AAV vector encoding diamine oxidase and its use

Abstract

Recombinant adeno-associated virus (rAAV) vectors, methods of treating a symptom (e.g., an ocular symptom), pharmaceutical compositions, and other compositions and methods are provided, wherein the rAAV vector comprises a polynucleotide encoding diamine oxidase (DAO1). The method of treatment may include administration to the lacrimal gland.

Description

【Technical Field】 【0001】 Cross - Reference to Related Applications This application claims the benefit of the filing dates 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, and the disclosures of these U.S. provisional patent applications are hereby incorporated by reference in their entireties. 【0002】 Sequence Listing This application incorporates by reference in its entirety the "037525 - 00558.xml" Sequence Listing XML file (71 KB), created on May 24, 2023, which is electronically submitted together with this specification. 【Background Art】 【0003】 Medical treatments for eye symptoms affecting the ocular surface, such as perennial and / or seasonal allergic conjunctivitis, atopic keratoconjunctivitis, giant papillary conjunctivitis, vernal keratoconjunctivitis (VKC), and allergic conjunctivitis including allergic keratoconjunctivitis, are known. These medical treatments include topical eye drops, oral medications, and others. 【0004】 Despite available treatments, conjunctivitis remains a problem for eye care practitioners (ophthalmologists and optometrists), and treatment strategies focus on the severity of the disease. Treatments for lower - severity cases may include anti - histamines, mast cell stabilizers, anti - inflammatory agents, and / or topical steroid eye drops. For more severe cases, oral anti - histamines or oral leukotriene inhibitor medications may be used. 【0005】 The delivery of antihistamines, mast cell stabilizers, calcineurin inhibitors, anti-inflammatory agents, and / or topical steroid eye drops for the treatment of conjunctivitis currently relies on repeated application of eye drops, which does not provide adequate treatment outcomes in many severe cases, such as vernal catarrh or atopic keratoconjunctivitis that can result in corneal ulceration, cataract formation, or glaucoma even with treatment. 【Summary of the Invention】 【0006】 Given the shortcomings of current medical treatment options for ocular surface disorders, there is a need for improved methods of delivering antihistamine treatment to the ocular surface without the need for repeated topical application, which can be difficult or impossible for some patients and can result in poor patient compliance. Additionally, pulsed administration of antihistamine treatment may only provide temporary or "episodic" relief and may not be suitable for chronic symptoms. The present disclosure provides recombinant constructs and methods that address these and other needs in some aspects, in addition to providing various other benefits as described herein and shown by the accompanying drawings. 【0007】 In some aspects, the present disclosure provides an adeno-associated virus (AAV) vector for the expression of diamine oxidase ("DAO1"; alternatively abbreviated herein as "DAO") in the eye after in vivo administration. 【0008】 In one aspect, the present disclosure is a recombinant AAV (rAAV) vector comprising an adeno-associated virus (AAV) capsid and an expression cassette, wherein the expression cassette is operably linked to a promoter and is known as DAO1, DAO, or histaminase Provided is an rAAV vector comprising a polynucleotide encoding diamine oxidase. In some embodiments, the polynucleotide encodes a protein that is at least 95% identical to SEQ ID NO: 1 or 2. In some embodiments, the polynucleotide comprises a nucleotide sequence that is at least 95% identical to a nucleotide sequence selected from SEQ ID NOs: 29-31. In some embodiments, the polynucleotide comprises a nucleotide sequence selected from SEQ ID NOs: 29-31. 【0009】 In another aspect, the present disclosure provides a composition comprising an rAAV vector, wherein the rAAV vector comprises (a) an AAV capsid, and (b) an expression cassette, the expression cassette comprising a polynucleotide comprising a nucleotide sequence that shares at least 95% identity with a nucleotide sequence selected from SEQ ID NOs: 29-31, and the polynucleotide being linked to a promoter. 【0010】 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 ITR is an AAV2 ITR. 【0011】 In some embodiments, the expression cassette comprises a nucleotide sequence that shares at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 23. 【0012】 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). 【0013】 In yet another aspect, the present disclosure provides a composition comprising an 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 that shares at least 95% identity with a nucleotide sequence selected from SEQ ID NOs: 29-31, and the polynucleotide is linked to a promoter. 【0014】 In some embodiments, the expression cassette comprises a polynucleotide sequence that shares at least 95% identity with SEQ ID NO: 23. 【0015】 In some embodiments, the AAV capsid is AAV2. In some embodiments, the AAV capsid is AAV5. In some embodiments, the AAV capsid is AAV9. 【0016】 In one aspect, the present disclosure provides a pharmaceutical composition comprising an rAAV vector or a composition described herein (e.g., a polypeptide comprising DAO1 or a fragment thereof) as described herein, and a pharmaceutically acceptable carrier. 【0017】 In some embodiments, the pharmaceutical composition comprises from about 1×10 5 to about 1×10 16 genomic copies per milliliter of rAAV vector. In some embodiments, the pharmaceutical composition comprises from about 1×1012 ~ about 6.2×10 12 genomic copies. 【0018】 In some embodiments, the pharmaceutical composition is in an amount or concentration sufficient to result in the expression of DAO1 within the range defined by 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, or any pair of values above, of DAO1 in the tear layer of a subject. The expression may 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 1, 2, 3, 4, or 5 days later. 【0019】 In some embodiments, the pharmaceutical composition may comprise a polypeptide comprising DAO1 (or a fragment thereof) in an amount within the range defined by 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, or any pair of values above. In some embodiments, the pharmaceutical composition may further comprise one or more excipients, carriers, and / or diluents suitable for administration to the eyes of a human or animal subject. 【0020】 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 ocular surface. In some embodiments, the pharmaceutical composition is formulated for use in the treatment of an eye disease, disorder, or condition or is adaptable for such use. 【0021】 In some aspects, the present disclosure provides a method of treating a condition in a subject in need thereof, the method comprising administering to the subject's eye an effective amount of the pharmaceutical composition described herein. 【0022】 In some embodiments, the pharmaceutical composition is delivered to the secretory glands of the subject's eye. In some embodiments, the pharmaceutical composition is delivered to the lacrimal gland. In some embodiments, the cells within the lacrimal gland are acinar cells. In some embodiments, the cells within the lacrimal gland are ductile epithelial cells. In some embodiments, the cells within the lacrimal gland are transduced by an rAAV vector. In some embodiments, the transduced cells within the lacrimal gland express an effective amount of DAO1 in the subject's tear film and optionally on the ocular surface. In some embodiments, the pharmaceutical composition is delivered to the accessory lacrimal glands (e.g., glands of Krause, glands of Wolfring, and / or glands of Popoff). 【0023】 In some embodiments, about 1×10 9 ~ about 1×10 10 、 about 1×10 10 ~ about 1×10 11 、 about 1×10 11 ~ about 1×10 12 、 about 1×10 12 ~ about 1×10 13 、 or about 1×10 13 ~ about 1×10 15 genomic copies are administered. 【0024】 In some embodiments, the condition is an ocular condition. In some embodiments, the condition or Eye symptoms are associated with increased histamine production and / or increased histamine signaling. In some embodiments, the symptoms or eye symptoms are inflammatory symptoms. In some embodiments, the symptoms or eye symptoms are autoimmune symptoms. In some embodiments, the symptoms or eye symptoms are allergic symptoms. In some embodiments, the symptoms or eye symptoms are an allergic reaction to a therapeutic agent. In some embodiments, the symptoms or eye symptoms are an allergic reaction to a microbial infection. In some embodiments, the symptoms or eye symptoms are vernal catarrh. In some embodiments, the symptoms or eye symptoms are atopic keratoconjunctivitis. In some embodiments, the symptoms or eye symptoms are seasonal or perennial allergic conjunctivitis. 【0025】 In some embodiments, administration results in the expression of DAO1 in cells of the lacrimal and / or accessory lacrimal glands. In some embodiments, administration results in the secretion of DAO1 into the tear film. In some embodiments, the secretion of DAO1 into the tear film is stimulated by electrical stimulation, mechanical stimulation, ultrasonic stimulation, and / or the administration of a drug. Examples of electrical stimulation are intranasal stimulation devices such as TrueTear® It is an intranasal tear neurostimulator. Examples of mechanical stimulation are vibrational energy provided by a device such as the iTEAR® 100. An example of ultrasonic stimulation is the application of a neuromodulation device such as the iTear system (Olympic Ophthalmics) developed to treat dry eye disease. In some embodiments, the drug that stimulates the secretion of DAO1 into the tear layer 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 mucoadhesive agent (e.g., diclofasol, rebamipide, or ecabet). In some embodiments, the drug that stimulates the secretion of DAO1 into the tear layer is administered into the eye. In some embodiments, the drug, such as a cholinergic agonist, is administered orally. In some embodiments, the secretion of DAO1 into the tear layer is stimulated by a drug administered into the nasal cavity, such as a cholinergic agonist or an nAChR agonist. In some aspects, the DAO1 described herein, or an rAAV vector, plasmid, or any other construct configured to express DAO1, may be administered to a subject as a combination therapy in combination with a tear-increasing stimulus and / or a drug (e.g., those described in this paragraph). In some aspects, such combination therapy may follow any protocol described in International Publication No. WO 2022 / 235786, the entire contents of which are incorporated herein by reference, or any component or parameter may be used. 