the disease, or reducing the increase of one or more symptoms of the disease; (c) alleviating, reducing one or more signs of the disease, or reducing the increase of one or more diseases of the disease; (d) causing regression of the disease. The therapeutic agent can be administered before, during, or after the onset of the disease or injury. Treatment of ongoing diseases is of particular interest, wherein the treatment stabilizes or reduces adverse clinical symptoms in the patient. It is desirable to perform such treatment before the function of the affected tissue is completely lost. It is desirable to administer the disclosed therapies during, and in some cases after, the symptomatic stage of the disease.

[0143] As used herein, "administer," "administering," "administration," and the like refer to providing a substance (e.g., a rAAV vector, or a polypeptide comprising DAO1 or a fragment thereof) to an individual in a pharmacologically useful manner (e.g., to treat a disease, disorder, or condition in the individual).

[0144] In some embodiments, the eye condition treated according to the methods described herein is associated with increased histamine production and / or increased histamine signaling. In some embodiments, the eye condition is an inflammatory condition. In some embodiments, the eye condition is an autoimmune reaction. In some embodiments, the eye condition is an autoimmune condition. In some embodiments, the eye condition is an allergic reaction. In some embodiments, the eye condition is an allergic reaction to a therapeutic agent. In some embodiments, the eye condition is an allergic condition. In some embodiments, the eye condition is vernal keratoconjunctivitis. In some embodiments, the eye condition is atopic keratoconjunctivitis. In some embodiments, the eye condition is seasonal or perennial allergic conjunctivitis.

[0145] In some embodiments, the present disclosure provides a method of treating an individual with an ocular disease or disorder comprising administering to the individual a rAAV vector described herein comprising nucleotides encoding human DAO1, wherein expression of human DAO1 in the individual is increased compared to expression of human DAO1 in an untreated individual or in the contralateral eye of the treated individual. Similarly, in various embodiments, methods of treatment are also described herein, comprising administering a polypeptide comprising DAO1 or a fragment thereof and administering an alternative vector (e.g., a plasmid) configured to express DAO1 or a fragment thereof.

[0146] In some embodiments, the present disclosure provides a rAAV vector for use in a method of treating an ocular condition in an individual in need thereof, the method comprising administering a recombinant adeno-associated virus (rAAV) vector to at least one eye of the individual or to at least one lacrimal gland of the eye of the individual, the rAAV vector comprising an AAV capsid and an expression cassette comprising a polynucleotide encoding human DAO1 operably linked to a promoter. In some embodiments, the rAAV vector used is administered to the lacrimal gland of the individual. In some embodiments, the rAAV vector used comprises an expression cassette encoding human DAO1.

[0147] In some embodiments, the present disclosure provides rAAV vectors for use or adapted to treat an individual with an ocular disease, disorder or condition. In some embodiments, the rAAV vector for use or adapted to use comprises an AAV capsid and an expression cassette comprising a polynucleotide encoding human DAO1 (or a fragment thereof) operably linked to a promoter.

[0148] In some embodiments, the present disclosure provides a method of treating an ocular condition in a subject in need thereof, comprising administering to at least one eye of the subject or to at least one lacrimal gland of an eye of the subject a recombinant adeno-associated virus (rAAV) vector described herein, a plasmid configured to express DAO1 or a fragment thereof, or a polypeptide comprising DAO1 or a fragment thereof. In some embodiments, the administered rAAV (or any other vector) may comprise nucleotides encoding DAO1 that share at least 90% (e.g., 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%) sequence identity with SEQ ID NO: 1 or 2. Similarly, in embodiments where DAO1 is administered directly, the polypeptide comprising DAO1 may share at least 90% (e.g., 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%) sequence identity with SEQ ID NO: 1 or 2.

[0149] In some embodiments, the present disclosure provides a rAAV vector for use in a method of treating an ocular condition in an individual in need thereof, the method comprising administering a recombinant adeno-associated virus (rAAV) vector to at least one eye of the individual or to at least one lacrimal gland of an eye of the individual, the rAAV vector comprising an expression cassette comprising a nucleic acid sequence of SEQ ID NO:23.

[0150] In some embodiments, the expression of human DAO1 is increased by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% compared to the expression of human DAO1 in an untreated individual or in the fellow eye of a treated individual. In some embodiments, the expression of human DAO1 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 expression of human DAO1 in an untreated individual or in the fellow eye of a treated individual.

[0151] In some embodiments, expression of human DAO1 is increased for about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 1 year compared to expression of human DAO1 in an untreated individual or in the fellow eye of a treated individual.

[0152] As used herein, "about" or "approximately" as applied to one or more values ​​of interest refers to a value similar to the reference value. In certain embodiments, unless otherwise specified or otherwise apparent from the context, the term "about" refers to a range of values ​​that fall within (greater or less than) 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less in either direction of the reference value (except for possible values ​​in which the values ​​will exceed 100%).

[0153] In some embodiments, a rAAV or other vector comprising nucleotides encoding a human DAO1 enzyme described herein is administered to a subject, wherein expression of the human DAO1 enzyme in the subject is increased compared to expression of human DAO1 in an untreated subject or in the fellow eye of the treated subject.

[0154] In some embodiments, the expression of the human DAO1 enzyme is increased by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% compared to the expression of the human DAO1 enzyme in an untreated individual or in the fellow eye of a treated individual. In some embodiments, the expression of the human DAO1 enzyme may be 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 expression of the human DAO1 enzyme in an untreated individual or in the fellow eye of a treated individual.

[0155] In some embodiments, expression of the human DAO1 enzyme is increased for about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 1 year compared to expression of the human DAO1 enzyme in an untreated individual or in the fellow eye of a treated individual.

[0156] In some embodiments, the present disclosure provides a method of treating an individual having an ocular disease or disorder, comprising administering DAO1 or a rAAV (or other vector) comprising nucleotides encoding a human DAO1 enzyme to the individual, wherein expression of the human DAO1 enzyme in the individual is increased compared to expression of the human DAO1 enzyme in an untreated individual or in the contralateral eye of the treated individual.

[0157] In some embodiments, the present disclosure provides a method for treating a condition associated with histamine intolerance in an individual in need thereof, the method comprising administering to the individual an effective amount of DAO1 or an effective amount of rAAV (or other vector) described herein. In some embodiments, the condition is associated with excessive production of histamine in one or more tissues of the individual. In some embodiments, the condition is associated with excessive production of histamine in one or both eyes of the individual. In some embodiments, the condition is associated with accumulation of histamine in one or more tissues of the individual. In some embodiments, the condition is associated with accumulation of histamine in one or both eyes of the individual.

[0158] In some embodiments, histamine intolerance is caused by an allergic condition. In some embodiments, the allergic condition is a seasonal or perennial allergic condition. In some embodiments, histamine intolerance is caused by an autoimmune condition.

[0159] In some embodiments, the condition is characterized by one or more allergic symptoms (e.g., sneezing, rhinitis, conjunctivitis, shortness of breath, coughing, chest tightness, hives, skin discoloration, stomach cramps, throat constriction, pain, swelling, vomiting, diarrhea, abdominal distension), wherein administration improves or prevents the one or more symptoms. In some embodiments, the condition is characterized by one or more symptoms of the eye (e.g., itching, swelling, tearing, and redness), wherein the administration improves or prevents the one or more symptoms. In some embodiments, the severity of one or more symptoms is assessed based on any method known in the art for characterizing allergy-related symptoms. In some embodiments, the severity of one or more symptoms is assessed based on any method known in the art for characterizing pathology associated with histamine intolerance. In some embodiments, one or more symptoms are assessed using the methods described herein (e.g., conjunctival allergen challenge, corneal staining test, dry eye score test, Schirmer score test, ocular surface disease index test).

[0160] In some embodiments, the method comprises administering a dose of DAO1 or rAAV (or another vector configured to express DAO1) to an individual with a condition associated with histamine intolerance (e.g., an allergic condition), wherein the condition is characterized by one or more symptoms described herein (e.g., one or more symptoms of allergy). In some embodiments, the dose is administered before the onset of one or more symptoms. In some embodiments, administration prevents the onset of one or more symptoms. In some embodiments, administration reduces the severity of one or more symptoms. For example, in some embodiments, the method comprises administering a dose of rAAV to an individual with a seasonal or perennial allergic condition characterized by one or more symptoms (e.g., rhinitis, keratoconjunctivitis, headache, fatigue, cough, sneezing), wherein the dose of rAAV is administered before the season in which the allergic condition occurs, wherein the administration prevents the onset of one or more symptoms and / or reduces the severity of one or more symptoms. In some embodiments, the dose is administered after the onset of one or more symptoms, wherein the administration reduces the severity of the one or more symptoms.

[0161] In some embodiments, the method comprises administering a dosing regimen of rAAV to an individual suffering from a condition associated with histamine intolerance (e.g., an allergic condition), wherein the condition is characterized by one or more symptoms described herein (e.g., one or more symptoms of allergy), wherein the dosing regimen comprises a first dose of rAAV and at least one additional dose, wherein the first dose of rAAV is administered to the individual before or after the onset of one or more symptoms, and wherein the at least one additional dose is administered to the individual after the first dose, thereby preventing the one or more symptoms or reducing the severity of the one or more symptoms. In some embodiments, the dosing regimen comprises a dosing frequency and / or dosing amount selected based on the pharmacokinetic parameters of rAAV. In some embodiments, the clinician will administer rAAV at a frequency and / or dose to achieve or maintain one or more desired effects. In some embodiments, one or more desired effects are to prevent one or more symptoms associated with the condition of the individual. In some embodiments, one or more desired effects are to reduce the severity of one or more symptoms associated with the condition of the individual. In some embodiments, the severity of one or more symptoms is measured using methods described herein or known in the art for assessing pathology associated with histamine intolerance. In some embodiments, one or more desired effects are achieved immediately after the first dose of rAAV is administered. In some embodiments, one or more desired effects occur at any time point after the first dose of rAAV is administered. In some embodiments, one or more desired effects are achieved after the administration of at least one additional dose of rAAV. In some embodiments, one or more desired effects are achieved at any time point during the dosing regimen. In some embodiments, one or more desired effects are achieved after the administration of the first dose of rAAV, and at least one additional dose of rAAV is administered to the individual to prevent the reversal of one or more desired effects. In some embodiments, the method comprises administering a first dose of rAAV to the individual immediately before, immediately after, or during the onset of one or more symptoms, and administering an additional dose after a duration of about 1 week, about 2 weeks, about 3 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 1 year, about 1.5 years, or about 2 years after the first dose. The above-described regimens and parameters can be used to design treatment regimens in which DAO1 is administered directly or via an alternative vector system (eg, plasmid-based expression).

[0162] In some embodiments, the present disclosure provides a method for treating an ocular inflammatory condition in a subject in need thereof, the method comprising administering to the subject one or more therapeutically effective amounts of DAO1 or rAAV or other vectors described herein.

[0163] In some embodiments, the present disclosure provides a method for treating an ocular autoimmune condition in an individual in need thereof, the method comprising administering to the individual one or more therapeutically effective amounts of DAO1 or rAAV or other vectors described herein.

[0164] In some embodiments, the present disclosure provides a method for treating an ocular allergic condition in a subject in need thereof, the method comprising administering to the subject one or more therapeutically effective amounts of DAO1 or rAAV or other vectors described herein.

[0165] In some embodiments, the present disclosure provides a method for treating vernal keratoconjunctivitis in an individual in need thereof, the method comprising administering to the individual one or more therapeutically effective amounts of DAO1 or rAAV or other vectors described herein.

[0166] In some embodiments, the present disclosure provides a method for treating atopic keratoconjunctivitis in an individual in need thereof, the method comprising administering to the individual one or more therapeutically effective amounts of DAO1 or rAAV or other vectors described herein.

[0167] In some embodiments, the present disclosure provides a method for treating seasonal or perennial allergic conjunctivitis in an individual in need thereof, the method comprising administering to the individual one or more therapeutically effective amounts of DAO1 or rAAV or other vectors described herein.

[0168] In some embodiments, the present disclosure provides a rAAV described herein for use in a method of treating a condition associated with histamine intolerance in an individual in need thereof, the method comprising administering to the individual an effective amount of a rAAV described herein.

[0169] In some embodiments, the present disclosure provides a rAAV described herein for use in the manufacture of a medicament for treating a condition associated with histamine intolerance in an individual in need thereof.

[0170] In some embodiments, the present disclosure provides a rAAV described herein for use in a method of treating an autoimmune condition in an individual in need thereof, the method comprising administering to the individual an effective amount of a rAAV described herein.

[0171] In some embodiments, the present disclosure provides a rAAV described herein for use in the manufacture of a medicament for treating an autoimmune condition in an individual in need thereof.

[0172] In some embodiments, the present disclosure provides a rAAV described herein for use in a method of treating an allergic condition in an individual in need thereof, the method comprising administering to the individual an effective amount of a rAAV described herein.

[0173] In some embodiments, the present disclosure provides a rAAV described herein for use in the manufacture of a medicament for treating an allergic condition in an individual in need thereof.

[0174] In some embodiments, the present disclosure provides a rAAV described herein for use in a method of treating vernal keratoconjunctivitis in an individual in need thereof, the method comprising administering to the individual an effective amount of a rAAV described herein.

[0175] In some embodiments, the present disclosure provides a rAAV described herein for use in the manufacture of a medicament for treating vernal keratoconjunctivitis in an individual in need thereof.

[0176] In some embodiments, the present disclosure provides a rAAV described herein for use in a method of treating atopic keratoconjunctivitis in an individual in need thereof, the method comprising administering to the individual an effective amount of a rAAV described herein.

[0177] In some embodiments, the present disclosure provides a rAAV described herein for use in the manufacture of a medicament for treating atopic keratoconjunctivitis in an individual in need thereof.

[0178] In some embodiments, the present disclosure provides a rAAV described herein for use in a method of treating seasonal or perennial allergic conjunctivitis in an individual in need thereof, the method comprising administering to the individual an effective amount of a rAAV described herein.

[0179] In some embodiments, the present disclosure provides a rAAV described herein for use in the manufacture of a medicament for treating seasonal or perennial allergic conjunctivitis in an individual in need thereof.

[0180] Combination therapy

[0181] In some embodiments, the treatment methods described herein comprise further administering one or more additional therapeutic agents to the individual. In some embodiments, the additional therapeutic agent is an agent that increases tear production. In some embodiments, the additional therapeutic agent is another AAV-based gene therapy construct. Suitable therapeutic agents suitable for use as combination therapies of the present disclosure are further described in PCT / US2022 / 027653, which is incorporated herein by reference.

[0182] In some embodiments, the present disclosure provides a method for treating a condition associated with histamine intolerance in an individual in need thereof, the method comprising administering an effective amount of DAO1 described herein or an effective amount of rAAV or other vector to one or both eyes of the individual, wherein the patient is receiving, has received, or will subsequently receive treatment to increase tear production and / or reestablish tear film homeostasis. In some embodiments, the individual has reduced tear production and / or impaired tear film homeostasis. In some embodiments, DAO1 or a vector (e.g., rAAV) is administered to one or both tear glands via intra-lacrimal injection. In some embodiments, DAO1 or a vector (e.g., rAAV) is administered by topical administration to the eye. In some embodiments, treatment is administered after DAO1 or a vector (e.g., rAAV) is administered to the individual. In some embodiments, treatment causes an increase in the amount of DAO1 or a vector (e.g., rAAV) delivered to the tear film of the individual's eyes within a predetermined time compared to the amount of DAO1 or a vector (e.g., rAAV) delivered to the tear film in the absence of treatment for the individual. In some embodiments, the predetermined time period is about 5 minutes to about 60 minutes.

[0183] 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.

[0184] nAChR is a type of pentamer ligand-gated ion channel that has high affinity and selectivity for both nicotine and acetylcholine (which is similar to the protonated form of nicotine) and comprises a combination of α and β subunits. Examples of nAChR subtypes include, but are not limited to, α3β4, α4β2, α3α5β4, and α4α6β2. For example, an important nAChR receptor subtype involved in stimulating the nasolacrimal reflex is the α4β2 subtype located on the trigeminal nerve endings in the nasal mucosa.

[0185] Administration of nAChR agonists can desensitize the receptors. Receptor desensitization results in a reduced response to the agonist even at higher agonist concentrations, which further results in reduced therapeutic efficacy. For example, short-term desensitization of nAChR receptors to agonists can occur within 24 hours after administration of the agonist. The possibility of receptor desensitization may potentially limit the frequency of dosing over a period of time to maintain an effective response to the agonist.

[0186] nAChR agonists can be characterized as full or partial agonists, as determined by their ability to activate a given receptor to produce a response compared to the response of acetylcholine (ACh) at the receptor. Generally, if an nAChR agonist causes a response equal to or greater than that of ACh when bound to a given receptor, it is a full agonist. If an nAChR agonist causes a lower response when bound to the receptor than that produced by ACh, it is a partial agonist.

[0187] The nAChR agonist response that can determine receptor activation can be produced, for example, using appropriate cell-based assays. Cells designed to express specific nAChR receptor subtypes and to produce current responses when combined with nAChR agonists and activated by them can be used to characterize the agonist characteristics of compounds and the amount of receptor activation thus determined. The example of general flow is described below.

[0188] First, cells expressing specific human nAChR subtypes are exposed to ACh. ACh binds and activates receptors, thereby causing electric current. The concentration of ACh is selected to cause the maximum response of receptors (e.g., 1280 micromoles of ACh). The current is recorded as ACh response, and used as 100% nAChR agonist response, and compared with it to the response of other nAChR agonists. After washing, cells are exposed to nAChR agonists of various concentrations (e.g., 0.1, 0.3, 1, 3, 10, 30, 100 and 300 micromoles). For each nAChR concentration, measure and record the electric current caused by being exposed to nAChR agonists. Then the nAChR agonist response data is normalized to one with respect to the maximum ACh induced current, and is plotted as a function of the logarithm of the nAChR agonist concentration. Then the nAChR agonist response is calculated as the percentage of ACh response.

[0189] In some embodiments, methods of determining the relative agonist activity of a nAChR agonist comprise conditions wherein an ACh response is evoked from a 1 or more millimolar ACh solution.

[0190] An nAChR agonist that induces a response equal to or greater than the maximum ACh response determined under the same receptor type is a full agonist. In some embodiments, taking into account experimental variability, nAChR agonists that induce a response of less than 100% of the ACh response can still be characterized as a full agonist. For example, variability between tests or measurement methods and statistical errors can explain differences in response results. In some embodiments, nAChR agonists that induce 80% to 120% of the ACh response are considered to be full agonists. In some embodiments, nAChR agonists that induce 99% or more of the ACh response are considered to be full agonists. In some embodiments, nAChR agonists that induce 95% or more of the ACh response are considered to be full agonists. In some embodiments, nAChR agonists that induce 90% or more of the ACh response are considered to be full agonists. In some embodiments, nAChR agonists that induce 85% or more of the ACh response are considered to be full agonists. In some embodiments, nAChR agonists that induce 80% or more of the ACh response are considered to be full agonists.

[0191] Taking into account experimental variability, if an nAChR agonist induces less than 100% of an ACh response, the agonist is generally considered to be a partial agonist. In some embodiments, an nAChR agonist that induces less than 95% of an ACh response is considered to be a partial agonist. In some embodiments, an nAChR agonist that induces less than 90% of an ACh response is considered to be a partial agonist. In some embodiments, an nAChR agonist that induces less than 85% of an ACh response is considered to be a partial agonist. In some embodiments, an nAChR agonist that induces less than 80% of an ACh response is considered to be a partial agonist.

[0192] In some embodiments, nAChR agonists that induce 5% to 95% of an ACh response are considered partial agonists. In some embodiments, nAChR agonists that induce 5% to 90% of an ACh response are considered partial agonists. In some embodiments, nAChR agonists that induce 5% to 85% of an ACh response are considered partial agonists. In some embodiments, nAChR agonists that induce 5% to 80% of an ACh response are considered partial agonists.

[0193] In some embodiments, nAChR agonists that induce 10% to 95% of an ACh response are considered partial agonists. In some embodiments, nAChR agonists that induce 10% to 90% of an ACh response are considered partial agonists. In some embodiments, nAChR agonists that induce 10% to 85% of an ACh response are considered partial agonists. In some embodiments, nAChR agonists that induce 10% to 80% of an ACh response are considered partial agonists.

[0194] nAChR agonists that produce low levels of electrical activity at relatively high concentrations of agonists can be described as weak partial agonists. In some embodiments, nAChR agonists that induce 30% or less of an ACh response are considered to be weak partial agonists. In some embodiments, nAChR agonists that induce 25% or less of an ACh response are considered to be weak partial agonists. In some embodiments, nAChR agonists that induce 20% or less of an ACh response are considered to be weak partial agonists. In some embodiments, the relatively high concentration of the nAChR agonist is at least 100 micromolar. In some embodiments, the relatively high concentration of the nAChR agonist is at least 200 micromolar. In some embodiments, the relatively high concentration of the nAChR agonist is at least 300 micromolar or higher. For example, a 300 micromolar concentration of an nAChR agonist that induces 25% of the maximum Ach-induced current is considered to be a weak partial agonist.

[0195] In some embodiments, the nAChR agonist is a full agonist. In some embodiments, the nAChR agonist is a partial agonist. In some embodiments, the nAChR agonist is a weak partial agonist.

[0196] In some embodiments, the nAChR agonist or a pharmaceutically acceptable salt thereof is an agonist of at least one nAChR subtype selected from α3β4, α3α5β4, α4β2, and α4α6β2. In some embodiments, the nAChR agonist or a pharmaceutically acceptable salt thereof is an agonist of at least two nAChR subtypes selected from α3β4, α3α5β4, α4β2, and α4α6β2. In some embodiments, the nAChR agonist or a pharmaceutically acceptable salt thereof is an agonist of at least three nAChR subtypes selected from α3β4, α3α5β4, α4β2, and α4α6β2. In some embodiments, the nAChR agonist or a pharmaceutically acceptable salt thereof is an agonist of nAChR subtype α3β4. In some embodiments, the nAChR agonist or a pharmaceutically acceptable salt thereof is an agonist of nAChR subtype α3α5β4. In some embodiments, the nAChR agonist or a pharmaceutically acceptable salt thereof is an agonist of nAChR subtype α4β2. In some embodiments, the nAChR agonist or a pharmaceutically acceptable salt thereof is an agonist of nAChR subtype α4α6β2. In some embodiments, the nAChR agonist or a pharmaceutically acceptable salt thereof is an agonist of nAChR subtype α7. In some embodiments, the nAChR agonist or a pharmaceutically acceptable salt thereof is not an agonist of nAChR subtype α7. In some embodiments, the nAChR agonist or a pharmaceutically acceptable salt thereof is not a full agonist of nAChR subtype α7. In some embodiments, the nAChR agonist is a full agonist of the aforementioned subtype. In some embodiments, the nAChR agonist is a partial agonist of the aforementioned subtype. In some embodiments, the nAChR agonist is a weak partial agonist of the aforementioned subtype.

[0197] The term "α7" or "α7" nAChR refers to the homomeric α7 subtype, wherein the pentameric subunit of the nAChR is composed entirely of α7 subunits. Thus, an nAChR agonist that binds to and activates nAChR α7 is an agonist that binds to and activates the nAChR homomeric α7 receptor. In some embodiments described herein, the nAChR agonist is not an α7 receptor agonist.

[0198] In some embodiments, the nAChR agonist or a pharmaceutically acceptable salt thereof selectively binds to at least one nAChR subtype selected from α3β4, α3α5β4, α4β2, and α4α6β2. As used herein, "selective binding" or "selective for..." means that for at least one reference nAChR subtype, the compound has a higher affinity for the nAChR subtype and / or has a lower half-maximal effective concentration ("EC50") for the nAChR subtype. Selectivity can be associated with at least a 5-fold affinity difference in EC50 values, at least a 10-fold affinity difference in EC50 values, at least a 20-fold affinity difference in EC50 values, or at least a 50-fold affinity difference in EC50 values. In some embodiments, the nAChR agonist or a pharmaceutically acceptable salt thereof selectively binds to the nAChR subtype α3β4. In some embodiments, the nAChR agonist or a pharmaceutically acceptable salt thereof selectively binds to the nAChR subtype α3α5β4. In some embodiments, the nAChR agonist or a pharmaceutically acceptable salt thereof selectively binds to nAChR subtype α4β2. In some embodiments, the nAChR agonist or a pharmaceutically acceptable salt thereof selectively binds to nAChR subtype α4α6β2. In some embodiments, the nAChR agonist or a pharmaceutically acceptable salt thereof selectively binds to nAChR subtype α7. In some embodiments, the nAChR agonist or a pharmaceutically acceptable salt thereof does not selectively bind to nAChR subtype α7.

[0199] nAChR agonists contemplated in the present disclosure include varenicline, a pharmaceutically acceptable salt thereof, and Compound 1 or a pharmaceutically acceptable salt thereof. In some embodiments, the nAChR agonist is not varenicline.

[0200] Varenicline is characterized as a full agonist of nAChR subtype α7 and a partial agonist of subtypes α3β4, α4β2, α6β2, α3α5β4, and α4α6β2. In some embodiments, the nAChR agonist is varenicline or a pharmaceutically acceptable salt thereof. Pharmaceutically acceptable salts of varenicline include varenicline tartrate. Additional relevant information about varenicline can be found, for example, in U.S. Pat. No. 9,504,644, U.S. Pat. No. 9,504,645, U.S. Pat. No. 9,532,944, U.S. Pat. No. 9,597,284, and U.S. Pat. No. 10,456,386.

[0201] As used herein, Compound 1 refers to the following structure:

[0202]

[0203] An alternative structural representation of compound 1 is shown here:

[0204]

[0205] Compound 1 may also be referred to by its chemical name. For example, Compound 1 is also known as (R)-5-((E)-2-pyrrolidin-3-ylvinyl)pyrimidine or variations thereof, including simpanicline, 5-{(E)-2-[(3R)-pyrrolidin-3-yl]vinyl}pyrimidine and (R,E)-5-((2-pyrrolidin-3-yl)vinyl)pyrimidine.

[0206] Compound 1 is a full agonist of nAChR subtypes α4β2, α3β4, α3α5β4, and α4α6β2. Compound 1 is a full agonist of nAChR subtypes α4β2 and α3β4.

[0207] Compound 1 is a partial agonist of the α3β2 subtype.

[0208] Compound 1 is a weak partial agonist of subtype α 7. In one example, a 300 micromolar concentration of Compound 1 citrate induced only 25% of the maximal ACh-evoked current.

[0209] In some embodiments, the nAChR agonist can be Compound 1 or a pharmaceutically acceptable salt thereof. Pharmaceutically acceptable salts of Compound 1 include galactarate (e.g., galactarate dihydrate) and citrate (e.g., monocitrate). Patent-related information on Compound 1 can be found in U.S. Patent No. 7,098,331, U.S. Patent No. 7,714,001, U.S. Patent No. 8,063,068, U.S. Patent No. 8,067,443, U.S. Patent No. 8,604,191, U.S. Patent No. 9,145,396, U.S. Patent No. 9,981,949, U.S. Patent No. 8,633,222, and PCT Publication No. WO 2017 / 177024.

[0210] In some embodiments, the nAChR agonist is (R)-5-((E)-2-pyrrolidin-3-ylvinyl)pyrimidine or a pharmaceutically acceptable salt thereof. In some embodiments, the nAChR agonist is (R)-5-((E)-2-pyrrolidin-3-ylvinyl)pyrimidine galactarate dihydrate. In some embodiments, the nAChR agonist is (R)-5-((E)-2-pyrrolidin-3-ylvinyl)pyrimidine monocitrate.

[0211] Mode of administration

[0212] In some embodiments, the present disclosure provides methods comprising administering to an eye of an individual or to the lacrimal gland of an eye of an individual a rAAV vector comprising an AAV capsid and an expression cassette comprising a polynucleotide encoding DAO1 operably linked to a promoter. In other aspects, methods according to the present disclosure may comprise administering to an eye of an individual or to the lacrimal gland of an eye of an individual a polypeptide comprising DAO1 or a fragment thereof or an alternative vector configured to express DAO1 or a fragment thereof (e.g., a plasmid-based expression system).

[0213] As mentioned above, the lacrimal functional unit is composed of the main lacrimal gland and accessory lacrimal gland, the ocular surface and interconnected nerve innervation. For each eye, the main lacrimal gland is located in the upper temporal part of the orbit in the tear fossa of the frontal bone. The accessory glands known as Wolfflin's glands and Kraus's glands are located in the eyelids. There are about 2 to 5 Wolfflin's glands and about 40 Kraus's glands in the upper eyelid. There are about 6 to 8 Kraus's glands in the lower eyelid. The specific location and anatomical structure of the lacrimal functional unit are well known (Conrady et al., Journal of Ophthalmology; 2016: 7542929 (2016)). The lacrimal glands together secrete the tear film onto the ocular surface through the tear duct. The lacrimal glands also express and secrete proteins and products necessary for corneal regeneration and promoting tear film transparency, such as transforming growth factor-β and retinol (Conradi et al., Journal of Ophthalmology; 2016:7542929 (2016); Pan et al., Optom Vis Sci.; 95:27-31 (2018)). In addition to secreting tears into the eye, the tear ducts also drain tears into the nasal cavity.

[0214] Administration of DAO1 or a vector (e.g., rAAV) to the lacrimal gland can be achieved by topical administration to the ocular surface, direct injection into the lacrimal gland, and / or local administration to the lacrimal gland. In some embodiments, DAO1 or a vector (e.g., rAAV) is administered to the lacrimal gland by topical administration. In some embodiments, DAO1 or a vector (e.g., rAAV) is administered to the lacrimal gland by direct injection. Access to the lacrimal gland can be achieved surgically or by manipulation of the eyelids. Manipulation of the eyelids provides access to the tissue for topical administration (e.g., by irrigating the tissue with a pharmaceutical composition comprising a viral vector). Direct injection into the lacrimal gland can be achieved by penetrating the skin over the lacrimal gland ( Figure 2A ) or by manipulating the eyelids to access the tear glands ( Figure 2B ). Administration of the viral vector to the lacrimal gland can be accomplished by transconjunctival injection. In some embodiments, the rAAV is administered to the lacrimal gland by direct injection, such as Figure 2A In some embodiments, rAAV is administered to the lacrimal gland by manipulating the eyelids, such as Figure 2B shown.

[0215] In some embodiments, cells in the eye, lacrimal gland, and / or nasolacrimal duct are transduced by rAAV vectors. Cells in the eye, lacrimal gland, and / or nasolacrimal duct include, but are not limited to, acinar cells, ductal cells, and / or myoepithelial cells. In some embodiments, transduced cells in the eye, lacrimal gland, and / or nasolacrimal duct express a therapeutically effective amount of the DAO1 enzyme into the tear film and optionally onto the ocular surface of the individual. In some embodiments, a therapeutically effective amount of the DAO1 enzyme is secreted into the nasal cavity of the individual. In some embodiments, a therapeutically effective amount of the DAO1 enzyme is secreted onto the ocular surface of the individual.

[0216] The delivery of rAAV vectors to the eye and / or lacrimal glands has been demonstrated in vivo to express transgenes into the tear film. In one example, the primary lacrimal glands of mice were directly injected with rAAV vectors encoding luciferase transgenes with serotypes AAV2, AAV4, AAV5, AAV 5w8, AAV x5, AAV 9, AAV12, and bovine AAV (BAAV). AAV9, AAV5w8, AAV5, and AAV2 are each capable of transducing the lacrimal duct and acinar cells of the lacrimal gland (Rocha et al., supra).

[0217] In some embodiments, the rAAV vector is administered to a lacrimal gland of an individual. In some embodiments, the lacrimal gland is a primary lacrimal gland. In some embodiments, the lacrimal gland is any one of a Wolfflin's gland or a Kraus's gland of an individual.

[0218] The compositions and rAAV vectors of the present disclosure can be administered to the lacrimal glands of an individual by any suitable method. For example, the compositions of the present disclosure can be administered by direct injection into the primary or accessory lacrimal glands.

[0219] Accessing the tear gland of a human subject can be achieved, for example, by manually lifting the upper eyelid to expose the palpebral lobe of the tear gland and delivering the therapeutic agent using a syringe (eg, with a 30G needle).

[0220] The viral vectors of the present disclosure are usually delivered to individuals as pharmaceutical compositions. The pharmaceutical composition comprises a pharmaceutically acceptable solvent (e.g., water, etc.) and one or more excipients. In some embodiments, the pharmaceutical composition comprises a buffer of about neutral pH (pH 5, 6, 7, 8 or 9). In some embodiments, the pharmaceutical composition comprises phosphate buffered saline (e.g., PBS with a pH of about 7). The pharmaceutical composition may comprise a pharmaceutically acceptable salt. The concentration of the salt may be selected to ensure that the pharmaceutical composition is isotonic or almost isotonic with the target tissue.

[0221] In some embodiments, the pharmaceutical composition of the present disclosure comprises about 1×10 8 genome copies (GC / mL), about 5×10 8 GC / mL, about 1×109 GC / mL, about 5×10 9 GC / mL, about 1×10 10 GC / mL, about 5×10 10 GC / mL, about 1×10 11 GC / mL, about 5×10 11 GC / mL, about 1×10 12 GC / mL, about 5×10 12 GC / mL, about 5×10 13 GC / mL or about 1×10 14 GC / mL. In some embodiments, the pharmaceutical composition of the present disclosure comprises about 1×10 GC / mL of viral vector (e.g., rAAV vector). 8 genome copies (GC / mL), about 5×10 8 GC / mL to about 1×10 9 GC / mL, about 1×10 9 GC / mL to about 5×10 9 GC / mL, about 5×10 9 GC / mL to about 1×10 10 GC / mL, about 1×10 10 GC / mL to about 5×10 10 GC / mL, about 5×10 10 GC / mL to about 1×10 11 GC / mL, about 1×10 11 GC / mL to about 5×10 11 GC / mL, about 5×10 11 GC / mL to about 1×10 12 GC / mL, about 1×10 12 GC / mL to about 5×10 12 GC / mL, about 5×10 12 GC / mL to about 5×10 13 GC / mL or about 5×10 13 GC / mL to about 1×10 14 GC / mL. In some embodiments, the pharmaceutical compositions of the present disclosure comprise about 5×10 8 GC / mL to about 5×10 9 GC / mL, about 5×10 9 GC / mL to about 5×10 10 GC / mL, about 5×10 10 GC / mL to about 5×10 11 GC / mL, about 5×10 11 GC / mL to about 5×10 12 GC / mL or about 5×1012 GC / mL to about 1×10 14 GC / mL of viral vector (e.g., rAAV vector). In yet other embodiments, the pharmaceutical composition of the present disclosure comprises about 5×10 8 GC / mL to about 5×10 10 GC / mL, about 5×10 10 GC / mL to about 5×10 12 GC / mL or about 5×10 12 GC / mL to about 1×10 14 GC / mL of viral vector (e.g., rAAV vector).

[0222] In some embodiments, the pharmaceutical compositions of the present disclosure comprise about 1×10 12 GC / mL to about 6.2×10 12 GC / mL of viral vector (e.g., rAAV vector). In some embodiments, the pharmaceutical composition of the present disclosure comprises about 1×10 12 GC / mL or about 6.2×10 12 GC / mL of viral vector (e.g., rAAV vector).

[0223] In some embodiments, the pharmaceutical compositions of the present disclosure are administered in a total volume of about 10 μL, about 20 μL, about 30 μL, about 40 μL, about 50 μL, about 60 μL, about 70 μL, about 80 μL, about 90 μL, about 100 μL, 110 μL, about 120 μL, about 130 μL, about 140 μL, about 150 μL, about 160 μL, about 170 μL, about 180 μL, about 190 μL, or about 200 μL. In some embodiments, the pharmaceutical compositions of the present disclosure are administered in a total volume of about 10 μL to about 20 μL, about 20 μL to about 30 μL, about 30 μL to about 40 μL, about 40 μL to about 50 μL, about 50 μL to about 60 μL, about 60 μL to about 70 μL, about 70 μL to about 80 μL, about 80 μL to about 90 μL, about 90 μL to about 100 μL, about 100 μL to 110 μL, 110 μL to about 120 μL, about 120 μL to about 130 μL, about 130 μL to about 140 μL, about 140 μL to about 150 μL, about 150 μL to about 160 μL, about 160 μL to about 170 μL, about 170 μL to about 180 μL, about 180 μL to about 190 μL, or about 190 μL to about 200 μL.

[0224] The number of genome copies per milliliter can be determined by quantitative polymerase chain reaction (qPCR) using a standard curve generated from reference samples with known viral polynucleotide genome concentrations. For AAV, the reference sample used is typically the transfer plasmid used to generate the rAAV vector, but other reference samples can also be used.

[0225] Alternatively or additionally, the concentration of the viral vector can be determined by measuring the titer of the vector on the cell line. Viral titer is generally expressed as viral particles per unit volume (vp) (e.g., vp / mL). In some embodiments, the pharmaceutical composition of the present disclosure comprises about 1×10 50 μg of viral vector (e.g., rAAV vector) per mL. 8 Virus particles (vp / mL), about 5×10 8 vp / mL, about 1×10 9 vp / mL, about 5×10 9 vp / mL, about 1×10 10 vp / mL, about 5×10 10 vp / mL, about 1×10 11 vp / mL, about 5×10 11 vp / mL, about 1×10 12 vp / mL, about 5×10 12 vp / mL, about 5×10 13 vp / mL or about 1×10 14 vp / mL. In some embodiments, the pharmaceutical composition of the present disclosure comprises about 1×10 vp / mL of viral vector (e.g., rAAV vector). 8 Virus particles (vp / mL) to about 5×10 8 vp / mL, about 5×10 8 vp / mL is about 1×10 9 vp / mL, about 1×10 9 vp / mL to about 5×10 9 vp / mL, about 5×10 9 vp / mL is about 1×10 10 vp / mL, about 1×10 10 vp / mL to about 5×10 10 vp / mL, about 5×10 10 vp / mL is about 1×10 11 vp / mL, about 1×10 11 vp / mL to about 5×10 11 vp / mL, about 5×10 11 vp / mL is about 1×10 12 vp / mL, about 1×10 12 vp / mL to about 5×10 12vp / mL, about 5×10 12 vp / mL to about 5×10 13 vp / mL or about 5×10 13 vp / mL is about 1×10 14 vp / mL.

[0226] A variety of tests can be used to evaluate individual eye conditions before, during and after treatment with any method or composition disclosed herein. In some embodiments disclosed herein, the effective treatment of an individual is indicated by one or more tests, which may be, for example, a) a dryness score test on a visual analog scale, b) a Hilmer's test, c) a corneal fluorescein staining test, and d) an ocular surface disease index test. Tests evaluating signs and symptoms of eye conditions can be performed under standardized or reproducible conditions to obtain individual test scores. Conditions include exposing an individual to an artificially generated environment to adversely challenge the individual or wherein monitoring and carefully controlling the environment (temperature, humidity, airflow).

[0227] Efficacy evaluation

[0228] The efficacy of the methods described herein can be assessed using any suitable method known in the art.

[0229] In some embodiments, the methods described herein cause a reduction in one or more symptoms of an ocular condition as compared to the symptoms of the ocular condition prior to administration of the rAAV vector. As used herein, "symptoms" include any diagnostic criteria or symptoms associated with a given ocular condition, including those described herein. Non-limiting examples of symptoms that can be alleviated by treatment according to the methods described herein include, for example, itching, swelling, tearing, and redness.

[0230] In some embodiments, after administration of the compositions and rAAVs of the present disclosure, symptoms (e.g., itching, swelling, tearing, and redness) may be alleviated. In some embodiments, one or more symptoms of an ocular condition are reduced compared to the symptoms of the ocular condition in an untreated control individual. In some embodiments, one or more symptoms of an ocular condition are reduced compared to the symptoms of the ocular condition in the fellow eye. "Fellow eye" refers to the eye of an individual opposite to the eye that has been treated with a composition according to the present disclosure. The fellow eye can be used as a control for treatment, as long as the individual suffers from bilateral disease, or in the case of a model animal, an experimental protocol leading to binocular treatment has been performed.

[0231] The efficacy of the treatment methods described herein can be assessed by evaluating the symptoms of the ocular condition treated according to the methods described herein on a clinical scale. Examples of clinical scales that can be used to assess the efficacy of the treatment methods described herein include, but are not limited to, the Conjunctival Itch Grading Scale (Table 4), the Conjunctival Redness Grading Scale (Table 5), and the Corneal Staining Grading Scale (Table 6). Figure 3 ).

[0232] As used herein, the term "statistically significant" refers to an analytical method selected by one of ordinary skill in the art based on the study design and the type of data generated to assess observed differences between two or more groups, wherein the analysis determines whether the difference is not random or due entirely to chance.

[0233] Conjunctival allergen challenge (CAC)

[0234] Conjunctival allergen challenge consisted of an initial visit (allergen titration visit) with bilateral instillation of the specific allergen that induced a positive skin test reaction at 10-minute intervals, increasing allergen concentrations until a positive reaction was induced. After challenge, ocular / conjunctival pruritus was assessed by the individual and ocular / conjunctival redness was assessed by a trained clinician; both assessments used a standardized rating scale ranging from 0 (none) to 4 (severe). A positive CAC reaction was defined as moderate to severe redness in two of the three vascular beds (conjunctiva, ciliary muscle, sclera) in each eye within 10 minutes of allergen instillation and moderate to severe itching in both eyes. Individuals who failed to test positive were excluded from the study. During a subsequent visit (allergen confirmation), patients were challenged with the final titer of allergen used in the first visit. If moderate to severe ocular allergic reactions were reproduced at two of the three time points after CAC, the diagnosis was confirmed and the individual was included in the study.

[0235] Conjunctival pruritus grading scale

[0236] In some embodiments, treating an ocular condition according to the methods described herein results in an improvement of 0.5, 1, 1.5, 2, 2.5, 3, 3.5, or 4 points on the Conjunctival Itch Rating Scale shown in Table 4.

[0237] Table 4: Conjunctival pruritus grading scale.