【0026】 In some embodiments, administration results in the improvement of one or more symptoms of the ocular condition. In some embodiments, the symptoms are selected from the group consisting of itching, swelling, tearing, and flushing. In some embodiments, administration results in an improvement of 0.5, 1, 1.5, 2, 2.5, 3, 3.5, or 4 points on the Conjunctival Itching Grading Scale. In some embodiments, administration results in an improvement of 1, 2, 3, or 4 points on the Conjunctival Redness Assessment Grading Scale. In some embodiments, administration results in an improvement of 1, 2, 3, or 4 points on the Ocular Tearing Score Grading Scale. In some embodiments, 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 greater than about 95% in at least one compartment of the Corneal Staining Grading Scale. 【0027】 In some embodiments, the improvement is measured 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. 【0028】 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 that is an agent that increases tear production is a cholinergic agonist. In some embodiments, the subject is human. 【0029】 In some embodiments, the present disclosure provides a DAO1, rAAV or other vector configured to express DAO1, composition, or pharmaceutical composition described herein for use in a method of treating a condition in a subject, the method comprising administering to the eye of the subject in need of treatment of the condition an effective amount of the pharmaceutical composition. 【0030】 In some embodiments, the present disclosure provides a DAO1, rAAV or other vector configured to express DAO1, composition, or pharmaceutical composition described herein for use in the production of a medicament for treating a condition in a subject in need of treatment of the condition. 【0031】 In some embodiments, the present disclosure provides a kit comprising a DAO1, rAAV or other vector configured to express DAO1, or composition described herein, and a pharmaceutically acceptable carrier, and instructions for use in treating a condition in a subject. In some embodiments, the instructions comprise administering the pharmaceutical composition to the eye of the subject. 【0032】 In some embodiments, the present disclosure provides a kit comprising a DAO1, rAAV or other vector configured to express DAO1, or composition described herein, and a pharmaceutically acceptable carrier, and instructions for use in treating a condition associated with histamine production and / or increased histamine signaling in a subject. In some embodiments, the instructions comprise administering the pharmaceutical composition to the eye of the subject. 【0033】 In some embodiments, the present disclosure provides a kit comprising DAO1 described herein, an rAAV or other vector configured to express DAO1, or a composition, and a pharmaceutically acceptable carrier, and instructions for use in treating autoimmune conditions in a subject. In some embodiments, the instructions include administering the pharmaceutical composition to the subject's eye. 【0034】 In some embodiments, the present disclosure provides a kit comprising DAO1 described herein, an rAAV or other vector configured to express DAO1, or a composition, and a pharmaceutically acceptable carrier, and instructions for use in treating allergic conditions in a subject. In some embodiments, the instructions include administering the pharmaceutical composition to the subject's eye. BRIEF DESCRIPTION OF THE DRAWINGS 【0035】 【Figure 1A】 FIG. 1A shows a vector map of an rAAV expression cassette having inverted terminal repeats (“ITR”), a promoter, and a DAO1 polynucleotide element. 【Figure 1B】 FIG. 1B provides a schematic depicting elements arranged between the ITRs of an rAAV expression cassette that, from 5' to 3', include a cytomegalovirus (“CMV”) enhancer / promoter, an N-terminal secretion signal, a nucleotide sequence encoding DAO1, a woodchuck hepatitis virus post-transcriptional regulatory element (“WPRE”), and a polyadenylation (“pA”) signal. 【Figure 2A】 FIGS. 2A and 2B show examples of delivery of a viral vector to the lacrimal gland of a human subject. 【Figure 2B】 FIGS. 2A and 2B show examples of delivery of a viral vector to the lacrimal gland of a human subject. 【Figure 3】 FIG. 3 shows an example of a corneal staining grading scale, which is a grading chart of the segmentation of the corneal surface based on the NEI / Industry Workshop scale. 【Figure 4】Figure 4A provides images obtained by confocal fluorescence microscopy of 293T cells transfected with an AAV plasmid encoding DAO1 at a magnification of 20× (top row) or 63× (middle row), compared to non-transfected control 293T cells at 20× magnification (bottom row). The leftmost column shows the detection of the fluorescence signal for the AF488-labeled secondary antibody used to detect labeling with the primary anti-DAO1 antibody, the middle column shows the fluorescence signal for nuclear staining using DAPI, and the rightmost column shows the overlay of the AF488 and DAPI fluorescence signals. Figure 4B provides images obtained by confocal fluorescence microscopy of 293T cells transfected with an AAV plasmid encoding DAO1 at a magnification of 20× (middle row) or 63× (bottom row), compared to non-transfected control 293T cells at 20× magnification (top row). The rightmost column shows the fluorescence signal for nuclear staining using DAPI, the middle column shows the fluorescence signal for the AF488-labeled secondary antibody used to detect labeling with the primary anti-DAO1 antibody, and the rightmost column shows the overlay of the AF488 and DAPI fluorescence signals. Figure 4C provides images obtained by confocal fluorescence microscopy of PANC-1 cells transfected with an AAV plasmid encoding DAO1 at 20× magnification (bottom row), compared to non-transfected control cells labeled with the secondary antibody at 40× magnification (top row) or unlabeled at 20× magnification (middle row). The fluorescence signals are as described in Figure 4B. Figure 4D provides images obtained by confocal fluorescence microscopy of PANC-1 cells transfected with an AAV plasmid encoding DAO1 at 40× magnification (top row), compared to non-transfected control cells at 20× magnification (bottom row). The fluorescence signals are as described in Figure 4B. 【Figure 5A】Figure 5A shows an image of a Western blot of whole cell lysates collected from 293T cells transiently transfected with AAV.DAO1 (lane labeled "AAV-DAO1") 48 hours after transfection, compared to control non-transfected 293T cells (lane labeled "293T"), with detection of DAO1 using rabbit anti-DAO1 antibody. The arrow indicates the protein band corresponding to the DAO1 polypeptide. 【Figure 5B】 Figure 5B shows an image of a Western blot of recombinant human DAO1 polypeptide with detection of DAO1 using mouse anti-his antibody. Figures 5C - 5D show images of Western blots for the detection of DAO1 in whole cell lysates collected from 293T cells transiently transfected with AAV.DAO1 (lane labeled "AAV-DAO") 24 hours after transfection, compared to control non-transfected 293T cells (lane labeled "293T") and recombinant human DAO1 polypeptide (lane labeled "recombinant DAO"). The arrow indicates the protein band corresponding to the DAO1 polypeptide (Figure 5C). A GAPDH Western blot (15 μg / well) was used as a loading control (Figure 5D). Figure 5E shows an image of a Western blot for the detection of DAO1 in conditioned media obtained from 293T cells transiently transfected with AAV.DAO1 ("AAV.DAO") or non-transfected 293T cells ("293T") 2, 4, and 6 hours after replacement with fresh media. The arrow indicates the protein band corresponding to the DAO1 polypeptide. 【Figure 6A】Figures 6A-6B show data generated using a colorimetric functional activity assay to measure DAO1 substrate cleavage. DAO1 cleaves a first substrate to yield H2O2 (hydrogen peroxide), and the hydrogen peroxide reacts with a second substrate to generate a signal that is detected using fluorescence spectroscopy (excitation wavelength of 535 nm and emission wavelength of 587 nm). Figure 6A shows a calibration curve generated by measuring the fluorescence signal of the second substrate as a function of the concentration of H2O2. Figure 6B shows a graph quantifying the H2O2 generated by the activity of DAO1 present in the medium or cell lysate of 293T cells transiently transfected with an AAV plasmid encoding DAO1, compared to the activity of the medium of non-transfected control cells. 【Figure 6B】 Figures 6A-6B show data generated using a colorimetric functional activity assay to measure DAO1 substrate cleavage. DAO1 cleaves a first substrate to yield H2O2 (hydrogen peroxide), and the hydrogen peroxide reacts with a second substrate to generate a signal that is detected using fluorescence spectroscopy (excitation wavelength of 535 nm and emission wavelength of 587 nm). Figure 6A shows a calibration curve generated by measuring the fluorescence signal of the second substrate as a function of the concentration of H2O2. Figure 6B shows a graph quantifying the H2O2 generated by the activity of DAO1 present in the medium or cell lysate of 293T cells transiently transfected with an AAV plasmid encoding DAO1, compared to the activity of the medium of non-transfected control cells. Figure 6C provides a graph quantifying the H2O2 generated at 30, 60, and 90 minutes by the activity of DAO1 present in a combination of conditioned medium and whole cell extracts obtained from 293T cells transiently transfected with an AAV plasmid encoding DAO1 or non-transfected control cells. Recombinant DAO1 was used as a positive control. 【Figure 7】Figure 7 shows a diagram depicting the elements between the ITRs of the AAV plasmid. The plasmid encodes EGFP (''secEGFP'') linked to a secretion signal at its N-terminus, under the control of the CMV promoter. The woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) functions to increase transgene expression and is proximal to the bovine growth hormone polyadenylation (pA) signal. 【Figure 8A】 Figures 8A - 8K are images of lacrimal gland tissue stained with an anti-eGFP antibody. The lacrimal gland was dosed by intraductal injection with an rAAV vector containing an expression cassette with the eGFP transgene. The lacrimal gland tissue was stained with an anti-eGFP antibody to evaluate eGFP expression. The black arrows indicate the staining showing eGFP expression. 【Figure 8B】 Figures 8A - 8K are images of lacrimal gland tissue stained with an anti-eGFP antibody. The lacrimal gland was dosed by intraductal injection with an rAAV vector containing an expression cassette with the eGFP transgene. The lacrimal gland tissue was stained with an anti-eGFP antibody to evaluate eGFP expression. The black arrows indicate the staining showing eGFP expression. 【Figure 8C】 Figures 8A - 8K are images of lacrimal gland tissue stained with an anti-eGFP antibody. The lacrimal gland was dosed by intraductal injection with an rAAV vector containing an expression cassette with the eGFP transgene. The lacrimal gland tissue was stained with an anti-eGFP antibody to evaluate eGFP expression. The black arrows indicate the staining showing eGFP expression. 【Figure 8D】 Figures 8A - 8K are images of lacrimal gland tissue stained with an anti-eGFP antibody. The lacrimal gland was dosed by intraductal injection with an rAAV vector containing an expression cassette with the eGFP transgene. The lacrimal gland tissue was stained with an anti-eGFP antibody to evaluate eGFP expression. The black arrows indicate the staining showing eGFP expression. 