[0238] score Descriptors for eye itching 0 none 0.5 Intermittent itching sensation that may be located in the corner of the eye 1.0 Intermittent itching that involves more than just the corners of the eyes 1.5 Intermittent itching sensation over the entire eye 2.0 Mild persistent itching (may be localized) with no desire to rub 2.5 Moderate, diffuse, persistent itching with a desire to rub 3.0 Severe itching, with a desire to rub 3.5 Severe itching can be improved by just gentle rubbing 4.0 Unbearable itching, with an irresistible desire to rub

[0239] In some embodiments disclosed herein, effective treatment is indicated by a statistically significant decrease in the subject's score on a conjunctival itch grading scale, and wherein the statistically significant decrease in the subject's score on the conjunctival itch grading scale is determined after administration of a first dose, or optionally one or more subsequent doses, of a rAAV vector described herein to the subject, wherein the subject's score on the conjunctival itch grading scale is compared to: a) the subject's score on the conjunctival itch grading scale prior to administration of the first dose of the rAAV vector; b) the subject's score on the conjunctival itch grading scale administered to a control; c) the subject's score on the conjunctival itch grading scale administered to a comparator compound; or d) the contralateral eye.

[0240] In some embodiments disclosed herein, the score on the conjunctival itch grading scale of the individual is compared to the corneal score of the individual before the rAAV vector is administered according to the methods described herein. In some embodiments disclosed herein, the score on the conjunctival itch grading scale of the individual is compared to the score on the conjunctival itch grading scale of the individual administered a control. In some embodiments disclosed herein, the score on the conjunctival itch grading scale of the individual is compared to the score on the conjunctival itch grading scale of the individual administered a comparison compound. In some embodiments disclosed herein, the score on the conjunctival itch grading scale of the individual is compared to the contralateral eye.

[0241] In some embodiments, a statistically significant reduction in an individual's ocular / conjunctival itch score is observed in an individual administered a rAAV vector described herein, as compared to a control. In some embodiments described herein, a statistically significant reduction in an individual's ocular / conjunctival itch score is at least 5%, 10%, 15%, 20%, 25%, 50%, 75%, 80%, 90%, 95%, 100%, 125%, 150%, 200%, 250%, 300%, or 350%. In some embodiments, a statistically significant reduction in an ocular / conjunctival itch score is observed in an individual administered a rAAV vector described herein compared to a control, wherein the statistically significant reduction in an ocular / conjunctival itch score in the individual is at least 5%, 10%, 15%, 20%, 25%, 50%, 75%, 80%, 90%, 95%, 100%, 125%, 150%, 200%, 250%, 300%, or 350%.

[0242] In some embodiments described herein, a statistically significant decrease in an individual's ocular / conjunctival itch score is characterized by a p-value of 0.05 or less, 0.01 or less, 0.005 or less, or 0.001 or less. In some embodiments described herein, a statistically significant decrease in an individual's ocular / conjunctival itch score is characterized by a p-value of 0.05 or less. In some embodiments described herein, a statistically significant decrease in an individual's ocular / conjunctival itch score is characterized by a p-value of 0.01 or less.

[0243] In some embodiments described herein, a statistically significant reduction in an individual's ocular / conjunctival itch score is within 1 day, within 2 days, within 3 days, within 4 days, within 1 week, within 2 weeks, within 3 weeks, within 4 weeks, within 1 month, within 2 months, within 3 months, within 4 months, within 5 months, or within 6 months following treatment with any disclosed method or composition.

[0244] In some embodiments, the subject's reduction in ocular / conjunctival itch score persists for about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 9 months, or 1 year following treatment with any method or composition disclosed herein.

[0245] In some embodiments described herein, the statistically significant decrease in the individual's ocular / conjunctival itch score is based on the individual's ocular / conjunctival itch score determined after administration of a first dose of the rAAV vector.

[0246] In some embodiments described herein, a statistically significant reduction is based on an individual's ocular / conjunctival itch score determined following treatment with any of the methods or compositions disclosed herein.

[0247] Conjunctival redness assessment grading scale

[0248] In some embodiments, treating an ocular condition according to the methods described herein results in an improvement of 1, 2, 3, or 4 points on the conjunctival redness assessment grading scale shown in Table 5.

[0249] Table 5: Grading scale for assessment of conjunctival redness

[0250]

[0251]

[0252] In some embodiments disclosed herein, effective treatment is indicated by a statistically significant decrease in the subject's score on a conjunctival redness assessment grading scale, and wherein the statistically significant decrease in the subject's score on the conjunctival redness assessment grading scale is determined after administration of a first dose, or optionally one or more subsequent doses, of a rAAV vector described herein to the subject, wherein the subject's score on the conjunctival redness assessment grading scale is compared to: a) the subject's score on the conjunctival redness assessment grading scale prior to administration of the first dose of the rAAV vector; b) the subject's score on the conjunctival redness assessment grading scale administered a control; c) the subject's score on the conjunctival redness assessment grading scale administered a comparator compound; or d) the contralateral eye.

[0253] In some embodiments disclosed herein, the individual's ocular / conjunctival redness score is compared to the individual's ocular / conjunctival redness score prior to administration of a first dose of a rAAV vector and a first dose of a treatment that increases tear production.

[0254] In some embodiments disclosed herein, the ocular / conjunctival itching score of a subject is compared to the ocular / conjunctival redness score of a subject administered a control.

[0255] In some embodiments disclosed herein, the ocular / conjunctival itch score of a subject is compared to the ocular / conjunctival redness score of a subject administered a comparator compound.

[0256] In some embodiments disclosed herein, the subject's ocular / conjunctival redness score is compared to the ocular / conjunctival itch score in the fellow eye.

[0257] In some embodiments, a statistically significant reduction in the ocular / conjunctival redness score of the individual is observed in the individual administered the rAAV vector described herein, as compared to the control. In some embodiments described herein, the statistically significant reduction in the ocular / conjunctival redness score of the individual is at least 5%, 10%, 15%, 20%, 25%, 50%, 75%, 80%, 90%, 95%, 100%, 125%, 150%, 200%, 250%, 300% or 350%. In some embodiments, a statistically significant reduction in an ocular / conjunctival redness score is observed in an individual administered a rAAV vector described herein compared to a control, wherein the statistically significant reduction in the ocular / conjunctival redness score in the individual is at least 5%, 10%, 15%, 20%, 25%, 50%, 75%, 80%, 90%, 95%, 100%, 125%, 150%, 200%, 250%, 300%, or 350%.

[0258] In some embodiments described herein, a statistically significant reduction in an individual's ocular / conjunctival redness score is characterized by a p-value of 0.05 or less, 0.01 or less, 0.005 or less, or 0.001 or less. In some embodiments described herein, a statistically significant reduction in an individual's ocular / conjunctival redness score is characterized by a p-value of 0.05 or less. In some embodiments described herein, a statistically significant reduction in an individual's ocular / conjunctival redness score is characterized by a p-value of 0.01 or less.

[0259] In some embodiments described herein, a statistically significant reduction in a subject's ocular / conjunctival redness score is within 1 day, within 2 days, within 3 days, within 4 days, within 1 week, within 2 weeks, within 3 weeks, within 4 weeks, within 1 month, within 2 months, within 3 months, within 4 months, within 5 months, or within 6 months following treatment with any disclosed method or composition.

[0260] In some embodiments, the subject's reduction in ocular / conjunctival redness score persists for about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 9 months, or 1 year following treatment with any of the methods or compositions disclosed herein.

[0261] In some embodiments described herein, the statistically significant decrease in the individual's ocular / conjunctival redness score is based on the individual's ocular / conjunctival redness score determined after administration of a first dose of the rAAV vector.

[0262] In some embodiments described herein, a statistically significant decrease is based on an individual's ocular / conjunctival redness score determined following treatment with any of the methods or compositions disclosed herein.

[0263] In some embodiments disclosed herein, the score on the conjunctival redness assessment grading scale of the individual is compared to the corneal score of the individual before the rAAV vector is administered according to the methods described herein. In some embodiments disclosed herein, the score on the conjunctival redness assessment grading scale of the individual is compared to the score on the conjunctival redness assessment grading scale of the individual administered a control. In some embodiments disclosed herein, the score on the conjunctival redness assessment grading scale of the individual is compared to the score on the conjunctival redness assessment grading scale of the individual administered a comparison compound. In some embodiments disclosed herein, the conjunctival redness assessment grading scale score of the individual is compared to the contralateral eye.

[0264] In some embodiments, treating an ocular condition according to the methods described herein results in an improvement of about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about 50%, about 50% to about 60%, about 60% to about 70%, about 70% to about 80%, about 80% to about 90%, or more than about 95% in at least one area on the corneal staining grading scale.

[0265] Corneal surface changes are associated with insufficient tear flow and excessive dryness, as well as the ocular conditions and ocular discomfort described herein. Corneal surface changes can include disruption of the mucin coating that protects surface epithelial cells, and / or damage to the epithelial cell wall.

[0266] Corneal staining test

[0267] Corneal surface changes are associated with insufficient tear flow and excessive dryness, as well as the ocular conditions and ocular discomfort described herein. Corneal surface changes can include disruption of the mucin coating that protects surface epithelial cells, and / or damage to the epithelial cell wall.

[0268] Corneal staining, including fluorescein staining, lissamine green staining and rose Bengal staining, is a diagnostic test for determining the health of the corneal surface and can indicate areas of damage on the corneal surface. Any suitable corneal staining method known in the art can be used to evaluate the efficacy of the treatment methods described herein, such as Begley et al., The Ocular Surface 17 (2019) 208-220. The normal corneal surface does not absorb water-soluble dyes instilled into the tear film. However, damaged epithelial cells allow water-soluble dyes to diffuse into surface cells. Dyes that stain damaged epithelial cells can be visualized on the corneal surface, indicating damage on the corneal surface.

[0269] To perform the corneal staining test, a staining dye is applied to one or both eyes. The dye is allowed to penetrate and stain the area between the surface cells. With this test, chains of necrotic cells and necrotic surface tissue (filaments) can be visualized. The test administrator then uses a developed corneal surface scoring system to assess the severity of the observed damage. This scoring system can be used to treat eye conditions over time. Figure 3 The NEI / Industry Workshop scale used in the scoring system is depicted. Other equivalent standardized scoring systems may be used. The test administrator grades the area of ​​corneal surface damaged and calculates a corneal score that reflects the severity of corneal surface damage.

[0270] The test administrator can use the corneal score to evaluate the effectiveness of a particular treatment in an individual. The test can be applied multiple times to monitor any changes in the severity of the individual's ocular surface over a period of time. Generally, the larger the number, the more severe the corneal surface damage, and the smaller the number, the less severe the corneal surface damage. A decrease in the corneal score over time is evidence of reduced corneal surface damage. A decrease in the corneal score generally indicates an improvement in an individual's condition. A decrease in the corneal score over time also demonstrates that the treatment is effective in treating dry eye, increasing tear production, or improving eye discomfort.

[0271] In some embodiments disclosed herein, effective treatment is indicated by a statistically significant decrease in the subject's corneal score, and wherein the statistically significant decrease in the subject's corneal score is determined after administering to the subject a first dose, or optionally one or more subsequent doses, of a composition described herein, wherein the subject's corneal score is compared to: a) the subject's corneal score prior to administration of the first dose of the rAAV vector and the first dose of treatment to increase tear production; b) the subject's corneal score administered a control; c) the subject's corneal score administered a comparator compound; or d) the contralateral eye.

[0272] In some embodiments disclosed herein, the corneal score of the individual is compared to the corneal score of the individual before the pharmaceutical composition is administered according to the methods described herein. In some embodiments disclosed herein, the corneal score of the individual is compared to the corneal score of the individual administered a control. In some embodiments disclosed herein, the corneal score of the individual is compared to the corneal score of the individual administered a comparison compound. In some embodiments disclosed herein, the corneal score of the individual is compared to the corneal score of the individual administered a comparison compound. In some embodiments disclosed herein, the corneal score of the individual is compared to the contralateral eye.

[0273] In some embodiments disclosed herein, an individual's corneal score can be used to measure the definition of corneal healing, which is defined as less than 0.5 mm fluorescein staining or no (zero) fluorescein staining (Bonini et al., Ophthalmology. 125: 1332-1343 (2018)).

[0274] In some embodiments, a statistically significant decrease in the corneal healing score of an individual is observed in an individual administered a rAAV vector described herein compared to a control.

[0275] In some embodiments described herein, the statistically significant reduction in the corneal healing score of the individual is at least 5%, 10%, 15%, 20%, 25%, 50%, 75%, 80%, 90%, 95%, 100%, 125%, 150%, 200%, 250%, 300% or 350%. In some embodiments, a statistically significant reduction in the OSDI score of the individual is observed in the individual administered with the rAAV vector described herein compared to the control, wherein the statistically significant reduction in the OSDI score of the individual is at least 5%, 10%, 15%, 20%, 25%, 50%, 75%, 80%, 90%, 95%, 100%, 125%, 150%, 200%, 250%, 300% or 350%.

[0276] In some embodiments described herein, the statistically significant reduction in an individual's corneal staining score is at least 5%, 10%, 15%, 20%, 25%, 50%, 75%, 80%, 90%, 95%, 100%, 125%, 150%, 200%, 250%, 300% or 350%.

[0277] In some embodiments described herein, a statistically significant decrease in a subject's corneal score is characterized by a p-value of 0.05 or less, 0.01 or less, 0.005 or less, or 0.001 or less.

[0278] In some embodiments described herein, a statistically significant reduction in a subject's corneal score is within 1 day, within 2 days, within 3 days, within 4 days, within 1 week, within 2 weeks, within 3 weeks, within 4 weeks, within 1 month, within 2 months, within 3 months, within 4 months, within 5 months, or within 6 months after treatment with any disclosed method or composition.

[0279] In some embodiments, the reduction in corneal score in a subject persists for at least 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, 9 months, or 1 year following treatment with any of the methods or compositions disclosed herein.

[0280] In some embodiments described herein, a statistically significant decrease is based on a corneal score of the individual determined after administration of one or more subsequent doses of a rAAV vector and optionally a first or one or more subsequent doses of a treatment that increases tear production.

[0281] Dry eyes score test

[0282] The eye dryness scoring test on a visual analog scale can be used to assess an individual's eye pathology, tearing, or eye discomfort. The test involves the use of a visual analog scale and comprises a 100 mm horizontal line with one endpoint at 0 marked as "no discomfort" and the other endpoint at 100 marked as "maximum discomfort". Figure 3 An example of a visual analogue scale is shown (not shown to actual scale). The individual is asked to rate their eye symptoms due to dry eyes by placing vertical marks on a horizontal line to indicate their level of discomfort. A dry eyes score is then obtained by identifying where the individual's response is on the 100 mm scale.

[0283] The eye dryness score measured in mm can be used to assess the severity of ocular symptoms and the effectiveness of a particular treatment for an individual. The test has the advantage of being administered to an individual at a frequency of every 5 minutes, which allows the test administrator to closely monitor changes in individual symptoms over time. The larger the number, the more uncomfortable the ocular symptom, and the smaller the number, the relatively weaker the discomfort. A decrease in the eye dryness score is evidence that the treatment is effective in treating ocular conditions, increasing tear production, or improving ocular discomfort. A decrease in the eye dryness score over time is evidence of a reduction or relief of ocular symptoms, and generally indicates an improvement in individual symptoms.

[0284] In some embodiments disclosed herein, effective treatment is indicated by a statistically significant decrease in the subject's eye dryness score, and wherein the statistically significant decrease in the subject's eye dryness score is determined after administering to the subject a first dose, or optionally one or more subsequent doses, of a rAAV vector described herein and a first dose, or optionally one or more subsequent doses, of a treatment that increases tear production, wherein the subject's eye dryness score is compared to: a) the subject's eye dryness score prior to administration of the first dose of the rAAV vector and the first dose of treatment that increases tear production; b) the subject's eye dryness score administered a control; c) the subject's eye dryness score administered a comparator compound; or d) the eye dryness score of the contralateral eye.

[0285] In some embodiments disclosed herein, the dry eye score of the individual is compared to the dry eye score of the individual prior to administration of a first dose of the rAAV vector and a first dose of treatment to increase tear production.

[0286] In some embodiments disclosed herein, the dryness of the eyes score of a subject is compared to the dryness of the eyes score of a subject administered a control.

[0287] In some embodiments disclosed herein, the dryness of eyes score of a subject is compared to the dryness of eyes score of a subject administered a comparator compound.

[0288] In some embodiments disclosed herein, the individual's eye dryness score is compared to the fellow eye's eye dryness score.

[0289] In some embodiments, a statistically significant reduction in the dry eye score of an individual is observed in an individual administered with a rAAV vector as described herein, compared to a control. In some embodiments described herein, the statistically significant reduction in the dry eye score of an individual is at least 5%, 10%, 15%, 20%, 25%, 50%, 75%, 80%, 90%, 95%, 100%, 125%, 150%, 200%, 250%, 300% or 350%. In some embodiments, a statistically significant reduction in the dry eye score of an individual is observed in an individual administered with a rAAV vector as described herein, compared to a control, wherein the statistically significant reduction in the dry eye score of the individual is at least 5%, 10%, 15%, 20%, 25%, 50%, 75%, 80%, 90%, 95%, 100%, 125%, 150%, 200%, 250%, 300% or 350%.

[0290] In some embodiments described herein, the statistically significant reduction in dry eye score of an individual is at least 3 mm, at least 5 mm, at least 10 mm, at least 15 mm, at least 20 mm, at least 25 mm, at least 30 mm, at least 35 mm, at least 40 mm, at least 45 mm, or at least 50 mm. In some embodiments, a statistically significant reduction in dry eye score of an individual is observed in an individual administered with a rAAV vector described herein compared to a control, wherein the statistically significant reduction in dry eye score of the individual is at least 3 mm, at least 5 mm, at least 10 mm, at least 15 mm, at least 20 mm, at least 25 mm, at least 30 mm, at least 35 mm, at least 40 mm, at least 45 mm, or at least 50 mm.

[0291] In some embodiments described herein, a statistically significant reduction in dry eye score for a subject is between 3 mm and 10 mm, between 3 mm and 20 mm, between 3 mm and 25 mm, between 3 mm and 30 mm, between 3 mm and 35 mm, between 3 mm and 40 mm, between 3 mm and 45 mm, between 3 mm and 50 mm, between 5 mm and 10 mm, between 5 mm and 20 mm, between 5 mm and 25 mm, between 5 mm and 30 mm, between 5 mm and 35 mm, between 5 mm and 40 mm, between 5 mm and 55 mm, between 5 mm and 50 mm, between 5 mm and 10 mm, between 5 mm and 20 mm, between 5 mm and 25 mm, between 5 mm and 30 mm, between 5 mm and 35 mm, between 5 mm and 40 mm, between 5 mm and 55 mm between 10mm and 45mm, between 5mm and 50mm, between 10mm and 15mm, between 10mm and 20mm, between 10mm and 25mm, between 10mm and 30mm, between 10mm and 35mm, between 10mm and 40mm, between 10mm and 45mm, between 10mm and 50mm, between 15mm and 20mm, between 20mm and 30mm, between 25mm and 35mm, between 30mm and 40mm, between 30mm and 45mm or between 30mm and 50mm.

[0292] In some embodiments described herein, a statistically significant decrease in the dry eyes score of an individual is characterized by a p-value of 0.05 or less, 0.01 or less, 0.005 or less, or 0.001 or less. In some embodiments described herein, a statistically significant decrease in the dry eyes score of an individual is characterized by a p-value of 0.05 or less. In some embodiments described herein, a statistically significant decrease in the dry eyes score of an individual is characterized by a p-value of 0.01 or less.

[0293] In some embodiments described herein, a statistically significant reduction in a subject's dry eye score is within 1 day, within 2 days, within 3 days, within 4 days, within 1 week, within 2 weeks, within 3 weeks, within 4 weeks, within 1 month, within 2 months, within 3 months, within 4 months, within 5 months, or within 6 months following treatment with any disclosed method or composition.

[0294] In some embodiments, the reduction in dry eye score in a subject persists for about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 9 months, or 1 year following treatment with any of the methods or compositions disclosed herein.

[0295] In some embodiments described herein, the statistically significant decrease in the dry eye score of the individual is based on the dry eye score of the individual determined after administration of a first dose of the rAAV vector.

[0296] In some embodiments described herein, a statistically significant decrease is based on a subject's dry eye score determined following treatment with any of the methods or compositions disclosed herein.

[0297] Schirmer's scoring test

[0298] Eye conditions may affect tear volume and tear production. The Schirmer's test can be used to assess an individual's tear production and evaluate the severity of dry eye, tear deficiency, or eye discomfort. The test measures the amount of tears produced in each eye. The test typically involves first placing an anesthetic into one or both eyes of the individual. These drops prevent the eyes from reacting to the test strips and shedding tears. The test administrator then places a piece of filter paper inside one or both lower eyelids and has the individual close their eyes. After 5 minutes, the test administrator removes the filter paper and assesses how well the tears have run on the filter paper. The Schirmer's test can be applied to one or both eyes.