【Figure 8E】 Figures 8A - 8K are images of lacrimal gland tissue stained with an anti-eGFP antibody. The lacrimal gland was dosed by intraductal injection with an rAAV vector containing an expression cassette with the eGFP transgene. The lacrimal gland tissue was stained with an anti-eGFP antibody to evaluate eGFP expression. The black arrows indicate the staining showing eGFP expression. 【Figure 8F】Figures 8A - 8K are images of lacrimal gland tissues stained with anti - eGFP antibody. The lacrimal glands were dosed by injecting an rAAV vector containing an expression cassette with the eGFP transgene into the lacrimal glands. The lacrimal gland tissues were stained with anti - eGFP antibody to evaluate eGFP expression. The black arrows indicate the staining showing eGFP expression. 【Figure 8G】 Figures 8A - 8K are images of lacrimal gland tissues stained with anti - eGFP antibody. The lacrimal glands were dosed by injecting an rAAV vector containing an expression cassette with the eGFP transgene into the lacrimal glands. The lacrimal gland tissues were stained with anti - eGFP antibody to evaluate eGFP expression. The black arrows indicate the staining showing eGFP expression. 【Figure 8H】 Figures 8A - 8K are images of lacrimal gland tissues stained with anti - eGFP antibody. The lacrimal glands were dosed by injecting an rAAV vector containing an expression cassette with the eGFP transgene into the lacrimal glands. The lacrimal gland tissues were stained with anti - eGFP antibody to evaluate eGFP expression. The black arrows indicate the staining showing eGFP expression. 【Figure 8I】 Figures 8A - 8K are images of lacrimal gland tissues stained with anti - eGFP antibody. The lacrimal glands were dosed by injecting an rAAV vector containing an expression cassette with the eGFP transgene into the lacrimal glands. The lacrimal gland tissues were stained with anti - eGFP antibody to evaluate eGFP expression. The black arrows indicate the staining showing eGFP expression. 【Figure 8J】 Figures 8A - 8K are images of lacrimal gland tissues stained with anti - eGFP antibody. The lacrimal glands were dosed by injecting an rAAV vector containing an expression cassette with the eGFP transgene into the lacrimal glands. The lacrimal gland tissues were stained with anti - eGFP antibody to evaluate eGFP expression. The black arrows indicate the staining showing eGFP expression. 【Figure 8K】 Figures 8A - 8K are images of lacrimal gland tissues stained with anti - eGFP antibody. The lacrimal glands were dosed by injecting an rAAV vector containing an expression cassette with the eGFP transgene into the lacrimal glands. The lacrimal gland tissues were stained with anti - eGFP antibody to evaluate eGFP expression. The black arrows indicate the staining showing eGFP expression. 【Figure 9】Figure 9 provides an image of a porcine lacrimal gland that was given an injection of AAV-secEGFP (serotype AAV2 or AAV9), harvested on day 103, and fixed in paraffin. IHC of 5 μM sections was performed using anti-GFP antibody and DAPI (nucleus) counterstaining. Images were captured using a confocal microscope at 100× magnification. Negative control animals were not given an injection. 【Figure 10】 Figure 10 provides an image of a porcine lacrimal gland that was given an injection of AAV9-secEGFP, harvested on day 103, and fixed in paraffin. IHC of 5 μM sections was performed using anti-GFP antibody and DAPI (nucleus) counterstaining. In addition to lacrimal gland acinar cells, ductal epithelial cells appear to have been transduced by AAV9 (white arrow). 【Figure 11】 Figure 11 provides an image of a porcine lacrimal gland that was given an injection of AAV, harvested on day 103, and fixed in paraffin. H&E staining of 5 μM paraffin sections at 100× reveals no inflammatory infiltrates, macroscopic abnormalities, or microscopic abnormalities. 【Figure 12】 Figure 12 provides a schematic of a treatment schedule for administering an AAV encoding a model protein via injection into the porcine lacrimal gland in combination with administration of OC-01 (varenicline) by nasal spray. Time points for tear collection and end of study are indicated. 【Figure 13】 Figure 13 shows a soft positive curve generated by measuring the fluorescence signal of a second substrate as a function of histamine concentration. This soft positive curve was used to evaluate the histamine degradation kinetics of recombinant porcine diamine oxidase (Figures 14 - 15) and histamine degradation by conditioned media from non-transfected control 293T cells and 293T cells transfected with plasmids engineered to express DAO1 (Figures 16 - 17). 【Figure 14】 Figures 14 - 15 provide graphs showing the histamine degradation kinetics of recombinant porcine diamine oxidase at 4 (Figure 14) and 24 hours (Figure 15). 【Figure 15】Figures 14-15 provide graphs showing the histamine degradation kinetics of recombinant butyramine oxidase at 4 (Figure 14) and 24 hours (Figure 15). 【Figure 16】 Figures 16-17 provide graphs showing histamine degradation by conditioned media obtained from non-transfected control 293T cells and 293T cells transfected with a plasmid engineered to express DAO1 at 4 (Figure 16) and 24 hours (Figure 17). 【Figure 17】 Figures 16-17 provide graphs showing histamine degradation by conditioned media obtained from non-transfected control 293T cells and 293T cells transfected with a plasmid engineered to express DAO1 at 4 (Figure 16) and 24 hours (Figure 17). 【Mode for Carrying Out the Invention】 【0036】 Detailed Description The present disclosure provides a method of treating an ocular symptom in a subject in need of treatment of an ocular symptom. Such a method includes, for example, the expression and subsequent secretion of diamine oxidase (DAO1) from the lacrimal gland, which can then be transported onto the surface of the eye and combined with other components of the tear film. In healthy subjects, the primary function of DAO1 is to break down excess histamine in the body. DAO1 is present in very low amounts in human blood circulation, but has been shown to be substantially increased in pregnant women, suggesting a protective mechanism against harmful histamine. It has also been shown that there is an imbalance between accumulated histamine and histamine breakdown in subjects with histamine intolerance and ocular conjunctivitis. After various noxious stimuli, excess histamine can be released from basophils and mast cells, creating tissue edema and initiating a cascade of events leading to inflammation and allergic responses. Elevated histamine levels in the tear film are hallmarks of perennial and / or seasonal allergic rhinitis, atopic keratoconjunctivitis, giant papillary conjunctivitis, vernal catarrh, and allergic keratoconjunctivitis. Histamine induces itching, flushing, and tearing through activation of H1 receptors on blood vessels and nociceptive nerves. In severe cases, patients can develop visual impairment due to corneal damage, cataracts, or glaucoma. 【0037】 As disclosed herein, AAV-based delivery of a transgene to the eye provides a method of treating an eye disorder. rAAV vectors have established advantages for transgene delivery. The transgene delivered by an rAAV vector is likely to integrate into the genome of the transduced cells, allowing for potential long-term expression of the transgene product. Additionally, rAAV vectors can be less immunogenic compared to other viral delivery vectors, such as adenoviruses. AAV-based delivery vectors that use subretinal and intravitreal injections to transduce cells in the posterior segment of the eye have been described and shown to be effective in vivo (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)). 【0038】 AAV serotypes used in AAV-based delivery of transgenes to the eye include AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, and AAV9, which are used to deliver the transgene (Lebherz et al. J Gene Med.; 10(4):375 - 382 (2008)). AAV serotypes used in 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)). 【0039】 In some embodiments, the rAAV vector is provided for the expression of DAO1. In some embodiments, at least one cell of the eye and / or the lacrimal gland is transduced with the rAAV vector of the present disclosure. The lacrimal gland is the main source of tears that contribute to the promotion of a healthy ocular surface and the maintenance of normal visual function. The main lacrimal gland includes the palpebral and orbital lobes, and They are continuous with each other at the lateral edge of the aponeurosis of the levator palpebrae superioris muscle. The lobules have numerous acini and intralobular ducts that form excretory ducts opening into the fornix of the conjunctiva. The main lacrimal gland is composed of acinar cells, ductal cells, and / or myoepithelial cells (Obata Cornea.;25(10 Suppl 1):S82-9(2006)). The main lacrimal gland secretes mucin onto the ocular surface of the eye of interest in addition to the aqueous layer of the tear film (see, for example, Paulsen, F., et al(2004)Cell and tissue research, 316(2),167-177). As used herein, the term "lacrimal gland" refers to the main lacrimal gland of the subject, as well as the glands of Wolfring and Krause. The accessory glands known as the glands of Wolfring and Krause are located within the eyelids. There are about 2-5 glands of Wolfring and about 40 glands of Krause in the upper eyelid. There are about 6-8 glands of Krause in the lower eyelid. The specific location and anatomical structure of the lacrimal gland functional unit are well known (Conrady et al.,J Ophthalmol. Article ID 7542929(2016)). 【0040】 Expression cassette The rAAV vectors of the present disclosure contain 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 inverted terminal repeats. In some embodiments, the expression cassette further comprises other polynucleotide sequences, such as a promoter, regulatory elements (e.g., one or more promoters), translation initiation sequences, coding sequences, and termination sequences (FIGS. 1A and 1B). 【0041】 In mammals, histamine is metabolized by oxidative deamination via DAO1 (also called histaminase, amiloride-binding protein, amine oxidase copper containing 1, AOC1) (see, for example, 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. 【0042】 Histamine intolerance is the accumulation of excessive levels of histamine due to decreased catabolism and / or increased production. Vakal et al., Molecules 2020, 25, 1293. Histamine is released between anaphylaxis and mast cell degranulation, and mast cell degranulation is suggested to increase DAO1 release, and DAO1 is increased in severe anaphylaxis in patients with adipositas (Boehm et al. Allergy. 2019, 74:58). Furthermore, some therapeutic agents, including, for example, berenil, pentamidine, aminoguanidine, metformin, have been shown to inhibit DAO1 (Vakal 2020). 【0043】 Excessive histamine causes a wide range of symptoms (see, for example, Schnedl et al., Food Sci Biotechnol(2019)28(6):1779-1784). Histamine is a pro-inflammatory mediator that contributes to allergic reactions (see, for example, White MV(1990),J Allergy Clin Immunol 86:599-605) and can contribute to vernal keratoconjunctivitis (VKC) (see, for example, Abelson et al., Opthamology 1995,102(12):1958-1963; Bonini et al.,(1992),Journal of allergy and clinical immunology,89(1),103-107). 【0044】 Thus, the present disclosure provides a method for inducing the decomposition of histamine (e.g., histamine present in tears). In some embodiments, the expression cassette is used in a method for treating a condition associated with increased histamine production (e.g., an ocular condition). 【0045】 DAO1 Enzyme In some embodiments, the expression cassette of the present disclosure comprises a polynucleotide sequence encoding DAO1 enzyme or its functional variant.As used herein, the term "DAO1 enzyme" refers to the DAO1 enzyme from any species.The term "functional variant" refers to a variant with sequence substitution, insertion, deletion, and / or N- or C-terminal truncation, and the functional variant retains one or more functions of reference protein, for example, native DAO1 enzyme.DAO1 is a 200kDa homomeric glycoprotein that catalyzes the oxidative deamination of the primary amino group of histamine. 【0046】 In some embodiments, the DAO1 enzyme is a human DAO1 enzyme. In some embodiments, the human DAO1 enzyme is identified in a public database. Human DAO1 isoforms known in the art are typically identified via public databases. For example, the National Library of Medicine National Center for Biotechnology Information (NCBI) Gene Database (accessible via the World Wide Web: ncbi.nlm.nih.gov / ) is a searchable database of genes that provides information on scientific names, chromosomal localization, gene products, gene attributes, associated markers, phenotypes, interactions, links to references, sequence information, information on sequence variants, genetic maps, expression reports, homologs, the content of protein domains, and access to external databases. As will be understood by those skilled in the art, sequence information for human DAO1 isoforms known in the art may be identified by entering an appropriate accession number into the NCBI Gene Database and selecting the sequence information in a 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 is identifiable via accession number NM_001272072.2 in the NCBI gene database and has the amino acid sequence shown in SEQ ID NO: 2. In another example, one isoform of human DAO1 is identifiable via accession number NM_001091 in the NCBI gene database and has the amino acid sequence shown by SEQ ID NO: 2. 【0047】 Exemplary amino acid sequences of human DAO1 and exemplary polynucleotide sequences (mRNA and ORF) encoding human DAO1 are shown 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. 【0048】 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. 【0049】 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 segments 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., segments 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 segments 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., segments spanning positions 39-125, 141-241, and / or 300-708 of SEQ ID NO: 2). 【0050】 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, each segment comprising at least 20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, or 500 consecutive amino acids of SEQ ID NO:1 or SEQ ID NO:2, and segments having lengths within the range defined by any pair of the above values. For example, a functional DAO1 enzyme may have 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 diversity in other parts 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 to segments spanning from position 20 to the final position of either SEQ ID NO:1 or SEQ ID NO:2 (e.g., omitting the predicted N-terminal signal peptide shared by both sequences). 【0051】 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 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. 【0052】 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. 【0053】 Polynucleotide encoding the DAO1 enzyme In some embodiments, the expression cassette comprises a polynucleotide encoding a DAO1 enzyme (e.g., a human DAO1 enzyme) described herein. In some embodiments, the polynucleotide comprises an ORF encoding a DAO1 enzyme (e.g., a human DAO1 enzyme) described herein. 【0054】 In some embodiments, the expression cassette provides increased expression of DAO1 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 subject or in the contralateral eye of a treated subject. As used herein, "subject" means any mammal, including mice, rabbits, non-human primates (NHPs), and humans. In some embodiments, the subject is a human or NHP. Further, "individual" or "patient" may be used interchangeably with "subject". In some embodiments, the expression of DAO1 may be increased by at least 1.5-fold, 2-fold, 3-fold, 4-fold, or 5-fold compared to the expression of DAO1 in an untreated subject or in the contralateral eye of a treated subject. In some embodiments, DAO1 may be expressed at any detectable level in the treated eye, while DAO1 may not be expressed or may be expressed at undetectable levels in an untreated subject or in the contralateral eye of a treated subject. In other words, the eye or lacrimal gland to which the rAAV vector described herein is administered may express DAO1 in a higher abundance than in an eye or lacrimal gland that has only endogenous (i.e., native) expression of DAO1, or in an eye that has lower or impaired secretion of endogenous (i.e., native) DAO1. 【0055】 The term "functional variant" refers to a variant having an array substitution, insertion, deletion, and / or N- or C-terminal truncation, and the functional variant retains one or more functions of the reference protein, e.g., native DAO1. 【0056】 In some embodiments, the expression cassette comprises a polynucleotide encoding a protein that is human DAO1 or a functional variant thereof. In some embodiments, the expression cassette comprises a polynucleotide comprising a nucleotide sequence encoding the amino acid sequence identified in Table 1. In some embodiments, the expression cassette comprises a polynucleotide comprising the nucleotide sequence identified in Table 1. 【0057】 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 the amino acid sequence shown in 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. 【0058】 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. 【0059】 In some embodiments, the expression cassette comprises a polynucleotide encoding DAO1 comprising a nucleotide sequence sharing 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 set forth in SEQ ID NO: 30. In some embodiments, the expression cassette comprises a polynucleotide encoding DAO1 comprising or consisting of a nucleotide sequence sharing 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 set forth in SEQ ID NO: 31. In some embodiments, the protein is human DAO1 or a functional variant thereof. 【0060】 Codon optimization In some embodiments, the expression cassette comprises a polynucleotide encoding a DAO1 enzyme, and the polynucleotide comprises or consists of a nucleotide sequence that is codon-optimized for expression in a target cell. In some embodiments, the target cell is a mammalian cell. In some embodiments, the target cell is a human cell, a mouse cell, or a non-human primate (NHP) cell. 【0061】 A codon-optimized nucleotide sequence, e.g., a codon-optimized nucleotide sequence encoding a DAO1 enzyme, is typically a sequence that contains at least one synonymous nucleic acid base substitution with respect to a reference sequence (e.g., the wild-type ORF encoding the DAO1 enzyme). The codon-optimized nucleotide sequence can be partially or completely different from the reference sequence. For example, a reference sequence encoding poly-serine that is uniformly encoded by the TCT codon can be sequence-optimized by replacing 100% of its nucleic acid bases (for each codon, the T at position 1 is replaced by A, the C at position 2 is replaced by G, and the T at position 3 is replaced by C) to obtain a sequence encoding poly-serine that is uniformly encoded by the AGC codon. The percentage of sequence identity obtained from a global pairwise alignment between the reference poly-serine nucleic acid sequence and the sequence-optimized poly-serine nucleic acid sequence is 0%. However, the protein products from both sequences are 100% identical. 【0062】 Codon optimization methods are known in the art and can be useful for achieving one or more desired results, for example, increasing the expression of a synthetic gene in a target cell. In some embodiments, the expression cassette contains a nucleotide sequence that has been sequence-optimized using a method of sequence optimization as compared to a reference sequence. Methods of sequence optimization are known in the art and include known sequence optimization tools, algorithms, and services. Non-limiting examples include services from GeneArt (Life Technologies), DNA2.0 (Menlo Park CA), Geneious®, and GeneGPS® (Atum, Newark, CA). 【0063】 In some embodiments, the expression cassette of the present disclosure comprises a polynucleotide encoding a DAO1 enzyme that is sequence-optimized relative to a reference sequence using a method of sequence optimization (e.g., GeneGPS™, e.g., Geneious™). In some embodiments, the method of sequence optimization includes codon optimization algorithms described in U.S. Patent No. 7,561,972; U.S. Patent No. 7,561,973; U.S. Patent No. 8,126,653; and U.S. Patent No. 8,401,798, each of which is incorporated herein by reference. In some embodiments, the polynucleotide sequence is sequence-optimized based on codon usage bias in a host cell (e.g., a mammalian cell, e.g., a human cell, a mouse cell, a non-human primate cell) relative to a reference sequence using a method of sequence optimization known in the art (e.g., GeneGPS™, e.g., Geneious™). In some embodiments, the polynucleotide sequence comprises or consists of a nucleotide sequence that is codon-optimized relative to a reference sequence using a method of sequence optimization, 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 that is 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. 【0064】 In some embodiments, the present 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. [Table 1] 【0065】 Expression cassette element In some embodiments, the expression cassette of the present disclosure includes a promoter. As used herein, the term "promoter" refers to a DNA sequence that directs the binding of RNA polymerase and thereby promotes RNA synthesis, i.e., the minimum sequence sufficient to direct transcription. Promoter and corresponding protein or polypeptide expression may be ubiquitous, i.e., strongly active in a wide range of cells, tissues, and species, or cell-type specific, tissue-specific, or species-specific. The promoter may be "constitutive," i.e., constantly active, or "inducible," i.e., one in which the promoter can be activated or inactivated by the presence or absence of a biotic or abiotic factor. Enhancer sequences, which may or may not be contiguous with the promoter sequence, are also included in the nucleic acid constructs or vectors of the present invention. Enhancer sequences affect promoter-dependent gene expression and may be located in the 5' or 3' region of a native gene. 