[0299] Generally, the less the amount of moisture on the filter paper, the less tears the individual produces. In a healthy eye, each strip of paper typically contains more than 10 millimeters of moisture. A Schirmer's score of less than 10 millimeters of moisture may indicate one or more of the following conditions, including an eye condition described herein, abnormally low tear volume, or eye discomfort.

[0300] The Schirmer's test can be used to assess the effectiveness of a particular treatment in an individual, and can be administered multiple times to monitor any changes in the severity of an individual's symptoms over a period of time. An increase in the Schirmer's score over time in an individual treated for an ocular condition, tear insufficiency, or ocular discomfort described herein is evidence of an increase in tear volume or tear production, and is generally indicative of an improvement in the individual's condition. An increase in the Schirmer's score over time is evidence that the treatment is effective in treating an ocular condition described herein, increasing tear production, or improving ocular discomfort.

[0301] In some embodiments disclosed herein, effective treatment is indicated by a statistically significant increase in the subject's Schirmer's score, and wherein the statistically significant increase in the subject's Schirmer's score is determined after administering any of the methods or compositions described herein to the subject, wherein the subject's Schirmer's score is compared to: a) the subject's Schirmer's score prior to administering the first dose of the rAAV vector and the first dose of treatment to increase tear production; b) the subject's Schirmer's score administered a control; c) the subject's Schirmer's score administered a comparator compound; or d) the contralateral eye.

[0302] In some embodiments disclosed herein, the Schirmer's score of the subject is compared to the Schirmer's score of the subject before administration of the pharmaceutical composition described herein. In some embodiments disclosed herein, the Schirmer's score of the subject is compared to the Schirmer's score of the subject administered a control. In some embodiments disclosed herein, the Schirmer's score of the subject is compared to the Schirmer's score of the subject administered a comparison compound. In some embodiments disclosed herein, the Schirmer's score of the subject is compared to the fellow eye.

[0303] In some embodiments, a statistically significant increase in the Schirmer's score of an individual is observed in an individual administered with a rAAV vector described herein, as compared to a control. In some embodiments described herein, the statistically significant increase in the Schirmer's score of an individual is at least 5%, 10%, 15%, 20%, 25%, 50%, 75%, 80%, 90%, 95%, 100%, 125%, 150%, 200%, 250%, 300%, or 350%. In some embodiments described herein, the increase in the Schirmer's score of an individual is at least 100%, 200%, or 300%. In some embodiments, a statistically significant increase in Schirmer's score in an individual is observed in an individual administered a rAAV vector described herein compared to a control, wherein the statistically significant increase in Schirmer's score in the individual is at least 5%, 10%, 15%, 20%, 25%, 50%, 75%, 80%, 90%, 95%, 100%, 125%, 150%, 200%, 250%, 300%, or 350%.

[0304] In some embodiments described herein, a statistically significant increase in a subject's Schirmer's score is at least 3 mm, at least 5 mm, at least 10 mm, at least 15 mm, at least 20 mm, at least 25 mm, at least 30 mm, at least 35 mm, at least 40 mm, at least 45 mm, or at least 50 mm.

[0305] In some embodiments described herein, the statistically significant increase in a subject's Schirmer's score is between 3 mm and 5 mm, between 3 mm and 10 mm, between 3 mm and 15 mm, between 3 mm and 20 mm, between 3 mm and 25 mm, between 3 mm and 30 mm, between 5 mm and 10 mm, between 5 mm and 15 mm, between 5 mm and 20 mm, between 5 mm and 25 mm, between 5 mm and 30 mm, between 10 mm and 15 mm, between 10 mm and 20 mm, between 10 mm and 25 mm, between 10 mm and 30 mm, between 15 mm and 20 mm, between 15 mm and 25 mm, between 15 mm and 30 mm, between 20 mm and 25 mm, or between 20 mm and 30 mm.

[0306] In some embodiments, a statistically significant increase in Schirmer's score in an individual is observed in an individual administered a rAAV vector described herein compared to a control, wherein the statistically significant increase in Schirmer's score in the individual is at least 3 mm, at least 5 mm, at least 10 mm, at least 15 mm, at least 20 mm, at least 25 mm, at least 30 mm, at least 35 mm, at least 40 mm, at least 45 mm, or at least 50 mm.

[0307] In some embodiments described herein, a statistically significant decrease in a subject's Schirmer's score is characterized by a p-value of 0.05 or less, 0.01 or less, 0.005 or less, or 0.001 or less.

[0308] In some embodiments described herein, a statistically significant reduction in a subject's Schirmer's score is within 1 day, 2 days, 3 days, 4 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months following treatment with any disclosed method or composition.

[0309] In some embodiments described herein, the statistically significant decrease in the subject's Schirmer's score is based on the subject's Schirmer's score determined following treatment with any of the methods or compositions disclosed herein.

[0310] In some embodiments described herein, a statistically significant decrease is based on a subject's Schirmer's score determined following treatment according to any of the methods or compositions disclosed herein.

[0311] Ocular Surface Disease Index Test

[0312] Individuals treated for ocular conditions and related symptoms can use questionnaires to provide important information for diagnosing their conditions and determining the severity of symptoms. A well-designed questionnaire can verify reproducibility and should consist of relevant questions that elicit responsive responses. An example of a questionnaire is the Ocular Surface Disease Index (OSDI), which is a 12-question survey for individuals with ocular conditions that has been shown to be a reliable and valid tool for directly assessing symptom frequency. The ocular symptoms assessed include, but are not limited to, burning / stinging, itching, foreign body sensation, eye discomfort, dry eyes, photophobia, and pain. Most people are familiar with questionnaires and understand that this is an effective way for healthcare providers to collect information. Questionnaires can also reduce bias because there are no verbal or visual cues that can inadvertently influence respondents. The test administrator collects responses from individuals and calculates the OSDI based on the individual's answers to the questions.

[0313] The OSDI score can be used to assess the severity of eye symptoms and the effectiveness of individual specific treatment. The higher the number, the higher the severity of eye symptoms. The reduction of the OSDI score over time is the evidence that eye symptoms reduce or alleviate, and generally indicates the improvement of individual symptoms. The reduction of the OSDI score is also the evidence that the treatment effectively treats eye symptoms and alleviates symptoms.

[0314] In some embodiments disclosed herein, effective treatment is indicated by a statistically significant decrease in the OSDI score of the subject, and wherein the statistically significant decrease in the OSDI score of the subject is determined after administering to the subject a first dose, or optionally one or more subsequent doses, of a rAAV vector described herein and a first dose, or optionally one or more subsequent doses, of a treatment that increases tear production, wherein the OSDI score of the subject is compared to: a) the OSDI score of the subject before administration of the first dose of the rAAV vector and the first dose of treatment that increases tear production; b) the OSDI score of the subject administered a control; c) the OSDI score of the subject administered a comparator compound; or d) the contralateral eye.

[0315] In some embodiments disclosed herein, the OSDI score of the individual is compared to the OSDI score of the individual before treatment according to the disclosed methods described herein. In some embodiments disclosed herein, the OSDI score of the individual is compared to the OSDI score of the individual administered a control. In some embodiments disclosed herein, the OSDI score of the individual is compared to the OSDI score of the individual administered a comparison compound. In some embodiments disclosed herein, the OSDI score of the individual is compared to the OSDI score of the contralateral eye.

[0316] In some embodiments, a statistically significant reduction in the OSDI score of the subject is observed in the subject administered a rAAV vector described herein compared to a control.

[0317] In some embodiments described herein, the statistically significant decrease in the OSDI score of the individual is at least 5%, 10%, 15%, 20%, 25%, 50%, 75%, 80%, 90%, 95%, 100%, 125%, 150%, 200%, 250%, 300% or 350%. In some embodiments, a statistically significant decrease in the OSDI score of the individual is observed in an individual administered with a rAAV vector described herein compared to a control, wherein the statistically significant decrease in the OSDI score of the individual is at least 5%, 10%, 15%, 20%, 25%, 50%, 75%, 80%, 90%, 95%, 100%, 125%, 150%, 200%, 250%, 300% or 350%.

[0318] In some embodiments described herein, a statistically significant decrease in an individual's OSDI score is characterized by a p-value of 0.05 or less, 0.01 or less, 0.005 or less, or 0.001 or less.

[0319] In some embodiments described herein, a statistically significant reduction in an individual's OSDI score is within 1 day, within 2 days, within 3 days, within 4 days, within 1 week, within 2 weeks, within 3 weeks, within 4 weeks, within 1 month, within 2 months, within 3 months, within 4 months, within 5 months, or within 6 months following treatment with any disclosed method or composition.

[0320] In some embodiments, the reduction in the subject's OSDI score persists for at least 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, 9 months, or 1 year following treatment with any method or composition disclosed herein.

[0321] In some embodiments described herein, the statistically significant decrease in the OSDI score of the individual is based on the OSDI score of the individual determined after administration of a first dose of the rAAV vector.

[0322] In some embodiments described herein, a statistically significant decrease is based on an individual's OSDI score determined following treatment according to a method or composition disclosed herein.

[0323] Maintaining effective treatment over time

[0324] The efficacy of the treatment methods described herein can be assessed at any suitable time point after administration, for example, improvements in the conjunctival itch grading scale, the conjunctival redness assessment grading scale, the ocular tearing score grading scale, and / or the corneal staining grading scale can be measured at about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 18 months, about 24 months, about 3 years, about 4 years, or about 5 years after administration of the rAAV vector described herein. In some embodiments, improvements in the conjunctival itch grading scale, the conjunctival redness assessment grading scale, the ocular tearing score grading scale, and / or the corneal staining grading scale are measured at two or more time points after administration of the rAAV vector described herein, for example, every 3 months, every 6 months, or every 12 months after administration of the rAAV vector.

[0325] The present disclosure can provide effective treatment over a period of time, wherein a statistically significant improvement in an individual's score is maintained. As used in the present disclosure and when it relates to maintaining a statistically significant improvement in an individual's score (EDS, Schirmer's, corneal staining, or OSDI), the term "maintain" means that the statistically significant improvement does not decrease below a certain threshold over time. The statistically significant improvement can be maintained even if the individual's score changes at a later time point. The improvement after treatment according to the disclosed methods can be maintained without additional administration or after one or more subsequent doses.

[0326] For example, a corneal staining score of 0.5-mm or 0-mm can be defined as corneal healing and represents significant improvement 30 or 60 days after starting therapy (Bonini et al., Ophthalmology. 125:1332-1343 (2018)).

[0327] For example, a dry eyes score "maintained within 10%" means that the reduction in the dry eyes score for the individual will not decrease by more than 10% during the specified time period. Further improvements in the dry eyes score for the individual will also be considered to maintain a statistically significant improvement (e.g., if the dry eyes score improves further by 15% during the specified time period, this will be considered "maintained within 10%").

[0328] In another example, if there is a statistically significant decrease (improvement) in the subject's dry eye score 30 days after treatment according to the methods described herein, and at a later time point, the subject's score is the same or lower (indicating benefit to the subject), then the statistically significant improvement is considered to be maintained. Alternatively, if at a later time point, the subject's dry eye score is greater than the dry eye score 30 days after treatment according to the methods described herein, the subject may still receive a benefit from the treatment, and the later determined score may still be a statistically significant improvement compared to the previous administration of the first dose or its pre-treatment baseline score.

[0329] In another example, if there is a statistically significant decrease (improvement) in the subject's dry eyes score 60 days after treatment according to the methods described herein, and at a later time point, the subject's score is the same or lower (indicating a benefit to the subject), then the statistically significant improvement is considered to be maintained. Alternatively, if at a later time point, the subject's dry eyes score is greater than the dry eyes score 60 days after treatment according to the methods described herein, the subject may still be receiving a benefit from the treatment, and the score determined later may still be a statistically significant improvement compared to before treatment according to the methods described herein.

[0330] Note that what constitutes improvement is different depending on the score being measured. For example, an improvement in the dry eyes score, cornea score, and OSDI score is a decrease in the numerical value of the score. For the Schirmer's test, an improvement is typically an increase in the numerical value of the Schirmer's score.

[0331] In some embodiments described herein, maintaining a statistically significant improvement in an individual score (e.g., EDS, Schirmer's, corneal staining, or OSDI) means that the statistically significant improvement does not decrease by more than 10%, 20%, 30%, 40%, 50%, or 60%.

[0332] In some embodiments described herein, the statistically significant improvement in the individual score (e.g., EDS, Schirmer's, cornea, or OSDI) is maintained for at least 1 week, at least 1 month, at least 3 months, at least 6 months, at least 9 months, or at least 12 months. In some embodiments described herein, the statistically significant improvement in the individual score is maintained for at least 30 days after treatment according to the methods described herein, wherein the statistically significant improvement is reduced by no more than 30%. In some embodiments, the statistically significant improvement in the individual score is maintained for at least 1 month from the administration of the first dose of the rAAV vector and the optional first dose of treatment to increase tear production, wherein the statistically significant improvement is reduced by no more than 30%.

[0333] In some embodiments described herein, a statistically significant improvement in an individual score (e.g., EDS, Schirmer's, cornea, or OSDI) is maintained for at least 1 week, at least 1 month, at least 3 months, at least 6 months, at least 9 months, or at least 12 months following treatment according to the methods described herein, wherein the statistically significant improvement is no more than 10%, 20%, 30%, 40%, 50%, or 60% lower than the individual score within 60 days following treatment according to the methods described herein.

[0334] In some embodiments described herein, a statistically significant improvement in a subject's score (e.g., EDS, Schirmer's, corneal staining, or OSDI) is maintained for at least 1 week, at least 1 month, at least 3 months, at least 6 months, at least 9 months, or at least 12 months following treatment according to the methods described herein, wherein the statistically significant improvement is no less than 20% less than the subject's corresponding dry eye score, corneal score, or OSDI score within 60 days following treatment according to the methods described herein.

[0335] In some embodiments, improvements measured on the Conjunctival Itch Rating Scale, the Conjunctival Redness Assessment Rating Scale, the Ocular Tearing Rating Scale, and the Corneal Staining Rating Scale are sustained for an extended period of time (e.g., at least 3 months, at least 6 months, at least 9 months, at least 12 months, at least 15 months, at least 18 months, at least 24 months, at least 3 years, at least 4 years, or at least 5 years) following administration of a rAAV vector provided herein.

[0336] Time and method of administration

[0337] The dosage regimen administered to an individual depends on various considerations, including the duration of effectiveness of each dose, the transduction efficiency of the rAAV vector, and the effect of the dose on the body. For example, in the event that the patient's condition does not improve, at the discretion of the healthcare provider, the method for treating an ocular condition as described herein may be adjusted in dosage or repeated administration in order to improve or otherwise control or limit the symptoms of the individual's ocular condition. For example, the time period between the administration of one or more doses is extended, or the time period between the number of days on which one or more doses are administered to an individual is extended. As a non-limiting example, the administration of one or more doses is modified to administer one or more doses after measuring the symptoms of the ocular condition.

[0338] As used herein, the term "dose" may refer to the dose of the pharmaceutical composition of the present disclosure, or a therapeutic dose that alleviates the symptoms of an ocular condition.

[0339] In some embodiments described herein, the dosage of the rAAV vector is the dosage of the rAAV vector carrying the expression cassette. In such cases, the delivery of the gene product of the appropriate dose (e.g., effective dose) is achieved by applying the appropriate amount / titer of the viral vector to the target site, which allows the expression of the gene product of the effective amount over a period of time. As used herein, "effective amount" refers to the amount or dosage of the rAAV, treatment or composition described herein that is sufficient to reduce the symptoms and / or signs of the ocular conditions described herein. As used herein, the term "amount" refers to an absolute amount (e.g., an absolute amount of a protein or rAAV particle) or a concentration (e.g., the concentration of a protein in a solution), and the amount mentioned in a given example refers to an absolute amount, a concentration, or both, which will be apparent to those skilled in the art.

[0340] In some embodiments, the viral vector is an rAAV vector. In some embodiments, the viral vector is applied to the lacrimal gland. In some embodiments, rAAV is applied to the lacrimal gland by topical application. In some embodiments, rAAV is applied to the lacrimal gland by direct injection. In some embodiments, the dose of rAAV causes the gene product to be stably produced for a period of time (e.g., about 1 day, about 2 days, about 4 days, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 9 months, about 12 months or longer). In some embodiments, a dose of rAAV causes the gene product to be stably produced for about 1 week. In some embodiments, a dose of rAAV causes the gene product to be stably produced for about 2 weeks. In some embodiments, a dose of rAAV causes the gene product to be stably produced for about 3 weeks. In some embodiments, a dose of rAAV causes the gene product to be stably produced for about 4 weeks. In some embodiments, a dose of rAAV causes the gene product to be stably produced for about 1 month. In some embodiments, a dose of rAAV causes the gene product to be stably produced for about 2 months. In some embodiments, a dose of rAAV causes stable production of gene products for about 3 months. In some embodiments, a dose of rAAV causes stable production of gene products for about 4 months. In some embodiments, a dose of rAAV causes stable production of gene products for about 5 months. In some embodiments, a dose of rAAV causes stable production of gene products for about 6 months. In some embodiments, a dose of rAAV causes stable production of gene products for about 9 months. In some embodiments, a dose of rAAV causes stable production of gene products for about 12 months.

[0341] In some embodiments, the method described herein comprises administering an effective amount of rAAV as described herein to an individual, wherein the rAAV comprises a polynucleotide encoding a gene product (e.g., DAO1). In some embodiments, the method comprises delivering the first dose and one or more subsequent doses of rAAV. One or more subsequent doses are administered after a period of time after the first dose. In some embodiments, the period of time between the first dose and the next subsequent dose is at least 1 day, at least 3 days, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 1 month, at least 2 months, at least 4 months, at least 6 months, at least 9 months, at least 12 months or longer. In some embodiments, the period of time between the first dose and the next subsequent dose is between 1 and 7 days, between 1 and 4 weeks, between 2 and 6 weeks, between 4 and 8 weeks, between 1 and 3 months, between 2 and 4 months, between 3 and 6 months, between 4 and 12 months, between 6 and 24 months. In some embodiments, the time period between one or more subsequent doses is at least 1 day, at least 3 days, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 1 month, at least 2 months, at least 4 months, at least 6 months, at least 9 months, at least 12 months or longer. In some embodiments, the time period between one or more subsequent doses is between 1 and 7 days, between 1 and 4 weeks, between 2 and 6 weeks, between 4 and 8 weeks, between 1 and 3 months, between 2 and 4 months, between 3 and 6 months, between 4 and 12 months, between 6 and 24 months.

[0342] In some embodiments, the methods described herein comprise administering an effective amount of rAAV to an individual, wherein the individual has received, is receiving, or will receive a treatment described herein for increasing tear production. In some embodiments, the treatment described herein for increasing tear production comprises administering one or more doses of a nAChR agonist described herein. In some embodiments, the nAChR agonist is administered by topical administration. In some embodiments, the nAChR agonist is administered as an intranasal spray.

[0343] In some embodiments, a treatment that increases tear production (e.g., a treatment comprising administering one or more doses of a nAChR agonist to an individual) is administered on a schedule separate from the administration of rAAV. In some embodiments, the treatment that increases tear production is administered throughout the period between multiple doses of rAAV. In some embodiments, the treatment that increases tear production is administered after the first dose of rAAV. In some embodiments described herein, the treatment that increases tear production is administered for 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 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least one year, or longer. In some embodiments described herein, the treatment to increase tear production is administered for 2-52 weeks, 2-40 weeks, 2-36 weeks, 2-24 weeks, 2-12 weeks, 2-8 weeks, 4-52 weeks, 4-40 weeks, 4-36 weeks, 4-24 weeks, 4-12 weeks, 4-8 weeks, 5-52 weeks, 5-40 weeks, 5-36 weeks, 5-24 weeks, 5-12 weeks, 5-8 weeks, 6-52 weeks, 6-40 weeks, 6-36 weeks, 6-24 weeks, 6-12 weeks, or 6-8 weeks.

[0344] In some embodiments, a treatment that increases tear production (e.g., a treatment comprising administering one or more doses of a nAChR agonist to a subject) is administered to a subject one to four times per day after the first day of rAAV administration, once per day after the first day of rAAV administration, twice per day after the first day of rAAV administration, or three times per day after the first day of rAAV administration.

[0345] In some embodiments, a dosing cycle of a nAChR agonist as described herein is administered to an individual, wherein the duration between doses of the nAChR agonist increases over time. For example, dosing every 4 hours is modified to dosing every 8 or 12 hours.

[0346] In some embodiments, the treatment that increases tear production is administered for at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, or at least one year. In some embodiments, the treatment that increases tear production is administered for 2-52 weeks, 2-40 weeks, 2-36 weeks, 2-24 weeks, 2-12 weeks, 2-8 weeks, 4-52 weeks, 4-40 weeks, 4-36 weeks, 4-24 weeks, 4-12 weeks, 4-8 weeks, 5-52 weeks, 5-40 weeks, 5-36 weeks, 5-24 weeks, 5-12 weeks, 5-8 weeks, 6-52 weeks, 6-40 weeks, 6-36 weeks, 6-24 weeks, 6-12 weeks, or 6-8 weeks.

[0347] In some embodiments, the method comprises a first dose and one or more subsequent doses of rAAV. In some embodiments, the one or more subsequent doses are administered after a period of time after the first dose. The period of time between the first dose and the next subsequent dose is at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, or at least 8 hours. The time period between the first dose and the next subsequent dose is 1-3 hours, 2-4 hours, 3-6 hours, or 4-8 hours. The time period between one or more subsequent doses is at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, or at least 8 hours. The time period between one or more subsequent doses is 1-3 hours, 2-4 hours, 3-6 hours, or 4-8 hours.

[0348] In some embodiments, the time period between administration of a dose of rAAV and an effective therapeutic dose for increasing tear production is less than 5 minutes, between 5-60 minutes, between 30-90 minutes, between 1-3 hours, between 1-8 hours, between 1-12 hours, between 1-24 hours, between 8-12 hours, between 8-24 hours, between 12-24 hours, between 1-3 days, between 1-7 days, between 1-14 days, between 1-28 days, between 3-7 days, between 3-14 days, between 3-28 days, between 7-14 days, or between 7-28 days.

[0349] In some embodiments, the method comprises administering a first dose and one or more subsequent doses of rAAV, and the first dose and one or more subsequent doses of treatment to increase tear production. One or more subsequent doses are administered after a period of time after the first dose. The period of time between the first dose and the next subsequent dose is at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, or at least 8 hours. The time period between the first dose and the next subsequent dose is 1-3 hours, 2-4 hours, 3-6 hours, or 4-8 hours. The time period between one or more subsequent doses is at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, or at least 8 hours. The time period between one or more subsequent doses is 1-3 hours, 2-4 hours, 3-6 hours, or 4-8 hours.

[0350] In some embodiments, the therapy to increase tear production and the rAAV are administered to an individual in need thereof in separate dosage forms. In some embodiments, the therapy to increase tear production and the rAAV are administered to an individual in need thereof in a combined dosage form.

[0351] Pharmaceutical compositions and kits

[0352] In some embodiments, the present disclosure provides a pharmaceutical composition comprising a rAAV vector described herein. In some embodiments, the pharmaceutical composition comprises a rAAV vector described herein and a pharmaceutically acceptable carrier, delivery agent or excipient. In some embodiments, the present disclosure provides a pharmaceutical composition comprising a polypeptide comprising a DAO1 enzyme or a fragment thereof, optionally wherein the polypeptide has an amino acid sequence having at least 90% identity to an amino acid sequence selected from SEQ ID NOs: 1 and 2; and b) a pharmaceutically acceptable carrier suitable for administration to the eye of a human individual. In some embodiments, the present disclosure provides a pharmaceutical composition comprising a) a vector comprising a polynucleotide encoding a polypeptide comprising a DAO1 enzyme or a fragment thereof, optionally wherein the polypeptide has an amino acid sequence having at least 90% identity to an amino acid sequence selected from SEQ ID NOs: 1 and 2; and b) a pharmaceutically acceptable carrier suitable for administration to the eye of a human individual. In some embodiments, the polypeptide has at least 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the polypeptide comprises at least 20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, or 500 amino acids.

[0353] In some embodiments, the pharmaceutical composition may comprise 0.25, 0.50, 0.75, 1.00, 1.25, 1.50, 1.75, 2.00, 2.25, 2.50, 2.75, 3.00, 3.25, 3.50, 3.75, 4.00, 4.25, 4.50, 4.75, 5.00, 5.25, 5.50, 5.75 or 6.00 μg of a polypeptide comprising DAO1 (or a fragment thereof), or an amount within the range defined by any pair of the foregoing values; and optionally one or more excipients, diluents and / or carriers. In some aspects, the polypeptide has an amino acid sequence having at least 90% identity to an amino acid sequence selected from SEQ ID NOs: 1 and 2. In some embodiments, the polypeptide comprises at least 20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, or 500 amino acids. In some embodiments, the pharmaceutical composition is formulated for treatment of an ocular disease, disorder, or condition.

[0354] In some embodiments, the present disclosure provides the use of the rAAV vector or pharmaceutical composition described herein in the preparation of a medicament for treating an ocular disease, disorder or condition. In some embodiments, the present disclosure provides the use of the rAAV vector or pharmaceutical composition herein for or suitable for treating an ocular disease, disorder or condition.

[0355] In some embodiments, the pharmaceutically acceptable carrier comprises phosphate buffered saline. In some embodiments, the pharmaceutical composition is formulated to be compatible with its intended route of administration (e.g., within the lacrimal gland). In some embodiments, the pharmaceutical composition is formulated for administration into the lacrimal gland. In some embodiments, the pharmaceutical composition is formulated for administration onto the surface of the eye. Examples of excipients, diluents, and / or carriers (which may be referred to as pharmaceutically acceptable carriers) suitable for administration to the eye include sterile, pyrogen-free water and sterile, pyrogen-free buffered saline (e.g., saline buffered with phosphate or other buffers such as HEPES to maintain pH at an appropriate physiological level), isotonic sodium chloride solutions, balanced salt solutions, emulsions (e.g., oil / water emulsions), and various types of wetting agents.

[0356] Any concentration of vector, such as rAAV vector, suitable for effectively transducing cells of the eye, lacrimal gland, and / or nasolacrimal duct can be prepared for contacting cells of the eye, lacrimal gland, and / or nasolacrimal duct in vitro or in vivo. For example, the rAAV vector can be administered at 10 5 Vector genomes / mL or higher concentrations, such as 5×10 5 vector genomes / mL; 10 6 vector genomes / mL; 5×10 6 vector genomes / mL; 10 7 vector genomes / mL; 5×10 7 vector genomes / mL; 10 8 vector genomes / mL; 5×10 8 vector genomes / mL; 10 9 vector genomes / mL; 5×10 9 vector genomes / mL; 10 10 vector genomes / mL; 5×10 10 vector genomes / mL; 10 11 vector genomes / mL; 5×10 11 vector genomes / mL; 10 12 vector genomes / mL; 5×10 12 vector genomes / mL; 10 13 Vector genomes / mL; 1.5×10 13 vector genomes / mL; 3×10 13vector genomes / mL; 5×10 13 vector genomes / mL; 7.5×10 13 vector genomes / mL; 9×10 13 vector genomes / mL; 10 14 vector genomes / mL; 5×10 14 vector genomes / mL; 10 15 vector genomes / mL; 5×10 15 vector genomes / mL; 10 16 vector genomes / mL; or more, but usually not more than 5×10 16 In some aspects, the rAAV vector can be formulated at a concentration within a range defined by any pair of concentrations described in this paragraph.

[0357] In some embodiments, the rAAV vector comprises an AAV capsid and an expression cassette configured to express DAO1, as described herein. The formulations according to the present disclosure may comprise any concentration of such rAAV vectors, for example, at a concentration suitable for effectively transducing cells of the eye, lacrimal gland, and / or nasolacrimal duct. In some embodiments, the formulation may comprise 10 5 ; 2.5×10 5 ; 5×10 5 ; 10 7 ; 2.5×10 7 ; 5×10 7 ; 7.5×10 7 ; 10 8 ; 2.5×10 8 ; 5×10 8 ; 7.5×10 8 ; 10 9 ; 2.5×10 9 ; 5×10 9 ; 7.5×10 9 ; 10 10 ; 2.5×10 10 ; 5×10 10 ; 7.5×10 10 ; 10 11 ; 2.5×10 11 ; 5×10 11 ; 7.5×10 11 ; 10 12 ; 2.5×10 12 ; 5×10 12 ; 7.5×10 12 ; 10 13 ; 2.5×10 13 ; 5×10 13 ; 7.5×1013 ; 1×10 14 ; 2.5×10 14 ; 5×10 14 ; 7.5×10 14 ; 1×10 15 ; 1×10 16 ; or 5×10 16 rAAV vector / mL, or a concentration of rAAV vector / mL within the range defined by any pair of concentrations described in this paragraph.

[0358] In some embodiments, the present disclosure provides alternative DAO1 vehicles, such as plasmids configured to express DAO1. Formulations according to the present disclosure may contain any concentration of such plasmids, for example, at a concentration suitable for effectively transfecting cells of the eye, lacrimal gland, and / or nasolacrimal duct. In some embodiments, the formulation may contain 10 5 ; 2.5×10 5 ; 5×10 5 ; 10 6 ; 2.5×10 6 ; 5×10 6 ; 7.5×10 6 ; 10 7 ; 2.5×10 7 ; 5×10 7 ; 7.5×10 7 ; 10 8 ; 2.5×10 8 ; 5×10 8 ; 7.5×10 8 ; 10 9 ; 2.5×10 9 ; 5×10 9 ; 7.5×10 9 ; 10 10 ; 2.5×10 10 ; 5×10 10 ; 7.5×10 10 ; 10 11 ; 2.5×10 11 ; 5×10 11 ; 7.5×10 11 ; 10 12 ; 2.5×10 12 ; 5×10 12 ; 7.5×10 12 ; 10 13 ; 2.5×10 13 ; 5×10 13 ; 7.5×10 13 ; 10 14 ; 2.5×10 14; 5×10 14 ; 7.5×10 14 ; 10 15 ; 2.5×10 15 ; 5×10 15 ; 7.5×10 15 ; 10 16 ; 2.5×10 16 ; 5×10 16 or 7.5×10 16 plasmid / mL, or a plasmid / mL concentration within the range defined by any pair of concentrations described in this paragraph.

[0359] Similarly, any total number of rAAV vectors suitable for providing appropriate transduction of the eye, lacrimal gland, and / or nasolacrimal duct cells to impart a desired effect or treat a disease can be administered to the eye of a mammal or primate. In some embodiments, at least 10 rAAV vectors are injected into each eye. 5 ; 2.5×10 5 ; 5×10 5 ; 7.5×10 5 ; 10 6 ; 2.5×10 6 ; 5×10 6 ; 7.5×10 6 ; 10 7 ; 2.5×10 7 ; 5×10 7 ; 7.5×10 7 ; 10 8 ; 2.5×10 8 ; 5×10 8 ; 7.5×10 8 ; 10 9 ; 2.5×10 9 ; 5×10 9 ; 7.5×10 9 ; 10 10 ; 2.5×10 10 ; 5×10 10 ; 7.5×10 10 ; 10 11 ; 2.5×10 11 ; 5×10 11 ; 7.5×10 11 ; 10 12 ; 2.5×10 12 ; 5×10 12 ; 7.5×10 12 ; 10 13 ; 2.5×10 13 ; 5×10 13 ; 7.5×10 13; 10 14 ,2.5×10 14 ; 5×10 14 ; 7.5×10 14 ; 10 15 ; 2.5×10 15 ; 5×10 15 or 7.5×10 15 ; 10 16 ; 2.5×10 16 ; 5×10 16 or 7.5×10 16 rAAV vectors or more, but usually no more than 1×10 15 For example, in some embodiments, about 1×10 of the rAAV vector is administered to each eye. 9 About 1×10 10 , about 1×10 10 About 1×10 11 , about 1×10 11 About 1×10 12 , about 1×10 12 About 1×10 13 or about 1×10 13 About 1×10 15 In some aspects, the total number of rAAV vectors administered to the eye of the treated human individual or animal may comprise a concentration within the range defined by any pair of concentrations described in this paragraph. Any suitable number of rAAV vector administrations may be performed to the mammalian or primate eye. In some embodiments, the method comprises a single administration; in other embodiments, multiple administrations are performed over time as deemed appropriate by the attending clinician.

[0360] In some embodiments, a suitable amount or concentration of a rAAV vector (or any other vector described herein) in a therapeutic formulation may be a concentration effective to express 100 pg / mL to 50 μg / mL of DAO1 in the tear film of a subject following administration of the composition to the subject. For example, the amount or concentration of the rAAV vector or any other vector described herein (e.g., a plasmid configured to express DAO1) can be a concentration that expresses 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 μg / mL of DAO1 in the tear film of a subject (or a concentration within a range defined by any pair of the foregoing values). Expression can be measured, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 hours after administration, or after a longer duration, for example after 1, 2, 3, 4, or 5 days.

[0361] The rAAV vector can be formulated into any suitable unit dose, including but not limited to 1×10 5 vector genomes or more, e.g. 1×10 6 , 1×10 7 , 1×10 8 , 1×10 9 , 1×10 10 , 1×10 11 , 1×10 12 , 1×10 13 , 1×10 14 or 1×10 15 vector genomes or more, in some cases 1 × 10 14 vector genomes, but usually no more than 4 × 10 15 In some embodiments, the rAAV vector is formulated into any suitable unit dose, including but not limited to 1×10 5 , 1×10 6 , 1x10 7 , 1×10 8 , 1×10 9 , 1×10 10 , 1×10 11 , 1×10 12 , 1×10 13 , 1×10 14or 1×10 15 In some embodiments, the unit dose is up to about 5×10 15 vector genomes, e.g. 1×10 14 vector genomes or less, e.g. 1×10 13 , 1×10 12 , 1×10 11 , 1×10 10 or 1×10 9 vector genomes or fewer, in some cases 1 × 10 8 vector genomes or less, and usually not less than 1 × 10 8 In some embodiments, the unit dose is up to about 5×10 15 vector genomes, e.g. 1×10 14 vector genomes or less, e.g. 1×10 13 , 1×10 12 , 1×10 11 , 1×10 10 , 1×10 9 , 1×10 8 or 1×10 7 Vector genomes or less. In some embodiments, the unit dose is 1×10 10 Up to 1×10 11 In some embodiments, the unit dose is 1×10 10 Up to 3×10 12 In some embodiments, the unit dose is 1×10 9 Up to 3×10 13 In some embodiments, the unit dose is 1×10 8 Up to 3×10 14 In some embodiments, the rAAV vector comprises an AAV capsid and an expression cassette configured to express DAO1, and the unit dose is at least, at most, exactly, or about 1×10 5 , 1×10 6 , 1×10 7 , 1×10 8 , 1×10 9 , 1×10 10 , 1×10 11 , 1×10 12 , 1×10 13 , 1×10 14 , 1×10 15 or 1×10 16 The rAAV vector is formulated at a concentration of, or a concentration within a range defined by any of the concentration pairs described in this paragraph.

[0362] In some embodiments, the present disclosure provides a vector comprising a plasmid configured to express DAO1, and the unit dose is at least, at most, exactly, or about 1×10 7 , 1×10 8 , 1×10 9 , 1×10 10 , 1×10 11 , 1×10 12 , 1×10 13 , 1×10 14 or 1×10 15 The concentration of the plasmid was adjusted.

[0363] In some embodiments, the unit dose of the pharmaceutical composition can be measured using the multiplicity of infection (MOI). MOI refers to the ratio or multiple of the vector or viral genome of the rAAV vector provided herein to the cells to which the nucleic acid can be delivered. In some embodiments, the MOI can be 1×10 6 In some embodiments, the MOI may be 1×10 5 Up to 1×10 7 In some embodiments, the MOI may be 1×10 4 Up to 1×10 8 In some embodiments, the recombinant virus of the present disclosure is at least about 1×10 1 , 1×10 2 , 1×10 3 , 1×10 4 , 1×10 5 , 1×10 6 , 1×10 7 , 1×10 8 , 1×10 9 , 1×10 10 , 1×10 11 , 1×10 12 , 1×10 13 , 1×10 14 , 1×10 15 , 1×10 16 , 1×10 17 and 1×10 18 In some embodiments, the MOI of the recombinant virus of the present disclosure is 1×10 8 Up to 3×10 14 In some embodiments, the MOI of the recombinant viruses of the present disclosure is at most about 1×10 1 , 1×10 2 , 1×10 3 , 1×10 4 , 1×10 5 , 1×106 , 1×10 7 , 1×10 8 , 1×10 9 , 1×10 10 , 1×10 11 , 1×10 12 , 1×10 13 , 1×10 14 , 1×10 15 , 1×10 16 , 1×10 17 and 1×10 18 MOI.

[0364] In some embodiments, the amount of the pharmaceutical composition comprises about 1×10 5 About 1×10 16 rAAV vectors, 1×10 8 About 1×10 15 rAAV vectors, about 1×10 9 About 1×10 14 rAAV vectors, about 1×10 10 About 1×10 13 rAAV vectors or about 1×10 11 About 3×10 12 rAAV vectors.

[0365] In preparing the rAAV compositions of the present disclosure, any host cell for producing rAAV vectors can be used, including, for example, mammalian cells (e.g., 293 cells), insect cells (e.g., SF9 cells), microorganisms, and yeast. The host cell can also be a packaging cell or a production cell, in which the AAV rep and cap genes are stably maintained in the host cell, and in which the rAAV vector genome is stably maintained and packaged. Exemplary packaging and production cells are derived from SF-9, 293, A549, or HeLa cells. The rAAV vector is purified and formulated using standard techniques known in the art.

[0366] In some embodiments, the present disclosure provides the use of the rAAV vectors described herein in the preparation of a medicament. In some embodiments, the present disclosure provides the use of the rAAV vectors described herein in the preparation of a medicament for use in the methods described herein.

[0367] In some embodiments, the present disclosure provides a kit comprising the rAAV herein and instructions. In some embodiments, the kit comprises the rAAV herein, and a package insert containing the kit instructions. In some embodiments, the kit comprises the rAAV herein and a pharmaceutically acceptable carrier, or a pharmaceutical composition comprising the rAAV and instructions for treating a disease, disorder, or condition described herein or delaying progression of a disease, disorder, or condition described herein in an individual in need thereof.

[0368] Exemplary Embodiments

[0369] The present disclosure relates to the following embodiments.

[0370] Example I-1. A recombinant adeno-associated virus (rAAV) vector comprising an AAV capsid and an expression cassette comprising a polynucleotide encoding DAO1 operably linked to a promoter.

[0371] Embodiment I-2. The rAAV vector according to Embodiment I-1, wherein the polynucleotide encodes an amino acid sequence having at least 95% identity to an amino acid sequence selected from SEQ ID NO: 1 and 2.

[0372] Embodiment I-3. The rAAV vector according to any one of embodiments I-1 to I-2, wherein the polynucleotide comprises a nucleotide sequence having at least 95% identity to a nucleotide sequence selected from SEQ ID NOs: 29-30.

[0373] Embodiment I-4. The rAAV vector according to any one of embodiments I-1 to I-3, wherein the polynucleotide comprises a nucleotide sequence selected from SEQ ID NO: 29-30.

[0374] Embodiment I-5. The rAAV vector according to any one of Embodiments I-1 to I-4, wherein the promoter is a CMV promoter comprising the nucleotide sequence shown in SEQ ID NO: 21 or a CAG promoter comprising the nucleotide sequence shown in SEQ ID NO: 5.

[0375] Embodiment I-6. The rAAV vector according to Embodiment I-5, wherein the expression cassette comprises the CMV promoter and the CMV enhancer.

[0376] Embodiment I-7. The rAAV vector of any one of embodiments I-1 to I-6, wherein the expression cassette comprises a polyadenylation (polyA) sequence.

[0377] Embodiment I-8. The rAAV vector according to Embodiment I-7, wherein the polyA sequence is a BGH polyA sequence.

[0378] Embodiment I-9. The rAAV vector according to any one of embodiments I-1 to I-8, wherein the expression cassette comprises a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE).

[0379] Embodiment I-10. The rAAV vector according to any one of embodiments I-1 to I-9, wherein the expression cassette comprises a Kozak sequence.

[0380] Embodiment I-11. A composition comprising a rAAV vector, wherein the rAAV vector comprises:

[0381] (a) AAV capsid, and

[0382] (b) an expression cassette, wherein the expression cassette comprises a polynucleotide comprising a nucleotide sequence having at least 95% identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 29-31, and wherein the polynucleotide is linked to a promoter.

[0383] Embodiment I-12. The rAAV vector of any one of Embodiments I-1 to I-10 or the composition of Embodiment I-11, wherein the expression cassette is flanked by two inverted terminal repeats (ITRs).

[0384] Embodiment I-13. The rAAV vector or composition of Embodiment I-12, wherein the ITR is AAV2 ITR.

[0385] Embodiment I-14. The rAAV vector of any one of Embodiments I-1 to I-10 and I-12 or the composition of Embodiments I-11 or I-12, wherein the expression cassette comprises a sequence that shares at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO:23.

[0386] Embodiment I-15. The rAAV vector of any one of Embodiments I-1 to I-10 and I-12 to I-14, or the composition of any one of Embodiments I-11 or I-14, wherein the AAV capsid comprises a VP3 that is at least 95%, 98% or 100% identical to AAV2VP3 (SEQ ID NO: 8), AAV5 VP3 (SEQ ID NO: 10), AAV8 VP3 (SEQ ID NO: 12) or AAV9VP3 (SEQ ID NO: 14).

[0387] Embodiment I-16. An rAAV vector according to any one of Embodiments I-1 to I-10 and I-12 to I-15, or a composition according to any one of Embodiments I-11 or I-15, wherein the AAV capsid comprises a VP3 that is at least 95%, 98% or 100% identical to AAV9VP3 (SEQ ID NO: 14).

[0388] Example 1-17. A composition comprising a rAAV vector, wherein the rAAV vector comprises:

[0389] (a) AAV2, AAV5, AAV8 or AAV9 capsid, and

[0390] (b) an expression cassette, wherein the expression cassette comprises a polynucleotide comprising a nucleotide sequence having at least 95% identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 29-31, and wherein the polynucleotide is linked to a promoter.