【0066】 Any suitable promoter region or promoter sequence therein can be used in the subject polynucleotide cassette as long as the promoter region promotes the expression of the polynucleotide sequence encoding the DAO1 enzyme in at least one eye and / or lacrimal gland. In some embodiments, the promoter promotes the expression of a gene in the mammalian eye and / or lacrimal gland. In some embodiments, the expression cassette includes a cell-type specific promoter. The promoter may specifically promote transcription in cells of the eye and / or cells of the lacrimal gland. 【0067】 In some embodiments, the promoter is the CAG promoter. In some embodiments, the promoter includes 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 includes SEQ ID NO: 5. 【0068】 In some embodiments, the promoter is the 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 consists of or comprises SEQ ID NO: 21. 【0069】 The expression cassette may contain a polyadenylation (polyA) sequence. In some embodiments, the expression cassettes described herein include a transcription termination signal. Elements that direct efficient termination and polyadenylation of the heterologous nucleic acid transcript increase heterologous gene expression. Transcription termination signals are generally found downstream of the polyadenylation signal. In some embodiments, the vector includes a polyadenylation sequence 3' of the polynucleotide encoding the polypeptide to be expressed. The terms "polyA site" or "polyA sequence", as used herein, refer to a DNA sequence that directs both termination and polyadenylation of the nascent RNA transcript by RNA polymerase II. The polyadenylation sequence can promote mRNA stability by addition of a polyA tail to the 3' end of the coding sequence and, thus, can contribute to an increase in translation efficiency. Cleavage and polyadenylation are directed by the poly(A) sequence in the RNA. The core poly(A) sequence for mammalian pre-mRNAs has two recognition elements flanking the cleavage-polyadenylation site. Typically, a nearly invariant AAUAAA hexamer is present 20-50 nucleotides upstream of a more variable element rich in U or GU residues. Cleavage of the nascent transcript occurs between these two elements and is linked to the addition of up to 250 adenosines to the 5' cleavage product. In particular embodiments, the core poly(A) sequence is an ideal polyA sequence (e.g., AATAAA, ATTAAA, AGTAAA). In particular embodiments, the poly(A) sequence is the SV40 polyA sequence, the bovine growth hormone polyA sequence (BGH polyA), the rabbit β-globin polyA sequence (rβgpA), variants thereof, or another suitable heterologous or endogenous polyA sequence known in the art. In some embodiments, the expression cassettes described herein include a polyA sequence. In some embodiments, the polyA sequence is the BGH polyA sequence. In some embodiments, the BGH polyA sequence comprises or consists of a nucleotide sequence having at least 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 18. In some embodiments, the BGH polyA sequence comprises or consists of SEQ ID NO: 18. 【0070】 Optionally, the rAAV vectors of the present disclosure include a woodchuck post-transcriptional regulatory element (WPRE). In some embodiments, the rAAV vector includes a WPRE comprising SEQ ID NO: 22. 【0071】 Recombinant AAV vector In some embodiments of the present disclosure, the subject expression cassette is used, for example, to deliver DAO1 or a functional variant thereof to at least one eye and / or lacrimal gland of a subject for treating an eye disorder. Thus, in some embodiments of the present disclosure, a composition that provides expression of DAO1 or a functional variant thereof in at least one eye and / or lacrimal gland of a subject is a gene delivery vector, and the gene delivery vector comprises the expression cassette described herein. 【0072】 In some embodiments, the gene delivery vector is an rAAV vector. In such embodiments, the expression cassette is flanked at the 5' and 3' ends by functional AAV inverted terminal repeat (ITR) sequences. By "functional AAV ITR sequence" is meant that the ITR sequence functions as intended for rescue, replication, and packaging of the AAV vector. Thus, the AAV ITRs for use in the gene delivery vectors of the present disclosure need not have a wild-type nucleotide sequence and may be altered by nucleotide insertions, deletions, or substitutions, or the AAV ITRs may be derived from any of several AAV serotypes, e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10. In some embodiments, the AAV ITR is derived from AAV1. In some embodiments, the AAV ITR is derived from AAV2. In some embodiments, the AAV ITR is derived from AAV3. In some embodiments, the AAV ITR is derived from AAV4 In some embodiments, the AAV ITR is derived from AAV5. In some embodiments, the AAV ITR is derived from AAV6. In some embodiments, the AAV ITR is derived from AAV7. In some embodiments, the AAV ITR is derived from AAV9. In some embodiments, the AAV ITR is derived from AAV1. In some embodiments, the AAV ITR is derived from AAV10. Certain rAAV vectors have wild-type REP and CAP genes that are wholly or partially deleted, but retain functional flanking ITR sequences. In some embodiments, the AAV ITRs are those identified in Table 2. 【Table 2】 【0073】 In such embodiments, the rAAV vector comprises an AAV capsid derived from any adeno-associated virus serotype known in the art or discovered in the future, including but not limited to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, etc. For example, the AAV capsid may be a wild-type (or "native") capsid. In some embodiments, the rAAV vector comprises an AAV capsid derived from AAV1. In some embodiments, The rAAV vector contains an AAV capsid derived from AAV2. In some embodiments, the rAAV vector contains an AAV capsid derived from AAV3. In some embodiments, the rAAV vector contains an AAV capsid derived from AAV4. In some embodiments, the rAAV vector contains an AAV capsid derived from AAV5. In some embodiments, the rAAV vector contains an AAV capsid derived from AAV6. In some embodiments, the rAAV vector contains an AAV capsid derived from AAV7. In some embodiments, the rAAV vector contains an AAV capsid derived from AAV8. In some embodiments, the rAAV vector contains an AAV capsid derived from AAV9. In some embodiments, the rAAV vector contains an AAV capsid derived from AAV10. AAV capsids of particular interest include AAV2, AAV5, AAV8, and AAV9. However, similar to the ITR, the capsid need not have a wild-type nucleotide sequence; rather, it may be altered by insertions, deletions, or substitutions of nucleotides in the VP1, VP2, or VP3 sequences as long as the capsid can transduce cells of the eye and / or lacrimal gland. In other words, the AAV capsid may be a variant AAV capsid. In some embodiments, the rAAV vector is a "pseudotyped" AAV created by using the capsid (cap) gene of one AAV and the rep gene and ITR from a different AAV, e.g., a pseudotyped AAV2 created by using rep from AAV2 and cap from AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, or AAV9 together with a plasmid containing a vector based on AAV2. For example, the rAAV vector may 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. 【0074】 In some embodiments, the rAAV is replication-defective and the rAAV vector cannot independently replicate and package its genome further. For example, when the eye and / or lacrimal gland are transduced with the rAAV vector, the gene is expressed in the transduced eye and / or lacrimal gland, but the transduced eye and / or lacrimal gland cannot replicate rAAV due to the fact that it lacks the rep and cap genes as well as accessory function genes. 【0075】 The rAAV vectors of the present disclosure comprising the expression cassettes described herein can be produced using helper-free production. rAAV is a replication-defective virus and normally requires components from a live helper virus, such as an adenovirus, in the host cell for packaging of the infectious rAAV vector. The rAAV helper-free production system enables the production of infectious rAAV vectors without the use of live helper viruses. In the helper-free system, the host packaging cell line is co-transfected with three plasmids. The first plasmid contains the adenoviral gene products (i.e., E2A, E4, and VA RNA genes) required for packaging of the rAAV vector. The second plasmid contains the required AAV genes (i.e., the 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 an infectious rAAV vector not supplied by the plasmid. In some embodiments, CA The P gene can encode, for example, the AAV capsid proteins described herein. In some embodiments, the promoter is the promoter sequence described herein. In some embodiments, the promoter sequence is the CAG sequence. In some embodiments, the encoded enzyme is DAO1. 【0076】 AAV serotypes that have been shown to infect the eye and / or lacrimal gland include AAV2, AAV5, AAV 5w8, and AAV9 (Rocha et al., supra). Exemplary amino acid and nucleotide sequences for AAV capsid proteins are identified in Table 3. 【0077】 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 having at least 95%, 96%, 97%, 98%, 99%, or 100% similarity 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). 【0078】 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 having at least 95%, 96%, 97%, 98%, 99%, or 100% similarity 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. 【0079】 In some embodiments, the AAV capsid protein comprises SEQ ID NO: 8. In some embodiments, the AAV capsid protein comprises a sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% similarity 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). 【0080】 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 having at least 95%, 96%, 97%, 98%, 99%, or 100% similarity 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. 【0081】 In some embodiments, the AAV serotype used for infecting 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 having at least 95%, 96%, 97%, 98%, 99%, or 100% similarity 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). 【0082】 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 having at least 95%, 96%, 97%, 98%, 99%, or 100% similarity 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. 【0083】 In some embodiments, the AAV serotype used for infecting 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 having at least 95%, 96%, 97%, 98%, 99%, or 100% similarity 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). 【0084】 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 having at least 95%, 96%, 97%, 98%, 99%, or 100% similarity 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. 【0085】 In some embodiments, the AAV serotype used for infecting 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 having at least 95%, 96%, 97%, 98%, 99%, or 100% similarity 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). 