[0391] Example 1-18. A composition comprising a rAAV vector, wherein the rAAV vector comprises:

[0392] (a) AAV2, AAV5, AAV8 or AAV9 capsid, and

[0393] (b) an expression cassette, wherein the expression cassette comprises a polynucleotide that shares at least 95% identity with SEQ ID NO:23.

[0394] Embodiment I-19. A composition according to embodiment I-17 or I-18, wherein the AAV capsid is AAV2.

[0395] Embodiment I-20. A composition according to embodiment I-17 or I-18, wherein the AAV capsid is AAV5.

[0396] Embodiment I-21. A composition according to embodiment I-17 or I-18, wherein the AAV capsid is AAV9.

[0397] Embodiment I-22. A pharmaceutical composition comprising the rAAV vector according to any one of Embodiments I-1 to I-10 and I-12 to I-15 or the composition according to any one of Embodiments I-11 to I-21, and a pharmaceutically acceptable carrier.

[0398] Embodiment I-23. The pharmaceutical composition according to Embodiment I-22, wherein the composition comprises about 1×10 7 About 1×10 14 genome copies.

[0399] Embodiment I-24. The pharmaceutical composition according to Embodiment I-22, wherein the composition comprises about 1×10 12 About 6.2×10 12 genome copies.

[0400] Embodiment I-25. The pharmaceutical composition of any one of embodiments I-22 to I-24, wherein the composition is formulated for administration into the lacrimal gland.

[0401] Embodiment 1-26. The pharmaceutical composition of any one of embodiments 1-22 to 1-25, wherein the composition is formulated for administration onto an ocular surface.

[0402] Embodiment 1-27. A pharmaceutical composition according to any one of embodiments 1-22 to 1-26, wherein the composition is formulated for or suitable for treating an ocular disease, disorder or condition.

[0403] Embodiment 1-28. A method of treating a condition in a subject in need thereof, the method comprising administering to an eye of the subject an effective amount of the pharmaceutical composition of any one of embodiments 1-22 to 1-27.

[0404] Embodiment 1-29. The method of Embodiment 1-28, wherein the pharmaceutical composition is delivered to an ocular gland of the subject.

[0405] Embodiment I-30. The method of Embodiment I-28 or I-29, wherein the pharmaceutical composition is delivered to the lacrimal gland.

[0406] Embodiment 1-31. The method of embodiment 1-30, wherein the cells within the lacrimal gland are transduced by the rAAV vector.

[0407] Embodiment 1-32. The method of Embodiment 1-31, wherein the transduced cells within the lacrimal gland express an effective amount of DAO1 into the tear film and optionally onto the ocular surface of the individual.

[0408] Embodiment I-33. The method of any one of embodiments I-28 to I-32, wherein the pharmaceutical composition is delivered to the accessory lacrimal gland.

[0409] Embodiment I-34. The method according to any one of embodiments I-28 to I-33, wherein about 1×10 9 About 1×10 10 , about 1×10 10 About 1×1011 , about 1×10 11 About 1×10 12 , about 1×10 12 About 1×10 13 or about 1×10 13 About 1×10 15 genome copies.

[0410] Embodiment 1-35. The method of any one of embodiments 1-28 to 1-34, wherein the condition is associated with increased histamine production and / or increased histamine signaling.

[0411] Embodiment 1-36. The method of any one of embodiments 1-28 to 1-35, wherein the condition is an inflammatory condition, optionally an inflammatory condition of the eye.

[0412] Embodiment 1-37. The method of any one of embodiments 1-28 to 1-36, wherein the condition is an autoimmune condition.

[0413] Embodiment 1-38. The method of any one of embodiments 1-28 to 1-36, wherein the condition is an allergic condition.

[0414] Embodiment 1-39. The method of Embodiment 1-38, wherein the condition is an allergic reaction to the therapeutic agent.

[0415] Embodiment 1-40. The method of embodiment 1-38, wherein the condition is an allergic reaction to a microbial agent.

[0416] Embodiment 1-41. The method of any one of embodiments 1-28 to 1-36, wherein the condition comprises vernal keratoconjunctivitis.

[0417] Embodiment 1-42. The method of any one of embodiments 1-28 to 1-36, wherein the condition comprises atopic keratoconjunctivitis.

[0418] Embodiment 1-43. The method of any one of embodiments 1-28 to 1-36, wherein the condition comprises seasonal or perennial allergic conjunctivitis.

[0419] Embodiment I-44. The method of any one of embodiments I-28 to I-43, wherein said administering results in expression of DAO1 in cells of said lacrimal gland and / or accessory lacrimal gland.

[0420] Embodiment I-45. The method of any one of embodiments I-28 to I-44, wherein the administering causes secretion of DAO1 into the tear film.

[0421] Embodiment I-46. The method of embodiment I-45, wherein secretion of DAO1 into the tear film is stimulated by a cholinergic agonist.

[0422] Embodiment 1-47. The method of any one of embodiments 1-28 to 1-46, wherein said administering results in improvement of one or more symptoms of said condition.

[0423] Embodiment 1-48. The method of embodiment 1-47, wherein the symptom is selected from the group consisting of itching, swelling, tearing and redness.

[0424] Embodiment 1-49. The method of any one of embodiments 1-28 to 1-46, wherein the administration causes an improvement of 0.5, 1, 1.5, 2, 2.5, 3, 3.5 or 4 points on the conjunctival itch grading scale.

[0425] Embodiment I-50. The method of any one of embodiments I-28 to I-46, wherein the administering results in an improvement of 1, 2, 3, or 4 points on a conjunctival redness assessment grading scale.

[0426] Embodiment I-51. A method according to any one of embodiments I-28 to I-46, wherein the administration causes an improvement of about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about 50%, about 50% to about 60%, about 60% to about 70%, about 70% to about 80%, about 80% to about 90%, or more than about 95% on the Schirmer's test.

[0427] Embodiment I-52. A method according to any one of Embodiments I-28 to I-46, wherein the administration causes an improvement of about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about 50%, about 50% to about 60%, about 60% to about 70%, about 70% to about 80%, about 80% to about 90%, or more than about 95% in at least one area on a corneal staining grading scale.

[0428] Embodiment I-53. The method of any one of embodiments I-47 to I-52, wherein the improvement is measured at about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 9 months, or about 12 months after the administration.

[0429] Embodiment I-54. A method according to any one of embodiments I-47 to I-52, wherein the improvement persists for at least 3 months, at least 6 months, at least 9 months, at least 12 months, at least 15 months, at least 18 months, at least 24 months, at least 3 years, at least 4 years or at least 5 years after said administration.

[0430] Embodiment I-55. The method of any one of embodiments I-28 to I-54, further comprising administering one or more additional therapeutic agents to the individual.

[0431] Embodiment 1-56. The method of Embodiment 1-55, wherein the one or more additional therapeutic agents increases tear production.

[0432] Embodiment I-57. The method of Embodiment I-56, wherein the one or more additional therapeutic agents that increase tear production are administered via topical nasal administration.

[0433] Embodiment 1-58. The method of embodiment 1-57, wherein the topical nasal administration is via an intranasal spray.

[0434] Embodiment 1-59. The method of any one of embodiments 1-56 to 1-58, wherein the one or more additional therapeutic agents that increase tear production comprise a nicotinic acetylcholine receptor (nAChR) agonist or a pharmaceutically acceptable salt thereof.

[0435] Embodiment I-60. The method of Embodiment I-59, wherein the nAChR agonist is a full agonist of an nAChR subtype selected from α4β2, α3β4, α3α5β4, α4α6β2, and combinations thereof.

[0436] Embodiment I-61. The method of any one of embodiments I-59 to I-60, wherein the nAChR agonist is varenicline or a pharmaceutically acceptable salt thereof.

[0437] Embodiment I-62. The method according to any one of embodiments I-59 to I-60, wherein the nAChR agonist is (R)-5-((E)-2-pyrrolidin-3-ylvinyl)pyrimidine or a pharmaceutically acceptable salt thereof.

[0438] Embodiment I-63. The method of any one of embodiments I-56 to I-62, wherein the one or more additional therapeutic agents that increase tear production are administered before or after administration of the pharmaceutical composition.

[0439] Embodiment 1-64. The method of any one of embodiments 1-56 to 1-63, wherein the one or more additional therapeutic agents that increase tear production are administered about 1 week after administration of the pharmaceutical composition.

[0440] Embodiment I-65. The method of any one of embodiments I-56 to I-64, which results in expression of DAO1 in the tear film and / or cornea of ​​the individual following administration of the pharmaceutical composition.

[0441] Embodiment 1-66. The method of Embodiment 1-65, wherein expression of DAO1 in the tear film and / or cornea is increased for a predetermined amount of time compared to administration of the pharmaceutical composition without the one or more additional therapeutic agents that increase tear production.

[0442] Embodiment 1-67. The method of embodiment 1-66, wherein the predetermined amount of time is about 5 minutes.

[0443] Embodiment 1-68. The method of embodiment 1-66, wherein the predetermined amount of time is about 1 hour.

[0444] Embodiment 1-69. The method of any one of embodiments 1-28 to 1-68, wherein the subject is a human.

[0445] Embodiment I-70. A pharmaceutical composition according to any one of embodiments I-22 to I-27 for use in a method of treating a condition in a subject in need thereof, comprising administering an effective amount of the pharmaceutical composition to an eye of the subject.

[0446] Embodiment I-71. A pharmaceutical composition according to any one of embodiments I-22 to I-27, for use in the manufacture of a medicament for treating a condition in a subject in need thereof.

[0447] Embodiment I-72. A kit comprising a pharmaceutical composition of the rAAV vector according to any one of Embodiments I-1 to I-10 and I-12 to I-15 or a composition according to any one of Embodiments I-11 to I-21 and a pharmaceutically acceptable carrier, and instructions for treating a condition in an individual, the kit comprising administering the pharmaceutical composition to the eye of the individual.

[0448] Embodiment I-73. A kit comprising a pharmaceutical composition of the rAAV vector of any of Embodiments I-1 to I-10 and I-12 to I-15 or a composition of any of Embodiments I-11 to I-21 and a pharmaceutically acceptable carrier, and instructions for treating a condition associated with increased histamine production and / or histamine signaling in an individual, the kit comprising administering the pharmaceutical composition to the eye of the individual.

[0449] Embodiment I-74. A kit comprising a pharmaceutical composition of the rAAV vector according to any one of Embodiments I-1 to I-10 and I-12 to I-15 or a composition according to any one of Embodiments I-11 to I-21 and a pharmaceutically acceptable carrier, and instructions for treating an autoimmune condition in an individual, the kit comprising administering the pharmaceutical composition to the eye of the individual.

[0450] Embodiment I-75. A kit comprising a pharmaceutical composition of the rAAV vector according to any one of Embodiments I-1 to I-10 and I-12 to I-15 or a composition according to any one of Embodiments I-11 to I-21 and a pharmaceutically acceptable carrier, and instructions for treating an allergic condition in an individual, the kit comprising administering the pharmaceutical composition to the eye of the individual.

[0451] Embodiment I-76. The kit of any one of embodiments I-72 to I-75, wherein the subject is receiving, has received, or subsequently received one or more additional therapeutic agents that increase tear production.

[0452] Embodiment 1-77. The kit of Embodiment 1-76, wherein the one or more additional therapeutic agents that increase tear production comprises a nicotinic acetylcholine receptor (nAChR) agonist or a pharmaceutically acceptable salt thereof.

[0453] Embodiment I-78. The kit of Embodiment I-76 or I-77, wherein the one or more additional therapeutic agents that increase tear production comprise varenicline or a pharmaceutically acceptable salt thereof.

[0454] Embodiment I-80. The kit of Embodiment I-76 or I-77, wherein the one or more additional therapeutic agents that increase tear production comprise (R)-5-((E)-2-pyrrolidin-3-ylvinyl)pyrimidine or a pharmaceutically acceptable salt thereof.

[0455] Embodiment I-81. The kit of Embodiment I-76 or I-80, wherein the subject receives the one or more additional therapeutic agents after administration of the pharmaceutical composition.

[0456] Example 1-82. A pharmaceutical composition comprising a) a polypeptide comprising a diamine oxidase ("DAO1") enzyme or a fragment thereof, optionally wherein the polypeptide has an amino acid sequence having at least 90% identity to an amino acid sequence selected from SEQ ID NOs: 1 and 2; and b) a pharmaceutically acceptable carrier suitable for administration to the eye of a human subject.

[0457] Example 1-83. A pharmaceutical composition comprising a) a vector comprising a polynucleotide encoding a polypeptide comprising a diamine oxidase ("DAO1") enzyme or a fragment thereof, optionally wherein the polypeptide has an amino acid sequence having at least 90% identity to an amino acid sequence selected from SEQ ID NOs: 1 and 2; and b) a pharmaceutically acceptable carrier suitable for administration to the eye of a human subject.

[0458] Embodiment I-84. A pharmaceutical composition according to Embodiment I-82 or I-83, wherein the pharmaceutically acceptable carrier comprises water; sterile water; pyrogen-free water; phosphate-buffered saline; HEPES-buffered saline; isotonic sodium chloride solution; balanced salt solution; a wetting agent; a surfactant; a tonicity agent; a pH adjuster; a viscosity modifier; a buffer; a disaccharide, optionally sucrose or trehalose; cellulose and / or its derivatives; an amino acid, optionally histidine; or any combination thereof.

[0459] Embodiment I-85. A pharmaceutical composition according to any one of embodiments I-82 to I-84, 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: 2.

[0460] Embodiment 1-86. A pharmaceutical composition according to any one of embodiments 1-82 to 1-85, wherein the polypeptide comprises at least 20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480 or 500 amino acids.

[0461] Embodiment 1-87. The pharmaceutical composition of any one of embodiments 1-82 to 1-86, wherein the formulation is a liquid formulated for administration to or into the ocular surface of the eye of the human subject or for intra-lacrimal injection.

[0462] Embodiment I-88. A pharmaceutical composition according to any one of Embodiments I-82 to I-87, comprising the carrier, wherein the carrier is present in the composition in an amount effective to express 100 pg / mL to 50 μg / mL of the polypeptide in the tear film of the individual after administering the composition to the individual.

[0463] Embodiment I-89. A pharmaceutical composition according to any one of embodiments I-82 to I-88, comprising the vector, wherein the polynucleotide is operably linked to a promoter.

[0464] Embodiment I-90. A pharmaceutical composition according to any one of embodiments I-82 to I-89, comprising the vector, wherein the vector is engineered to constitutively express the polypeptide having an amino acid sequence that is at least 90% identical to an amino acid sequence selected from SEQ ID NO: 1 and 2.

[0465] Embodiment I-91. A pharmaceutical composition according to any one of Embodiments I-82 to I-90, comprising the vector, wherein the vector comprises a virus, optionally an adenoviral vector or a lentiviral vector; a plasmid; an episome; or an artificial chromosome; and optionally comprises one or more lipids, polycations, DNA-carrier proteins, histones, pseudocapsids, chimeric proteins or endocytic receptor proteins.

[0466] Embodiment I-92. The pharmaceutical composition according to any one of embodiments I-82 to I-87, comprising the polypeptide, wherein the polypeptide is present in the pharmaceutical composition at a concentration of 100 pg / mL to 50 μg / mL.

[0467] Embodiment I-93. The pharmaceutical composition according to any one of embodiments I-82 to I-87 or I-93, comprising the polypeptide, wherein the polypeptide is present in the pharmaceutical composition in an amount of 500 ng to 5 μg.

[0468] Embodiment I-94. A pharmaceutical composition according to any one of embodiments I-82 to I-87, I-92 or I-93, comprising the polypeptide, wherein the polypeptide is present in the pharmaceutical composition in a unit dose.

[0469] Embodiment 1-95. A method of treating a condition in a subject in need thereof, the method comprising administering to an eye of the subject an effective amount of a pharmaceutical composition according to any one of Embodiments 1-82 to 1-94.

[0470] Embodiment 1-96. The method of Embodiment 1-95, wherein the ocular disease, disorder or condition is associated with increased histamine production and / or increased histamine signaling.

[0471] Embodiment 1-97. The method of Embodiment 1-95 or 1-96, wherein the condition is an inflammatory condition, optionally an inflammatory condition of the eye.

[0472] Embodiment 1-98. The method of any one of embodiments 1-95 to 1-97, wherein the condition is an autoimmune condition.

[0473] Embodiment 1-99. The method of any one of embodiments 1-95 to 1-98, wherein the condition is an allergic condition.

[0474] Embodiment I-100. The method of any one of embodiments I-95 to I-99, wherein the condition is an allergic reaction to the therapeutic agent.

[0475] Embodiment I-101. The method of any one of embodiments I-95 to I-100, wherein the condition is an allergic reaction to a microbial agent.

[0476] Embodiment I-102. The method of any one of embodiments I-95 to I-101, wherein the condition comprises vernal keratoconjunctivitis.

[0477] Embodiment I-103. The method of any one of embodiments I-95 to I-102, wherein the condition comprises atopic keratoconjunctivitis.

[0478] Embodiment 1-104. The method of any one of embodiments 1-95 to 1-103, wherein the condition comprises seasonal or perennial allergic conjunctivitis.

[0479] Embodiment I-105. The method of any one of embodiments I-95 to I-104, wherein said administering results in expression of a functional diamine oxidase in one or more cells of the lacrimal gland and / or accessory lacrimal gland of said individual.

[0480] Embodiment I-106. The method of any one of embodiments I-95 to I-105, wherein the administering causes secretion of functional diamine oxidase into the tear film of the individual.

[0481] Embodiment I-107. The method of any one of embodiments I-95 to I-106, wherein the secretion of the functional diamine oxidase into the tear film is stimulated by a cholinergic agonist.

[0482] Embodiment I-108. The method of Embodiment I-107, wherein the administering is to or into the ocular surface of the subject, and / or to the lacrimal gland of the subject.

[0483] Embodiment I-109. The method according to any one of embodiments I-95 to I-108, wherein the symptom is selected from the group consisting of itching, swelling, tearing and redness.

[0484] Embodiment I-110. The method of any one of embodiments I-95 to I-109, wherein the administering causes an improvement of 0.5, 1, 1.5, 2, 2.5, 3, 3.5 or 4 points on the conjunctival itch grading scale.

[0485] Embodiment I-111. The method of any one of embodiments I-95 to I-110, wherein the subject is a human subject.

[0486] Embodiment I-112. A kit comprising a pharmaceutical composition according to any one of embodiments I-82 to I-94 and instructions for treating a condition in a human subject, the kit comprising administering the pharmaceutical composition to an eye of the human subject.

[0487] Embodiment I-113. A pharmaceutical composition according to any one of embodiments I-82 to I-94 for use in the manufacture of a medicament for treating a condition in a human subject in need thereof.

[0488] Examples

[0489] The following specific examples are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.

[0490] Example 1: In vitro transfection of 293T cells with AAV transfer plasmid encoding DAO1

[0491] Conduct research to evaluate the use of Figure 1A Expression and activity of the DAO1 polypeptide (referred to as "AAV.DAO1") after in vitro transfection of 293T cells with the AAV transfer plasmid depicted in . The AAV transfer plasmid contains 5' and 3' AAV2 ITRs (SEQ ID NOs: 16 and 17, respectively). Between the 5' and 3' ITRs is a cDNA encoding the human DAO1 polypeptide and elements for providing optimal expression of the DAO1 polypeptide. A schematic diagram depicting the construct is shown in Figure 1BThe encoded human DAO1 polypeptide has the amino acid sequence shown in SEQ ID NO: 2. The cDNA encoding the human DAO1 polypeptide is codon-optimized to improve expression in human cells and has the nucleotide sequence shown in SEQ ID NO: 29. The nucleotide sequence from 5' ITR to 3' ITR is shown in SEQ ID NO: 23.

[0492] According to the manufacturer's rapid transformation protocol, plasmid amplification was performed by adding 10ng of AAV.DAO1 plasmid to 50μl New England Biolabs (NEB) stable competent Escherichia coli (high efficiency grade) (NEB C3040). Single bacterial colonies were selected and kanamycin (kanamycin) (50μg / mL) was used as the positive selection of related colony clones for inoculation of 5mL Luria broth (LB). The starter culture was cultivated at 30°C for 16 hours and then used to inoculate 250mL LB / kanamycin with a 1 / 500 dilution. This culture was cultivated at 30°C for 16 hours and then centrifuged at 4,000×g for 20 minutes to precipitate bacteria. Qiagen Hi-Speed ​​Maxi Kit (Qiagen 12662) was used to purify plasmid DNA according to the manufacturer's protocol. Based on fragment size, plasmids were verified by restriction enzyme digestion and agarose gel electrophoresis.

[0493] 293T cells were cultured at 6.5 × 10 5 Cells / well were seeded in 6-well plates and transfected with 2.5 μg of AAV.DAO1 plasmid DNA using Lipofectamine 3000 according to the manufacturer's protocol. Control cells were transfected with empty vector. The cells were grown and then incubated at 5.5 × 10 5 Seed on 35 mm glass bottom tissue culture plates.

[0494] Immunofluorescence

[0495] Immunofluorescence imaging was performed to detect DAO1 expression in transfected cells.

[0496] 293T cells were cultured at 5.5 × 10 5 PANC-1 cells were seeded at 4×10 5Seeded on 33mm glass bottom TC culture dishes (MatTek P35G-0-10-C) coated with poly-L-lysine (VWR103701-192). Twelve to twenty-four (12-24) hours after seeding the cells, 3.75μl of lipofectamine 3000 and 2.5μg of pAAV-DAO plasmid were used per well to transfect the cells according to the lipofectamine 3000 (ThermoFisher L300015) transfection protocol. 24 hours after transfection, the culture medium was aspirated and the cells were washed twice with preheated PBS (37°C). 500μl of 4% paraformaldehyde was added and incubated at room temperature for 10 minutes. The cells were washed twice with PBS. 500μl of blocking buffer was added and the cells were incubated at room temperature for 45 minutes. The blocking buffer consisted of PBS, 0.1% Triton X and 1% bovine serum albumin (BSA). The cells were stained with a primary antibody to diamine oxidase and a fluorescently labeled secondary antibody. The cells were counterstained with DAPI and then imaged on a fluorescence microscope. Specifically, the primary antibody was diluted in blocking buffer, and 200 μl of the diluted mixture was added to the center glass portion of the culture dish and incubated at 37°C for 2 hours. The primary antibody concentration was optimized experimentally and a 1:50 dilution was used. The cells were washed 3 times with PBS for 15 minutes. The fluorescently labeled secondary antibody and DAPI were diluted 1:400 in PBS, added to the glass portion of the culture dish, and incubated at room temperature for 1 hour (protected from light). The cells were then washed 3 more times with PBS for 15 minutes. The cells were then imaged on a Keyence BZ-X series microscope.

[0497] like Figure 4 As shown in A-4B, the expression of DAO1 protein was measured in 293T cells transfected with AAV transfer plasmids (as indicated by bright and punctate signals), but not in control cells. Figure 4 As shown in C-4D, the expression of DAO1 protein was further detected in PANC-1 cells transiently transfected with AAV.DAO plasmid.

[0498] Western blotting

[0499] Western blot was performed to detect DAO1 expression in the conditioned medium of cells transfected with AAV.DAO1 compared to control cells, wherein rabbit anti-DAO1 antibody and labeled anti-rabbit secondary antibody were used for detection of DAO1. For conditioned medium collection, the cell culture medium was aspirated 24 hours after transfection and washed twice with 2mL preheated Opti-MEM (Thermo Fisher 31985-070). Then 1.4ml OPTI-MEM was added and conditioned medium was collected at intervals of 2, 4 and 6 hours. 1× protease inhibitor was added immediately after collection, and the conditioned medium was placed on ice for 5 minutes and centrifuged at 12,000×g for 5 minutes at 4°C to remove culture debris. The conditioned medium supernatant was then concentrated 40 times and stored at -20°C using an Amicon Ultra-2 centrifugal filter unit (Millipore UFC201024) according to the manufacturer's protocol.

[0500] Whole cell extracts were also used for Western blotting. Whole cell extracts were prepared by aspirating cell culture medium 24 or 48 hours after transfection. Cells were washed twice with 2 mL of 4 ° C PBS and then lysed using 300 μl RIPA buffer (Sigma R0278) containing protease / phosphatase inhibitors (Cell Signaling 5872S) diluted from 100X stock solution to 1×. Whole cell extracts were then centrifuged at 12,000 × g for 5 minutes at 4 ° C to remove any cell debris, and the supernatant was then aliquoted and stored at -20 ° C. Whole cell extracts (according to the above method) were quantitatively collected using a micro-bicinchoninic acid assay (micro-bicinchoninic acid assay, BCA) protein analysis kit (Thermo Fisher 23235) according to the manufacturer's recommendations. Sample concentrations were normalized by diluting with double distilled water (ddH20) and Laemmli buffer. The extract was then heated to 100 ° C for 10 minutes. The samples were electrophoresed on a 10% SDS-PAGE gel and then transferred to a PVDF membrane using an Invitrogen iBlot 2 dry blotting system. The membrane was blocked for 1 hour at room temperature on a rotating platform using a 5% blotting grade blocking agent (BioRad 1706404). The primary antibody was diluted in a 5% blocking buffer containing 0.1% Tween 20 and incubated overnight at 4°C on a shaking platform. The optimized primary antibody concentration was determined experimentally. On a rotating platform, the membrane was washed in PBS containing 0.05% Tween 20 for 10 minutes. The washing step was repeated 4 times. The HRP-conjugated secondary antibody was diluted (1:10,000) in a 5% blocking agent containing 0.1% Tween20 and incubated for 1 hour on a rotating platform at room temperature. The previous washing steps were repeated, with the last wash being PBS alone. The membrane was incubated in SuperSignal West Pico Plus (Thermo Fisher 34577) for 1 min and chemiluminescent detection was performed on a Bio-Rad ChemiDoc MP.

[0501] As controls, Western blotting was also performed using human DAO1 recombinant protein (40 ng) and mouse anti-his antibody. Western blotting of GAPDH (30 μg / well) served as an internal reference.

[0502] like Figure 5A As shown, DAO1 polypeptide was observed in the culture medium obtained from the transfected cells, but not in the control cells. The DAO1 polypeptide had the same molecular weight as the human DAO1 recombinant protein used as a positive control ( Figure 5B5C-5D ). As shown in FIG. 5C-5D , DAO1 of the expected size (kDa) was detected in whole cell extracts obtained from transfected 293T cells. Recombinant DAO1 served as a positive control ( FIG. 5C ) and GAPDH served as an internal reference ( FIG. 5D ).

[0503] Additionally, DAO1 expression in conditioned media was measured 2, 4, and 6 hours after fresh media exchange. As shown in Figure 5E, significant levels of DAO1 were detected in as little as 2 hours after media exchange.

[0504] Together these data demonstrate that DAO1 is expressed and secreted in cell culture medium.

[0505] DAO1 enzyme activity analysis

[0506] In order to evaluate the functional activity of the expressed DAO1 protein, an activity assay was performed. The principle of the assay is to provide a substrate that causes a reaction that releases hydrogen peroxide (H2O2) in the presence of active DAO1. The H2O2 released by the cleavage substrate reacts with a second substrate and produces a fluorescent signal that excites at 535nm and emits at 587nm. The amount of fluorescence recorded at 587nm indicates the amount of enzyme activity.

[0507] The experimental method involves seeding 293T cells into a 6-well plate format and transfecting with AAV.DAO. Negative control cells were not transfected. Twenty-four hours after transfection, the culture medium was removed and supplemented with 2 mL / well maintenance medium. Twenty-four, conditioned medium and whole cell extract (WCE) were collected. After collection, protease inhibitors were added, and the conditioned medium / WCE was placed on ice for 5 minutes and centrifuged at 12,000 × g for 5 minutes at 4°C to remove culture debris. The culture medium / WCE was aliquoted and stored at -20°C. Micro BCA analysis was performed to quantify the protein level of WCE. Standards were prepared using 10mM H2O2 provided by the diamine oxidase activity assay kit (Sigma MAK351). Serially diluted standards, undiluted AAV.DAO conditioned medium, 100ng WCE, and control samples were added to a 96-well reaction plate. The reaction mixture was prepared using DAO assay buffer, substrate, enzyme mixture, and probe. The reaction plate was then incubated at 37°C for 90 minutes and kinetic measurements were performed at 30, 60 and 90 minutes after incubation. Measurements were performed using a Molecular Devices ID3 plate reader at 535 nm excitation wavelength and 587 nm emission wavelength. DAO1 activity was measured in nmol H2O2 (experiment 1) or pmol / min / μl (experiment 2, using kinetic measurements) produced within 90 minutes.

[0508] like Fig. 6AAs shown, a calibration curve was generated by measuring the fluorescence signal (535 nm / 587 nm) in the presence of increasing concentrations of H2O2. The assay was used to measure the activity of DAO1 in conditioned medium obtained from 293T cells transfected with AAV.DAO1 or in cell lysates combined with conditioned medium and compared with the activity measured in conditioned medium obtained from untransfected 293T cells. Figure 6B As shown, significantly higher H2O2 was produced in conditioned medium alone or in combination with cell lysate obtained from 293T cells transfected with AAV.DAO1 compared to conditioned medium obtained from untransfected 293T cells. As shown in FIG6C , kinetic measurements of DAO1 activity in conditioned medium from transfected 293T cells at 30, 60, and 90 minutes indicated the target regulatory capacity and functional activity of DAO1 expressed by AAV-DAO.

[0509] This indicates that the DAO1 protein present in the cells and expressed into the cell culture medium is able to cleave the given substrate and exhibit functional enzymatic activity.

[0510] Example 2: Intra-lacrimal gene therapy using rAAV vectors

[0511] This example shows a 9-day pilot study in which a single dose of rAAV vector was administered as an intra-lacrimal injection followed by a single dose of varenicline as an intranasal dose to Dutch-Belted rabbits. It evaluated the efficacy and tolerability of a panel of rAAV vector embodiments administered once via injection to the lacrimal gland. At two concentrations (1×10 12 GC / mL and 6.2×10 12 Each rAAV vector composition in the test group was tested at 100 GC / mL. The rAAV vector group included examples of capsid proteins with AAV2, AAV5, AAV8, and AAV9 serotypes. The expression cassette delivered by the rAAV vector encodes an enhanced green fluorescent protein (eGFP) transgene ( Figure 7 ). On day 9 after the injection of the rAAV vector, the animals were given an intranasal dose of varenicline. Varenicline induces tear production in the animals so that eGFP delivered to the lacrimal gland by the rAAV vector and expressed under the control of the CMV promoter in the lacrimal gland cells will be secreted into the tear film and onto the ocular surface of the animals. Two main goals will be achieved using this approach:

[0512] (1) To test the feasibility of capsid serotypes AAV2, AAV5, AAV8, and AAV9 to deliver transgenes to cells in the lacrimal gland, resulting in measurable CMV promoter-driven expression of the transgene within the cells.

[0513] (2) To evaluate the feasibility of increasing the relative amount of a transgene encoded by an expression cassette that enters the tear film and onto the ocular surface of animals.

[0514] Animal studies were performed at Charles River Laboratories (CRL) by CRL researchers.

[0515] Animal testing systems, husbandry and life monitoring

[0516] The animals used in this study were male Dutch rabbits, aged between 4 and 5 months, and weighing 1.3 to 2.3 kg. The animals were acclimated for 10 days before treatment began. Each animal was housed individually and cared for using a standard care regimen, including regular environmental conditions, feeding schedules, and veterinary care.

[0517] rAAV vector compositions and formulations

[0518] In this study, a composition group comprising a rAAV vector containing an expression cassette encoding an eGFP transgene operably linked to a CMV promoter was provided for intra-lacrimal injection under the conditions in Table 6. Each composition contained a rAAV vector having a different AAV capsid protein serotype. The compositions were labeled OC-100a-d, each corresponding to a different AAV capsid protein serotype. The dosage formulations for intra-lacrimal injection were prepared using a clean procedure at the target concentrations described in Table 8 below by diluting with phosphate buffered saline solution.

[0519] Table 6. Summary of rAAV compositions

[0520]

[0521] Intra-lacrimal injection of rAAV compositions

[0522] Animals were dosed via intra-lacrimal injection on day 1 of the study. An overview of the formulation concentration, dose volume, dose frequency, and number of animals and lacrimal glands for each composition tested is shown in Table 7. Prior to injection, animals were anesthetized by intramuscular injection of dexmedetomidine (0.25 mg / kg) with an isoflurane / oxygen mixture injected via mask to maintain anesthesia as necessary. Following dosing, topical antibiotics were applied to each eye. On day 9 of the study, animals were administered varenicline tartrate intranasally (50 μL of 1.2 mg / mL varenicline per nostril) to induce tear production.

[0523] Table 7. Overview of experimental design

[0524]

[0525] Biological analysis

[0526] Blood was collected from the ear vessels of all animals on the 1st day before dosing, and again on the 8th and 9th days (about 1 hour after intranasal administration). The blood samples were placed on ice until plasma was separated by centrifugation. The plasma samples were divided into 250 μL aliquots and frozen at -80°C for subsequent analysis.

[0527] A Schirmer's tear test was performed on days 8 and 9 to collect eye moisture from the animals. The test strip was placed inside the lower eyelid for approximately 1 minute. The test strip was removed and placed in individual tubes and frozen at -80°C for subsequent analysis.

[0528] Plasma samples and Schirmer's test strips were analyzed for eGFP concentration and eGFP mRNA concentration using validated procedures from Syneos Analytical Laboratories.

[0529] Immunohistochemistry of lacrimal gland tissue

[0530] Animals were euthanized on day 9 after blood and eye water were collected by intravenous injection of sodium pentobarbital. Five sagittal sections of the left eye and sections of the left and right lacrimal glands were prepared for immunohistochemistry (IHC) according to laboratory standard operating procedures. Lacrimal gland IHC samples were stained for eGFP and evaluated microscopically.

[0531] result

[0532] Microscopic evaluation was performed to determine the efficiency of eGFP expression in lacrimal gland tissues administered in vivo with rAAV compositions. Isolated positive acinar cells in IHC samples had pink to red cytoplasmic staining indicative of GFP expression ( Figures 8A-8K ; Exemplary staining is indicated by black arrows). 12 GC / mL of rAAV composition of AAV2 capsid protein (OC-100a) ( Fig. 8A ), containing 1×10 12 GC / mL of rAAV5 capsid protein composition ( Figure 8B ) and 6.2×10 12 GC / mL of rAAV5 capsid protein composition ( Figures 8C-8H ) and contains 6.2×10 12 GC / mL of rAAV9 capsid protein composition ( Figures 8I-8K ) was observed to have positive eGFP expression.

[0533] in conclusion

[0534] The results of this example show that rAAV vectors can be used to deliver expression cassettes to the lacrimal gland by direct injection. The results also show that rAAV vectors containing capsid proteins having at least AAV2, AAV5, or AAV9 serotypes can be used to deliver expression cassettes to cells within the lacrimal gland. In addition, the results demonstrate that delivery of an expression cassette containing a transgene operably linked to a CAG promoter sequence results in expression of the transgene in lacrimal gland cells.

[0535] Example 3: Expression of EGFP transgene in porcine lacrimal glands via intra-lacrimal injection delivery of rAAV

[0536] The aim of the study was to evaluate whether the lacrimal gland could be used as a method to modify or enrich the tear film with a protein of interest in pigs. Subsequently, in vivo studies were performed to test whether EGFP could be produced in the acinar cells of the lacrimal gland and then secreted into the tear film following delivery of an adenoviral vector consisting of a plasmid encoding eGFP. To introduce the cDNA encoding EGFP into the acinar cells, an adeno-associated virus (AAV) containing the cDNA encoding secreted EGFP (secEGFP) was injected into the lacrimal gland. To produce each of the 2 different serotypes (2 and 9) of AAV for secEGFP, an AAV transfer plasmid was generated that contained the essential elements for secEGFP expression between the inverted terminal repeats (ITRs). The DNA sequence between the ITRs was packaged into the manufactured AAV ( Figure 7 ).

[0537] Design Analysis and Methods

[0538] Research-grade AAV for secreted EGFP (serotypes 2 and 9) was synthesized at Sirion and cultured at 5 × 10 12 AAV was provided at a stock concentration. AAV was tested in vitro by CJ solution using HEK 293T cells and ELISA to ensure that the manufactured AAV would transduce cells. At Texas A&M University (Texas A&M), eight domestic pigs received a single intra-lacrimal injection of EGFP, with the right (OD; right eye (oculus dexter)) lacrimal gland receiving a low dose and the left (OS; left eye (oculus sinister)) lacrimal gland receiving a high dose. Six weeks after the first injection, a second high-dose injection of AAV2 and AAV9 was performed. The study evaluated EGFP expression on day 35. After confirmation of tear EGFP levels, the study was terminated 8 weeks after the second injection to evaluate the presence of EGFP in the lacrimal gland and to evaluate any potential inflammation or glandular abnormalities. (Tables 8 and 9). In the study, nasal spray administration was applied between weeks 3 and 4 (Table 10).

[0539] Table 8: Research plan for in vivo studies of AAV2-secEGFP and AAV9-secEGFP in domestic pigs.

[0540]

[0541]

[0542] Table 9: AAV injection dose and volume.

[0543]

[0544] vg = viral genome

[0545] Table 10: OC-01 nasal spray administration from Day 21 to Day 28.

[0546]

[0547] mcg = microgram

[0548] After the second AAV-secEGFP injection, tears were collected from each eye via Schirmer's strips on day 82. Tears were collected by placing a Schirmer's tear test strip in the inferior conjunctival fornix and leaving it in place for 2 minutes. Tear proteins were extracted from the Schirmer's tear test strips and subjected to mesoscale discovery (MSD) analysis to detect the presence of EGFP protein in tears.

[0549] Lacrimal glands were collected on day 103 for ocular histopathology, and samples were sent to Zyagen (San Diego, CA) for EGFP immunohistochemistry (IHC).

[0550] ELISA results showed that the manufactured AAV serotypes could transduce HEK 293T cells in vitro and produce secreted EGFP. EGFP expression in tear samples was confirmed by MSD analysis and by IHC 82 days after AAV transduction with eGFP, with some levels >400 pg / mL ( Fig. 9 ). IHC showed that EGFP was expressed in acinar cells, and AAV2 was observed to be more infective of acinar cells compared with AAV9. In addition, transduction of ductile epithelial cells was observed in the lacrimal gland injected with AAV9 ( Fig.10 ). Hematoxylin and eosin staining of porcine lacrimal glands after repeated AAV injections did not reveal any inflammatory infiltration, atrophy, or edema ( Fig.11 ).

[0551] Porcine lacrimal glands injected with AAV2-secEGFP or AAV9-secEGFP expressed the EGFP transgene product in acinar cells as well as in ductile epithelial cells. EGFP expressed in the lacrimal gland was found to be secreted into the tear film. Additionally, no safety signals or inflammatory infiltrates were observed in any of the animals following repeated injections of AAV2 or AAV9, regardless of whether they initially received a low or high dose of AAV during the first injection. The results of the described study suggest that acinar cells of the lacrimal gland are a target for gene therapy approaches to modify and / or enrich the tear film.

[0552] Example 4: Expression of rAAV transgenes in porcine lacrimal glands in combination with OC-1 nasal spray

[0553] This example describes a study conducted in pigs to evaluate the expression of rAAV encoding a model protein after a single lacrimal gland injection. The purpose of this study is to determine the expression of mRNA encoding a model protein (referred to as "protein_A" in the examples and figures) in the lacrimal gland and the level of transgenic protein in tears after a single injection of an AAV encoding protein_A (referred to as "AAV-protein_A" in the examples and figures) to transduce the lacrimal glands of pigs. The study is further designed to confirm the expression and secretion of protein_A after intra-lacrimal injection, and to evaluate the relative amount of protein present on the ocular surface after stimulating tear production with varenicline nasal spray. Varenicline ("OC-1") is the following compound:

[0554]

[0555] The AAV-Protein_A plasmid encodes AAV2 5′ITR, CMV enhancer / promoter, Kozak-containing intron sequence, open reading frame encoding Protein_A, woodchuck hepatitis virus posttranscriptional regulatory elements, polyA sequence and AAV2 3′ITR from 5′ to 3′.

[0556] Study parameters are detailed in Table 11. Administration and dosage are shown in Table 12.

[0557] Table 11: Study plan for in vivo evaluation of intra-lacrimal injection of rAAV in domestic pigs

[0558]

[0559] Table 12: AAV injection dose and volume

[0560]

[0561] The concentration of AAV-Protein A stock solution was 5×10 12 vg / mL and were provided in 500 μL aliquots and stored at -20°C (short term, <2 years) or -80°C (long term).11 vg).

[0562] Timeline of pig research Fig.12 As shown. For all pigs (N=14), day 0 was the injection day. Tears were collected from each eye via Schirmer's strips (applied for about 2 minutes and then removed) on days 7, 14, 21, 28, 35, 42, 60 / 61, and 90. The Schirmer's strips were immediately cut above the fluid line or dye line with a pair of scissors. The bottom of the Schirmer's strip (tear saturated) was then placed in a microcentrifuge tube and kept on ice until transferred to a refrigerator (-80°C). On days 14 and 42, tear collection was performed first, and then the nasal spray dose was administered. About 2 minutes after the nasal spray dose was administered, tear collection was performed on the second day. For days 22 to 28, OC-01 nasal spray was applied to both nostrils twice a day (at least 6 hours between administrations) on days 22 to 27, and once on day 28. Tear collection on day 28 will be performed 2 minutes after the nasal spray is administered.

[0563] At day 90, gross pathology was performed and body weight was assessed. In addition, one lacrimal gland from each animal was collected for ocular histopathology by immunohistochemistry (IHC). After the lacrimal glands were collected, they were fixed in 10% formalin for 24 to 48 hours (at room temperature), then fixed in 70% EtOH and stored at 4°C. The volume in the tube was kept 5 times the volume of the tissue, and the tissue was completely immersed.