【0086】 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 having at least 95%, 96%, 97%, 98%, 99%, or 100% similarity 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. [Table 3] 【0087】 Exemplary rAAV In some embodiments, the 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, the expression cassette comprising a polynucleotide operably linked to a promoter, the polynucleotide comprising a nucleotide sequence encoding DAO1. In some embodiments, the promoter is any one 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, from 5' to 3', a 5' ITR (e.g., an AAV2 5' ITR), a promoter (e.g., a CMV promoter), a 5' untranslated region (5' UTR), a polynucleotide sequence comprising a nucleotide sequence encoding a DAO1 enzyme, a polyA sequence, and a nucleotide sequence comprising a 3' ITR (e.g., an AAV2 3' ITR). In some embodiments, the expression cassette comprises, from 5' to 3', a 5' ITR (e.g., an AAV2 5' ITR), an enhancer (e.g., a CMV enhancer), a promoter (e.g., a CMV promoter), a 5' UTR, a polynucleotide sequence comprising a nucleotide sequence encoding a DAO1 enzyme, a WPRE sequence, a polyA sequence, and a nucleotide sequence comprising a 3' ITR (e.g., an AAV2 3' ITR). In some embodiments, the 5' UTR comprises a Kozak sequence located immediately upstream of the start codon in the nucleotide sequence encoding the DAO1 enzyme. 【0088】 In some embodiments, rAAV comprises an expression cassette comprising an AAV capsid and a polynucleotide operably linked to a promoter, wherein the polynucleotide comprises a nucleotide sequence encoding a human DAO1 enzyme as described herein. In some embodiments, rAAV comprises an expression cassette comprising an AAV capsid and a polynucleotide operably linked to a CAG promoter or a CMV promoter, wherein the polynucleotide comprises a nucleotide sequence encoding a DAO1 enzyme as described herein. 【0089】 In some embodiments, rAAV comprises an expression cassette comprising an AAV capsid and 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, and the DAO1 enzyme comprises or consists of an amino acid 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: 1. In some embodiments, the DAO1 enzyme comprises or consists of the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, rAAV comprises an expression cassette comprising an AAV capsid and a polynucleotide operably linked to a promoter (e.g., a CAG promoter), wherein the polynucleotide comprises a nucleotide sequence encoding a DAO1 enzyme, and 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. 【0090】 In some embodiments, rAAV comprises an expression cassette comprising an AAV capsid and a polynucleotide operably linked to a promoter (e.g., a CAG promoter or a CMV promoter), the polynucleotide comprising a nucleotide sequence encoding a DAO1 enzyme, the DAO1 enzyme comprising or consisting of an amino acid sequence sharing at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 2. In some embodiments, the DAO1 enzyme comprises or consists of the amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, rAAV comprises an expression cassette comprising an AAV capsid and a polynucleotide operably linked to a promoter (e.g., a CAG promoter or a CMV promoter), the polynucleotide comprising a nucleotide encoding a DAO1 enzyme, the nucleotide sequence comprising 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, rAAV comprises an expression cassette comprising an AAV capsid and a polynucleotide operably linked to a promoter (e.g., a CAG promoter or a CMV promoter), the polynucleotide comprising a nucleotide encoding a DAO1 enzyme, the nucleotide sequence comprising 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. 【0091】 In some embodiments, the rAAV comprises 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 the AAV capsid and SEQ ID NO: 23. In some embodiments, the expression cassette comprises or consists of SEQ ID NO: 23. 【0092】 In some embodiments, the rAAV comprises an expression cassette comprising an AAV2 capsid and a polynucleotide operably linked to a promoter, the polynucleotide comprising a nucleotide sequence encoding the human DAO1 enzyme described herein. In some embodiments, the rAAV comprises an expression cassette comprising an AAV2 capsid and a polynucleotide operably linked to a CAG promoter or a CMV promoter, the polynucleotide comprising a nucleotide sequence encoding the DAO1 enzyme described herein. 【0093】 In some embodiments, the rAAV comprises an expression cassette comprising an AAV2 capsid and a polynucleotide (e.g., a CAG promoter or a CMV promoter) operably linked to a promoter, the polynucleotide comprising a nucleotide sequence encoding a DAO1 enzyme, the DAO1 enzyme comprising or consisting of an amino acid sequence sharing 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: 1. In some embodiments Moreover, the DAO1 enzyme comprises or consists of the amino acid sequence shown in SEQ ID NO: 1. In some embodiments, the rAAV comprises an expression cassette comprising a polynucleotide operably linked to an AAV2 capsid and a promoter (e.g., a CAG promoter or a CMV promoter), the polynucleotide comprising nucleotides encoding the DAO1 enzyme, the nucleotide sequence comprising 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. 【0094】 In some embodiments, the rAAV comprises an expression cassette comprising a polynucleotide operably linked to an AAV2 capsid and a promoter (e.g., a CAG promoter or a CMV promoter), the polynucleotide comprising a nucleotide sequence encoding a DAO1 enzyme, the DAO1 enzyme comprising or consisting of an amino acid sequence sharing at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 2. In some embodiments, the DAO1 enzyme comprises the amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, the rAAV comprises an expression cassette comprising a polynucleotide operably linked to an AAV2 capsid and a promoter (e.g., a CAG promoter or a CMV promoter), the polynucleotide comprising a nucleotide encoding a DAO1 enzyme, the nucleotide sequence comprising 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 expression cassette comprising a polynucleotide operably linked to an AAV2 capsid and a promoter (e.g., a CAG promoter or a CMV promoter), the polynucleotide comprising a nucleotide encoding a DAO1 enzyme, the nucleotide sequence comprising 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. 【0095】 In some embodiments, the rAAV comprises 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 the AAV2 capsid and SEQ ID NO: 23. In some embodiments, the rAAV comprises an expression cassette comprising or consisting of an expression cassette comprising the AAV2 capsid and SEQ ID NO: 23. 【0096】 In some embodiments, the rAAV comprises an expression cassette comprising an AAV5 capsid and a polynucleotide operably linked to a promoter, the polynucleotide comprising a nucleotide sequence encoding a human DAO1 enzyme as described herein. In some embodiments, the rAAV comprises an expression cassette comprising an AAV5 capsid and a polynucleotide operably linked to a CAG promoter, the polynucleotide comprising a nucleotide sequence encoding a DAO1 enzyme as described herein. 【0097】 In some embodiments, the rAAV comprises an expression cassette comprising an AAV5 capsid and a polynucleotide operably linked to a promoter (e.g., a CAG promoter or a CMV promoter), the polynucleotide comprising a nucleotide sequence encoding a DAO1 enzyme, the DAO1 enzyme comprising or consisting of an amino acid sequence sharing 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: 1. In some embodiments, the DAO1 enzyme comprises the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the rAAV comprises an expression cassette comprising an AAV5 capsid and a polynucleotide operably linked to a promoter (e.g., a CAG promoter or a CMV promoter), the polynucleotide comprising a nucleotide encoding a DAO1 enzyme, the nucleotide sequence comprising 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. 【0098】 In some embodiments, the rAAV comprises an expression cassette comprising a polynucleotide operably linked to an AAV5 capsid and a promoter (e.g., a CAG promoter or a CMV promoter), the polynucleotide comprising a nucleotide sequence encoding a DAO1 enzyme, the DAO1 enzyme comprising an amino acid sequence sharing at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 2. In some embodiments, the DAO1 enzyme comprises the amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, the rAAV comprises an expression cassette comprising a polynucleotide operably linked to an AAV5 capsid and a promoter (e.g., a CAG promoter or a CMV promoter), the polynucleotide comprising nucleotides encoding a DAO1 enzyme, the nucleotide sequence comprising 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 expression cassette comprising a polynucleotide operably linked to an AAV5 capsid and a promoter (e.g., a CAG promoter or a CMV promoter), the polynucleotide comprising nucleotides encoding a DAO1 enzyme, the nucleotide sequence comprising 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. 【0099】 In some embodiments, the rAAV comprises an AAV5 capsid and a sequence similar to that of SEQ ID NO:23. In some embodiments, the rAAV comprises an expression cassette comprising or consisting of 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 the AAV5 capsid. 【0100】 In some embodiments, the rAAV comprises an expression cassette comprising an AAV9 capsid and a polynucleotide operably linked to a promoter, the polynucleotide comprising a nucleotide sequence encoding the human DAO1 enzyme described herein. In some embodiments, the rAAV comprises an expression cassette comprising an AAV9 capsid and a polynucleotide operably linked to a CAG promoter, the polynucleotide comprising a nucleotide sequence encoding the DAO1 enzyme described herein. 【0101】 In some embodiments, the rAAV comprises an expression cassette comprising a polynucleotide operably linked to an AAV9 capsid and a promoter (e.g., a CAG promoter or a CMV promoter), the polynucleotide comprising a nucleotide sequence encoding a DAO1 enzyme, the DAO1 enzyme comprising an amino acid sequence sharing 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: 1. In some embodiments, the DAO1 enzyme comprises the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the rAAV comprises an expression cassette comprising a polynucleotide operably linked to an AAV9 capsid and a promoter (e.g., a CAG promoter or a CMV promoter), the polynucleotide comprising nucleotides encoding a DAO1 enzyme, the nucleotide sequence comprising 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. 【0102】 In some embodiments, the rAAV comprises an expression cassette comprising a polynucleotide operably linked to an AAV9 capsid and a promoter (e.g., a CAG promoter or a CMV promoter), the polynucleotide comprising a nucleotide sequence encoding a DAO1 enzyme, the DAO1 enzyme comprising or consisting of an amino acid sequence sharing at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 2. In some embodiments, the DAO1 enzyme comprises the amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, the rAAV comprises an expression cassette comprising a polynucleotide operably linked to an AAV9 capsid and a promoter (e.g., a CAG promoter or a CMV promoter), the polynucleotide comprising nucleotides encoding a DAO1 enzyme, the nucleotide sequence comprising 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 a promoter ( For example, it includes an expression cassette comprising a polynucleotide operably linked to a CAG promoter or a CMV promoter), wherein the polynucleotide comprises nucleotides encoding the DAO1 enzyme, and 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. 