[0564] The second lacrimal gland was collected, rinsed with phosphate buffered saline, and immediately placed in RNA-Later. The samples were snap frozen in liquid nitrogen. Approximately 0.5-1 cm square tissue pieces from the apex, kidney, and liver were collected and immediately placed in 2 mL cryovials and snap frozen in liquid nitrogen. The cryovials were then stored at -80°C until shipped for mRNA analysis.

[0565] mRNA analysis was performed using the developed one-step duplex RT-qPCR method to analyze the expression of AAV-derived protein_A gene in the lacrimal gland of domestic pigs. Each lacrimal gland was homogenized and the lysate was loaded into QIASymphony for automatic RNA extraction using silica-based RNA purification, magnetic separation and enzyme-removed DNA. The AAV-protein_A vector contains bovine growth hormone (bGH) polyA sequences in the 3' untranslated region of the transgene. Protein_A mRNA was analyzed using primers and probes targeting the bGH polyA sequence, and the sequences are shown in Table 13. 76bp of the bGH sequence was amplified. The Ct values ​​of bGH mRNA copies and pig endogenous Hprt1 mRNA of the extracted total RNA samples were analyzed on 96-well plates using the QuantStudio 7Flex real-time PCR system and one-step duplex RT-qPCR method (see Table 14). Each plate includes a standard curve, a negative control and a quality control sample, which are prepared separately to avoid cross contamination. Each standard curve includes 108, 107, 105, 104, 103, 102, 50, 25 and 0 copies of bGH standard DNA levels per well. RT-qPCR of RNA samples was performed in duplicate wells up to 100 ng / well. The number of bGH mRNA copies in each RT-qPCR well was interpolated from the bGH DNA standard curve (acceptance criteria: R2≥0.980). The interpolated single-stranded (ss) mRNA from the double-stranded (ds) standard curve was adjusted using a two-fold multiplication step, and the average copy number of the two replicate wells will be reported as the number of copies of ss bGH mRNA per 100 ng RNA sample. In addition, each RNA sample was tested using qScript XLT one-step RT-qPCR (without adding reverse transcriptase) to monitor potential carrier DNA contamination in the RNA sample.

[0566] Table 13: Primer and probe sequences for bGH sequences

[0567]

[0568]

[0569] Table 14: RT-qPCR analysis conditions

[0570]

[0571] Example 5: Expression of functional DAO1 in 293T cells

[0572] This example describes a study evaluating the expression of the DAO1 transgene in 293T cells using a colorimetric histamine quantification assay. The purpose of the study was to confirm whether the transfection resulted in the expression of functional DAO1.

[0573] background

[0574] Five definite biogenic amine neurotransmitters have been identified so far, including dopamine, norepinephrine, epinephrine, histamine and serotonin formed by decarboxylation of amino acids or amination events of ketones or aldehydes. In particular, histamine is formed by decarboxylation of histidine and was described more than 115 years ago (Windaus A, Vogt W. "Synthese des Imidazolyl-athylamins." Ber. Dtsch. Chem. Ges. 1907; 40: 3691-3695). The degradation of histamine by DAO1 mainly occurs outside the cell, which is most relevant to the treatment method when attempting to regulate the histamine response caused by histamine uptake or local mast cell degranulation leading to histamine release into the extracellular matrix and systemic circulation. One mechanism by which histamine is degraded by DAO1 is by oxidative deamination of histamine. Oxidation is the first step in the degradation / metabolism of histamine, ultimately releasing byproducts out of the body as urine components.

[0575] DAO1 enzyme activity analysis

[0576] In this assay, 293T cells transfected with a plasmid engineered to express DAO1 and untransfected 293T cells (negative control) were generated and / or cultured using standard protocols, and conditioned media from each sample was collected and used. Protein concentrator was used for concentration. A modified protocol was used as described below. Histamine MAK432 Histamine Quantification Assay Kit assesses DAO1 enzyme activity.

[0577] The MAK432 kit provides a colorimetric method for detecting total histamine from various sources. This kit is based on the enzymatic oxidation of histamine, which is combined with the reduction of the included formazan WST reagent. The intensity of the product color measured at 450nm is proportional to the histamine concentration in the sample. The MAK432 kit includes a "reaction enzyme" component for oxidizing the test sample and a histamine standard that can be used to prepare a histamine standard curve. For the purpose of determining whether the DAO1 transgene is functional and capable of oxidizing histamine, the manufacturer's protocol is modified by eliminating the use of the provided reaction enzyme. In brief, 25 μg / ml histamine and phosphate-buffered saline are combined with 600 μL of conditioned medium (from 293T cells transfected with DAO1 plasmid, or negative control 293T cells) or recombinant porcine DAO1 (used as a positive control) in a 5 mL reaction volume. The reaction time is 4 or 24 hours at 37°C on a 150rpm rotary shaker. Then the manufacturer's detection solution is added and stopped after 35 minutes. Fig.13 The standard curve of histamine production is shown, R 2The value is 0.998. Figure 14-15 It shows that by Fig.14 ) and 24 hours ( Fig.15 ) Kinetics of histamine degradation with increasing concentrations of recombinant porcine DAO1. Figure 16-17 Compared with the untransfected negative control, the conditioned medium of 293T cells transfected with DAO1 plasmid showed a significant increase in the expression of DAO1 in the conditioned medium after 4 hours of exposure ( Fig.16 )、24 hours( Fig.17 ) after significantly degrading histamine.

[0578] As shown in the histamine level data provided, transfection with the DAO1 expression plasmid resulted in a significant decrease in histamine levels compared to the negative control (e.g., >50% decrease after 4 hours and >90% decrease after 24 hours), confirming that transfection of 293T cells with the DAO1 plasmid resulted in the expression of functional DAO1.

[0579] ****

[0580] Although embodiments of the present invention have been shown and described herein, it will be appreciated by those skilled in the art that such embodiments are provided by way of example only. Those skilled in the art will now appreciate a large number of variations, changes, and substitutions without departing from the present invention. It should be understood that each alternative to the embodiments of the present invention described herein can be used to practice the present invention. It is contemplated that the following claims define the scope of the present invention, and therefore encompass methods and structures within the scope of these claims and their equivalents.

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Claims

1. A recombinant adeno-associated virus (rAAV) vector comprising an AAV capsid and an expression cassette comprising a polynucleotide encoding diamine oxidase (DAO1) operably linked to a promoter.

2. The rAAV vector of claim 1, wherein the polynucleotide encodes an amino acid sequence having at least 95% identity to an amino acid sequence selected from SEQ ID NOs: 1 and 2.

3. The rAAV vector according to any one of claims 1 to 2, wherein the polynucleotide comprises a nucleotide sequence having at least 95% identity to a nucleotide sequence selected from SEQ ID NOs: 29-31.

4. The rAAV vector of any one of claims 1 to 3, wherein the polynucleotide comprises a nucleotide sequence selected from SEQ ID NOs: 29-31.

5. The rAAV vector according to any one of claims 1 to 4, wherein the promoter is a CMV promoter comprising the nucleotide sequence shown in SEQ ID NO: 21 or a CAG promoter comprising the nucleotide sequence shown in SEQ ID NO:

5.

6. The rAAV vector of claim 5, wherein the expression cassette comprises the CMV promoter and the CMV enhancer.

7. The rAAV vector of any one of claims 1 to 6, wherein the expression cassette comprises a polyadenylation (polyA) sequence.

8. The rAAV vector according to claim 7, wherein the polyA sequence is a BGH polyA sequence.

9. The rAAV vector of any one of claims 1 to 8, wherein the expression cassette comprises a Woodchuck Hepatitis Virus Post-transcriptional Regulatory Element (WPRE).

10. The rAAV vector of any one of claims 1 to 9, wherein the expression cassette comprises a Kozak sequence.

11. A composition comprising a rAAV vector, wherein the rAAV vector comprises: (a) AAV capsid, and (b) an expression cassette, wherein the expression cassette comprises a polynucleotide comprising a nucleotide sequence sharing at least 95% identity with a nucleotide sequence selected from the group consisting of SEQ ID NOs: 29-31, and wherein the polynucleotide is linked to a promoter.

12. The rAAV vector of any one of claims 1 to 10 or the composition of claim 11, wherein the expression cassette is flanked by two inverted terminal repeats (ITRs).

13. The rAAV vector or composition of claim 12, wherein the ITR is an AAV2 ITR.

14. The rAAV vector of any one of claims 1 to 10 and 12 or the composition of claim 11 or 12, wherein the expression cassette comprises a nucleotide sequence that shares at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO:

23.

15. The rAAV vector of any one of claims 1 to 10 and 12 to 14 or the composition of any one of claims 11 to 14, 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).

16. The rAAV vector of any one of claims 1 to 10 and 12 to 15 or the composition of any one of claims 11 to 15, wherein the AAV capsid comprises a VP3 that shares at least 95%, 98% or 100% identity with AAV9 VP3 (SEQ ID NO: 14).

17. A composition comprising a rAAV vector, wherein the rAAV vector comprises: (a) AAV2, AAV5, AAV8 or AAV9 capsid, and (b) an expression cassette, wherein the expression cassette comprises a polynucleotide comprising a nucleotide sequence sharing at least 95% identity with a nucleotide sequence selected from the group consisting of SEQ ID NOs: 29-31, and wherein the polynucleotide is linked to a promoter.

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

23.

19. The composition of claim 17 or 18, wherein the AAV capsid is AAV2.

20. The composition of claim 17 or 18, wherein the AAV capsid is AAV5.

21. The composition of claim 17 or 18, wherein the AAV capsid is AAV9.

22. A pharmaceutical composition comprising the rAAV vector according to any one of claims 1 to 10 and 12 to 15 or the composition according to any one of claims 11 to 21, and a pharmaceutically acceptable carrier.

23. The pharmaceutical composition of claim 22, wherein the composition comprises about 1×10 7 About 1×10 14 genome copies.

24. The pharmaceutical composition of claim 22, wherein the composition comprises about 1×10 12 About 6.2×10 12 genome copies.

25. The pharmaceutical composition of any one of claims 22-24, wherein the composition is formulated for administration into the lacrimal gland.

26. The pharmaceutical composition of any one of claims 22-25, wherein the composition is formulated for administration onto an ocular surface.

27. The pharmaceutical composition of any one of claims 22 to 26, wherein the composition is formulated for or suitable for treating an ocular disease, disorder or condition.

28. A method of treating a condition in a subject in need thereof, the method comprising administering to an eye of the subject an effective amount of a pharmaceutical composition according to any one of claims 22 to 27.

29. The method of claim 28, wherein the pharmaceutical composition is delivered to an ocular secretory gland of the subject.

30. The method of claim 28 or 29, wherein the pharmaceutical composition is delivered to the lacrimal gland.

31. The method of claim 30, wherein cells within the lacrimal gland are transduced by the rAAV vector.

32. The method of claim 31 , wherein the transduced cells within the lacrimal gland express an effective amount of DAO1 into the tear film and optionally onto the ocular surface of the individual.

33. The method of any one of claims 28-32, wherein the pharmaceutical composition is delivered to the accessory lacrimal gland.

34. The method of any one of claims 28 to 33, wherein about 1×10 9 About 1×10 10 , about 1×10 10 About 1×10 11 , about 1×10 11 About 1×10 12 , about 1×10 12 About 1×10 13 or about 1×10 13 About 1×10 15 genome copies.

35. The method of any one of claims 28-34, wherein the condition is an optical condition.

36. The method of any one of claims 28-35, wherein the condition is associated with increased histamine production and / or increased histamine signaling.

37. The method of any one of claims 28 to 36, wherein the condition is an inflammatory condition, optionally an inflammatory condition of the eye.

38. The method of any one of claims 28-37, wherein the condition is an autoimmune condition.

39. The method of any one of claims 28-37, wherein the condition is an allergic condition.

40. The method of claim 39, wherein the condition is an allergic reaction to a therapeutic agent.

41. The method of claim 39, wherein the condition is an allergic reaction to a microbial agent.

42. The method of any one of claims 28-37, wherein the condition comprises vernal keratoconjunctivitis.

43. The method of any one of claims 28-37, wherein the condition comprises atopic keratoconjunctivitis.

44. The method of any one of claims 28-37, wherein the condition comprises seasonal or perennial allergic conjunctivitis.

45. The method of any one of claims 28-44, wherein the administration results in expression of DAO1 in cells of the lacrimal gland and / or accessory lacrimal gland.

46. ​​The method of any one of claims 28-45, wherein the administering results in secretion of DAO1 into the tear film.

47. The method of claim 46, wherein secretion of DAO1 into the tear film is stimulated by a cholinergic agonist.

48. The method of any one of claims 28-47, wherein the administration results in improvement of one or more symptoms of the condition.

49. The method of claim 48, wherein the symptom is selected from the group consisting of itching, swelling, tearing and redness.

50. The method of any one of claims 28-49, wherein the administering results in an improvement of 0.5, 1, 1.5, 2, 2.5, 3, 3.5, or 4 points on a conjunctival itch grading scale.

51. The method of any one of claims 28-50, wherein the administering results in an improvement of 1, 2, 3, or 4 points on a conjunctival redness assessment grading scale.

52. The method of any one of claims 28-51, wherein the administration results in an improvement of about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about 50%, about 50% to about 60%, about 60% to about 70%, about 70% to about 80%, about 80% to about 90%, or more than about 95% on a Schirmer test.

53. The method of any one of claims 28 to 52, wherein the administration results in an improvement of about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about 50%, about 50% to about 60%, about 60% to about 70%, about 70% to about 80%, about 80% to about 90%, or more than about 95% in at least one area on a corneal staining grading scale.

54. The method of any one of claims 48-53, wherein the improvement is measured at about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 9 months, or about 12 months after the administering.

55. The method of any one of claims 48-54, wherein the improvement persists for at least 3 months, at least 6 months, at least 9 months, at least 12 months, at least 15 months, at least 18 months, at least 24 months, at least 3 years, at least 4 years, or at least 5 years after the administering.

56. The method of any one of claims 28-55, further comprising administering one or more additional therapeutic agents to the subject.

57. The method of any one of claims 28-56, wherein the subject is a human.

58. The pharmaceutical composition of any one of claims 22 to 27 for use in a method of treating a condition in a subject in need thereof, the method comprising administering an effective amount of the pharmaceutical composition to an eye of the subject.

59. A pharmaceutical composition according to any one of claims 22 to 27 for use in the manufacture of a medicament for treating a condition in a subject in need thereof.

60. A kit comprising a pharmaceutical composition of a rAAV vector according to any one of claims 1 to 10 and 12 to 16 or a composition according to any one of claims 11 to 21 and a pharmaceutically acceptable carrier, and instructions for treating a condition in a subject, the kit comprising administering the pharmaceutical composition to the eye of the subject.

61. A kit comprising a pharmaceutical composition of a rAAV vector according to any one of claims 1 to 10 and 12 to 16 or a composition according to any one of claims 11 to 21 and a pharmaceutically acceptable carrier, and instructions for treating a condition associated with increased histamine production and / or histamine signaling in an individual, the kit comprising administering the pharmaceutical composition to the eye of the individual.

62. A kit comprising a pharmaceutical composition of a rAAV vector according to any one of claims 1 to 10 and 12 to 16 or a composition according to any one of claims 11 to 21 and a pharmaceutically acceptable carrier, and instructions for treating an autoimmune condition in an individual, the kit comprising administering the pharmaceutical composition to the eye of the individual.

63. A kit comprising a pharmaceutical composition of a rAAV vector according to any one of claims 1 to 10 and 12 to 16 or a composition according to any one of claims 11 to 21 and a pharmaceutically acceptable carrier, and instructions for treating an allergic condition in an individual, the kit comprising administering the pharmaceutical composition to the eye of the individual.

64. A pharmaceutical composition comprising a) a polypeptide comprising a diamine oxidase ("DAO1") enzyme or a fragment thereof, optionally wherein the polypeptide has an amino acid sequence having at least 90% identity to an amino acid sequence selected from SEQ ID NOs: 1 and 2; and b) a pharmaceutically acceptable carrier suitable for administration to the eye of a human subject.

65. A pharmaceutical composition comprising a) a vector comprising a polynucleotide encoding a polypeptide comprising a diamine oxidase ("DAO1") enzyme or a fragment thereof, optionally wherein the polypeptide has an amino acid sequence having at least 90% identity to an amino acid sequence selected from SEQ ID NOs: 1 and 2; and b) a pharmaceutically acceptable carrier suitable for administration to the eye of a human subject.

66. A pharmaceutical composition according to claim 64 or 65, wherein the pharmaceutically acceptable carrier comprises water; sterile water; pyrogen-free water; phosphate-buffered saline; HEPES-buffered saline; isotonic sodium chloride solution; balanced salt solution; a wetting agent; a surfactant; a tonicity agent; a pH adjuster; a viscosity modifier; a buffer; a disaccharide, optionally sucrose or trehalose; cellulose and / or its derivatives; an amino acid, optionally histidine; or any combination thereof.

67. The pharmaceutical composition of any one of claims 64 to 66, 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:

2.

68. The pharmaceutical composition of any one of claims 64-67, wherein the polypeptide comprises at least 20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, or 500 amino acids.

69. The pharmaceutical composition of any one of claims 64-68, wherein the formulation is a liquid formulated for administration to or into the ocular surface of the eye of the human subject or for intra-lacrimal injection.

70. The pharmaceutical composition of any one of claims 65 to 69, comprising the vector, wherein the vector is present in the composition in an amount effective to express 100 pg / mL to 50 μg / mL of the polypeptide in the tear film of the individual after administration of the composition to the individual.

71. The pharmaceutical composition of any one of claims 65 to 70, comprising the vector, wherein the polynucleotide is operably linked to a promoter.

72. The pharmaceutical composition of any one of claims 65 to 71, comprising the vector, wherein the vector is engineered to constitutively express the polypeptide having an amino acid sequence having at least 90% identity to an amino acid sequence selected from SEQ ID NOs: 1 and 2.

73. The pharmaceutical composition of any one of claims 65 to 72, comprising the vector, wherein the vector comprises a virus, optionally an adenoviral vector or a lentiviral vector; a plasmid; an episome; or an artificial chromosome; and optionally comprises one or more lipids, polycations, DNA-carrier proteins, histones, pseudocapsids, chimeric proteins, or endocytic receptor proteins.

74. The pharmaceutical composition of any one of claims 64 or 66 to 69, comprising the polypeptide, wherein the polypeptide is present in the pharmaceutical composition at a concentration of 100 pg / mL to 50 μg / mL.

75. The pharmaceutical composition of any one of claims 64, 66-69 or 74, comprising the polypeptide, wherein the polypeptide is present in the pharmaceutical composition in an amount of 500 ng to 5 μg.

76. The pharmaceutical composition of any one of claims 64, 66-69, 74 or 75, comprising the polypeptide, wherein the polypeptide is present in the pharmaceutical composition in a unit dose.

77. A method of treating an ocular disease, disorder or condition in a subject in need thereof, the method comprising administering to an eye of the subject an effective amount of a pharmaceutical composition according to any one of claims 64 to 76.

78. The method of claim 77, wherein the ocular disease, disorder or condition is associated with increased histamine production and / or increased histamine signaling.

79. The method of claim 77 or 78, wherein the condition is an inflammatory condition, optionally an inflammatory condition of the eye.

80. The method of any one of claims 77-79, wherein the condition is an autoimmune condition.

81. The method of any one of claims 77-80, wherein the condition is an allergic condition.

82. The method of any one of claims 77-81, wherein the condition is an allergic reaction to a therapeutic agent.

83. The method of any one of claims 77-82, wherein the condition is an allergic reaction to a microbial agent.

84. The method of any one of claims 77-83, wherein the condition comprises vernal keratoconjunctivitis.

85. The method of any one of claims 77-84, wherein the condition comprises atopic keratoconjunctivitis.

86. The method of any one of claims 77-85, wherein the condition comprises seasonal or perennial allergic conjunctivitis.

87. The method of any one of claims 77-86, wherein the administration results in expression of a functional diamine oxidase in one or more cells of the lacrimal gland and / or accessory lacrimal gland of the subject.

88. The method of any one of claims 77-87, wherein the administering results in secretion of functional diamine oxidase into the tear film of the subject.

89. The method of claim 88, wherein secretion of the functional diamine oxidase into the tear film is stimulated by a cholinergic agonist.

90. The method of any one of claims 77-89, wherein the administering is to or into the ocular surface of the subject, and / or to the lacrimal gland of the subject.

91. The method of any one of claims 77-90, wherein the symptom is selected from the group consisting of itching, swelling, tearing, and redness.

92. The method of any one of claims 77-91, wherein the administering results in an improvement of 0.5, 1, 1.5, 2, 2.5, 3, 3.5, or 4 points on a conjunctival itch grading scale.

93. The method of any one of claims 77-92, wherein the subject is a human subject.

94. A kit comprising a pharmaceutical composition according to any one of claims 64 to 76 and instructions for treating a condition in a human subject, the kit comprising administering the pharmaceutical composition to an eye of the human subject.

95. A pharmaceutical composition according to any one of claims 64 to 76 for use in the manufacture of a medicament for treating a condition in a human subject in need thereof.

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