【0103】 In some embodiments, the rAAV comprises or consists of an expression cassette comprising an AAV9 capsid and 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 or consists of an expression cassette comprising an AAV9 capsid and SEQ ID NO: 23. 【0104】 Method of use The methods and compositions described herein can be used to treat ocular symptoms and to reduce symptoms associated with ocular symptoms. Terms such as "treating" and "treat" are used herein to generally mean obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic, e.g., in terms of preventing a disease or its symptoms completely or partially, e.g., reducing the likelihood that a disease or its symptoms will occur in a subject, and / or therapeutic in terms of partial or complete cure of a disease and / or adverse effects attributable to the disease. "Treatment" as used herein covers any treatment of a disease in a mammal and includes, but is not limited to, (a) inhibition of the progression of the disease; (b) reduction, alleviation, or reduction of the increase of one or more symptoms of the disease; (c) reduction, alleviation, or reduction of the increase of one or more signs of the disease; (d) induction of regression of the disease. A therapeutic agent may be administered before, during, or after the onset of a disease or injury. Treatment of a disease in progress, where the treatment stabilizes or reduces an undesirable clinical symptom of the patient, is of particular interest. Such treatment is preferably carried out prior to complete loss of function in the affected tissue. The subject treatment is preferably administered during the symptomatic stage of the disease and, in some cases, after the symptomatic stage of the disease. 【0105】 As used herein, "administer", "administering", and "administration" refer to providing a substance (e.g., an rAAV vector, or a polypeptide comprising DAO1, or a fragment thereof) to a subject in a manner that is pharmacologically useful (e.g., for treating a disease, disorder, or symptom in the subject). 【0106】 In some embodiments, the eye symptoms treated according to the methods described herein are associated with increased histamine production and / or increased histamine signaling. In some embodiments, the eye symptoms are inflammatory symptoms. In some embodiments, the eye symptoms are autoimmune reactions. In some embodiments, the eye symptoms are autoimmune symptoms. In some embodiments, the eye symptoms are allergic reactions. In some embodiments, the eye symptoms are allergic reactions to therapeutic agents. In some embodiments, the eye symptoms are allergic symptoms. In some embodiments, the eye symptoms are vernal catarrh. In some embodiments, the eye symptoms are atopic keratoconjunctivitis. In some embodiments, the eye symptoms are seasonal or perennial allergic conjunctivitis. 【0107】 In some embodiments, the present disclosure provides a method of treating a subject having an eye disease or disorder, the method comprising administering to the subject an rAAV vector comprising a nucleotide encoding human DAO1 as described herein, wherein the expression of human DAO1 is increased in the subject as compared to the expression of human DAO1 in an untreated subject or in the contralateral eye of the treated subject. Similarly, treatment methods are also described herein in various embodiments that include administration of an alternative vector (e.g., a plasmid) configured to express DAO1 or a fragment thereof in addition to administration of a polypeptide comprising DAO1 or a fragment thereof. 【0108】 In some embodiments, the present disclosure provides an rAAV vector for use in a method of treating an ocular symptom in a subject in need of treatment of the ocular symptom, the method comprising administering a recombinant adeno-associated virus (rAAV) vector to at least one eye of the subject or to at least one lacrimal gland of the subject's eye, the rAAV vector comprising an expression cassette comprising a polynucleotide encoding human DAO1 operably linked to an AAV capsid and a promoter. In some embodiments, the rAAV vector for use is administered to the lacrimal gland of the subject. In some embodiments, the rAAV vector for use comprises an expression cassette encoding human DAO1. 【0109】 In some embodiments, the present disclosure provides an rAAV vector for use in, or adaptable for use in, treating a subject having an ocular disease, disorder, or symptom. In some embodiments, the rAAV vector for use in, or adaptable for use in, comprises an expression cassette comprising a polynucleotide encoding human DAO1 (or a fragment thereof) operably linked to an AAV capsid and a promoter. 【0110】 In some embodiments, the present disclosure provides a method of treating an ocular symptom in a subject in need thereof, the method comprising administering to at least one eye of the subject or to at least one lacrimal gland of the subject's eye 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 rAAV (or any other vector) administered 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. 【0111】 In some embodiments, the present disclosure provides an rAAV vector for use in a method of treating an ocular symptom in a subject in need thereof, the method comprising administering to at least one eye of the subject or to at least one lacrimal gland of the subject's eye a recombinant adeno-associated virus (rAAV) vector comprising an expression cassette comprising the nucleic acid sequence of SEQ ID NO: 23. 【0112】 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 subject or in the contralateral eye of a treated subject. 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 subject or in the contralateral eye of a treated subject. 【0113】 In some embodiments, the expression of human DAO1 is increased by about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 1 year compared to the expression of human DAO1 in an untreated subject or in the contralateral eye of a treated subject. 【0114】 As used herein, "about" or "approximately" when applied to one or more values of interest refers to a value similar to the recited reference value. In certain embodiments, "about" refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of the recited reference value in either direction (greater than or less than) of the recited reference value, unless otherwise stated or otherwise apparent from the context (except where such numbers would exceed 100% of the possible values). 【0115】 In some embodiments, a subject is administered an rAAV or other vector comprising a nucleotide encoding the human DAO1 enzyme described herein, and the expression of the human DAO1 enzyme is increased in the subject compared to the expression of human DAO1 in an untreated subject or in the contralateral eye of a treated subject. 【0116】 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 subject or in the contralateral eye of a treated subject. 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 subject or in the contralateral eye of a treated subject. 【0117】 In some embodiments, the expression of 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 the expression of human DAO1 enzyme in an untreated subject or in the contralateral eye of a treated subject. 【0118】 In some embodiments, the present disclosure provides a method of treating a subject having an eye disease or disorder, the method comprising administering to the subject an rAAV (or other vector) comprising DAO1 or a nucleotide encoding the human DAO1 enzyme, wherein the expression of the human DAO1 enzyme is increased in the subject compared to the expression of the human DAO1 enzyme in an untreated subject or in the contralateral eye of a treated subject. 【0119】 In some embodiments, the present disclosure provides a method of treating a symptom associated with histamine intolerance in a subject in need of treatment for the symptom, the method comprising administering to the subject an effective amount of DAO1 or an effective amount of an rAAV (or other vector) described herein. In some embodiments, the symptom is associated with overproduction of histamine in one or more tissues of the subject. In some embodiments, the symptom is associated with overproduction of histamine in one or both eyes of the subject. In some embodiments, the symptom is associated with accumulation of histamine in one or more tissues of the subject. In some embodiments, the symptom is associated with accumulation of histamine in one or both eyes of the subject. 【0120】 In some embodiments, histamine intolerance is caused as a result of allergic symptoms. In some embodiments, the allergic symptoms are seasonal or year-round allergic symptoms. In some embodiments, histamine intolerance is caused as a result of autoimmune symptoms. 【0121】 In some embodiments, the symptoms are characterized by one or more allergic symptoms (e.g., sneezing, rhinitis, conjunctivitis, shortness of breath, cough, chest constriction, hives, skin discoloration, stomach cramps, throat constriction, pain, swelling, vomiting, diarrhea, fullness), and administration alleviates or prevents one or more symptoms. In some embodiments, the symptoms are characterized by one or more symptoms of the eyes (e.g., itching, swelling, tearing, and flushing), and administration alleviates or prevents one or more symptoms. In some embodiments, the severity of one or more symptoms is evaluated based on any method known in the art for characterizing allergy-related symptoms. In some embodiments, the severity of one or more symptoms is evaluated based on any method known in the art for characterizing conditions associated with histamine intolerance. In some embodiments, one or more symptoms are evaluated using the methods described herein (e.g., conjunctival allergen challenge, corneal staining test, ocular dryness score test, Schirmer score test, ocular surface disease index test). 【0122】 In some embodiments, the method comprises administering a dose of DAO1, or rAAV (or another vector configured to express DAO1), to a subject having symptoms associated with histamine intolerance (e.g., allergic symptoms), where the symptoms are characterized by one or more syndromes described herein (e.g., one or more allergic syndromes). In some embodiments, the dose is administered prior to the onset of one or more syndromes. In some embodiments, the administration prevents the onset of one or more syndromes. In some embodiments, the administration reduces the severity of one or more syndromes. For example, in some embodiments, the method comprises administering a dose of rAAV to a subject having seasonal or perennial allergic symptoms characterized by one or more syndromes (e.g., rhinitis, conjunctivitis, headache, fatigue, cough, sneeze), where the dose of rAAV is administered prior to the season in which the allergic symptoms occur, and the administration prevents the onset of one or more syndromes and / or reduces the severity of one or more syndromes. In some embodiments, the dose is administered after the onset of one or more syndromes, and the administration reduces the severity of one or more syndromes. 【0123】 In some embodiments, the method comprises administering a dosing regimen of rAAV to a subject having symptoms associated with histamine intolerance (e.g., allergic symptoms), the symptoms being characterized by one or more syndromes described herein (e.g., one or more syndromes of allergy), the dosing regimen comprising a first dose and at least one additional dose of rAAV, the first dose of rAAV being administered to the subject prior to or subsequent to the onset of one or more syndromes, and the at least one additional dose being administered to the subject after the first dose, thereby preventing or reducing the severity of one or more syndromes. In some embodiments, the dosing regimen comprises the frequency and / or dose of administration selected based on the pharmacokinetic parameters of rAAV. In some embodiments, a clinician administers rAAV at a frequency and / or dose to achieve or maintain one or more desired effects. In some embodiments, the one or more desired effects are to prevent one or more syndromes associated with the subject's symptoms. In some embodiments, the one or more desired effects are to reduce the severity of one or more syndromes associated with the subject's symptoms. In some embodiments, the severity of one or more syndromes is measured using methods described herein or known in the art for assessing conditions associated with histamine intolerance. In some embodiments, the one or more desired effects are achieved immediately after administration of the first dose of rAAV. In some embodiments, the one or more desired effects The result occurs at any point in time after administration of the first dose of rAAV. In some embodiments, one or more desired effects are achieved after administration of at least one additional dose of rAAV. In some embodiments, one or more desired effects are achieved at any point in time during the dosing regimen. In some embodiments, one or more desired effects are achieved after administration of the first dose of rAAV, and the subject is administered at least one additional dose of rAAV to prevent reversal of the one or more desired effects. In some embodiments, the method comprises administering a first dose of rAAV to the subject prior to, 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 protocols and parameters described above may be used to design a regimen for a treatment in which DAO1 is administered directly or via an alternative vector system (e.g., plasmid-based expression). 【0124】 In some embodiments, the present disclosure provides a method of treating ocular inflammatory symptoms in a subject in need thereof, the method comprising administering to the subject one or more therapeutically effective doses of DAO1, or an rAAV or other vector described herein. 【0125】 In some embodiments, the present disclosure provides a method of treating ocular autoimmune symptoms in a subject in need thereof, the method comprising administering to the subject one or more therapeutically effective doses of DAO1, or an rAAV or other vector described herein. 【0126】 In some embodiments, the present disclosure provides a method of treating ocular allergic symptoms in a subject in need of treatment of ocular allergic symptoms, the method comprising administering to the subject one or more therapeutically effective doses of DAO1, or an rAAV or other vector described herein. 【0127】 In some embodiments, the present disclosure provides a method of treating vernal catarrh in a subject in need of treatment of vernal catarrh, the method comprising administering to the subject one or more therapeutically effective doses of DAO1, or an rAAV or other vector described herein. 【0128】 In some embodiments, the present disclosure provides a method of treating atopic keratoconjunctivitis in a subject in need of treatment of atopic keratoconjunctivitis, the method comprising administering to the subject one or more therapeutically effective doses of DAO1, or an rAAV or other vector described herein. 【0129】 In some embodiments, the present disclosure provides a method of treating seasonal or perennial allergic conjunctivitis in a subject in need of treatment of seasonal or perennial allergic conjunctivitis, the method comprising administering to the subject one or more therapeutically effective doses of DAO1, or an rAAV or other vector described herein. 【0130】 In some embodiments, the present disclosure provides an rAAV described herein for use in a method of treating symptoms associated with histamine intolerance in a subject in need of treatment of symptoms associated with histamine intolerance, the method comprising administering to the subject an effective amount of the rAAV described herein. 【0131】 In some embodiments, the present disclosure provides the rAAV described herein for use in the production of a medicament for treating symptoms associated with histamine intolerance in a subject in need of treatment of symptoms associated with histamine intolerance. 【0132】 In some embodiments, the present disclosure provides the rAAV described herein for use in a method of treating an autoimmune symptom in a subject in need of treatment of an autoimmune symptom, the method comprising administering to the subject an effective amount of the rAAV described herein. 【0133】 In some embodiments, the present disclosure provides the rAAV described herein for use in the production of a medicament for treating an autoimmune symptom in a subject in need of treatment of an autoimmune symptom. 【0134】 In some embodiments, the present disclosure provides the rAAV described herein for use in a method of treating an allergic symptom in a subject in need of treatment of an allergic symptom, the method comprising administering to the subject an effective amount of the rAAV described herein. 【0135】 In some embodiments, the present disclosure provides the rAAV described herein for use in the production of a medicament for treating an allergic symptom in a subject in need of treatment of an allergic symptom. 【0136】 In some embodiments, the present disclosure provides the rAAV described herein for use in a method of treating vernal catarrh in a subject in need of treatment of vernal catarrh, the method comprising administering to the subject an effective amount of the rAAV described herein. 【0137】 In some embodiments, the present disclosure provides the rAAV described herein for use in the production of a medicament for treating vernal catarrh in a subject in need of treatment of vernal catarrh. 【0138】 In some embodiments, the present disclosure provides the rAAV described herein for use in a method of treating allergic keratoconjunctivitis in a subject in need of treatment of allergic keratoconjunctivitis, the method comprising administering to the subject an effective amount of the rAAV described herein. 【0139】 In some embodiments, the present disclosure provides the rAAV described herein for use in the production of a medicament for treating allergic keratoconjunctivitis in a subject in need of treatment of allergic keratoconjunctivitis. 【0140】 In some embodiments, the present disclosure provides the rAAV described herein for use in a method of treating seasonal or perennial allergic conjunctivitis in a subject in need of treatment of seasonal or perennial allergic conjunctivitis, the method comprising administering to the subject an effective amount of the rAAV described herein. 【0141】 In some embodiments, the present disclosure provides the rAAV described herein for use in the production of a medicament for treating seasonal or perennial allergic conjunctivitis in a subject in need of treatment of seasonal or perennial allergic conjunctivitis. 【0142】 Combination therapy In some embodiments, the treatment methods described herein include further administering to a subject one or more additional therapeutic agents. In some embodiments, the additional therapeutic agent is an agent that increases tear production. In some embodiments, the additional therapeutic agent is another AAV-based gene therapy construct. Suitable therapeutic agents for use as combination therapies in the present disclosure are further described in PCT / US2022 / 027653, which is incorporated herein by reference. 【0143】 In some embodiments, the present disclosure provides a method of treating a symptom associated with histamine intolerance in a subject in need of treatment of a symptom associated with histamine intolerance, the method comprising administering to one or both eyes of the subject an effective amount of DAO1, or an effective amount of an rAAV or other vector described herein, wherein the patient has been, is being, or will subsequently be given a treatment that increases tear production and / or re-establishes tear film homeostasis. In some embodiments, the subject has decreased tear production and / or impaired tear film homeostasis. In some embodiments, DAO1, or the vector (e.g., rAAV), is administered to one or both lacrimal glands via intraductal injection. In some embodiments, DAO1, or the vector (e.g., rAAV), is administered to the eye by topical application. In some embodiments, the treatment is administered to the subject after administration of DAO1, or the vector (e.g., rAAV). In some embodiments, the treatment results in an increase in the amount of DAO1, or the vector (e.g., rAAV), delivered to the tear film of the subject's eye within a predetermined time compared to the amount of DAO1 or the vector (e.g., rAAV) delivered to the tear film when the subject is not given the treatment. In some embodiments, the predetermined period is from about 5 minutes to about 60 minutes. 【0144】 In some embodiments, a treatment for increasing 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 as described herein. 【0145】 nAChRs are pentameric ligand-gated ion channels that have high affinity and selectivity for both nicotine and acetylcholine (which is similar to nicotine in its protonated form), and that include combinations of alpha and beta subunits. Examples of nAChR subtypes include, but are not limited to, alpha3beta4, alpha4beta2, alpha3alpha5beta4, and alpha4alpha6beta2. For example, an important nAChR receptor subtype involved in the induction of the nasolacrimal reflex is the alpha4beta2 subtype located on trigeminal nerve terminals in the nasal mucosa. 【0146】 Administration of an nAChR agonist can desensitize the receptor. Receptor desensitization results in a reduction in the response to the agonist, even at higher agonist concentrations, which in turn results in a decrease in the effectiveness of the treatment. For example, short-term desensitization of the nAChR receptor to an agonist can occur over a 24-hour period following administration of the agonist. The potential for receptor desensitization can potentially limit the dosing frequency over a certain time period in order to preserve an effective response to the agonist. 【0147】 An nAChR agonist may be characterized as a full or partial agonist as determined by its ability to activate the receptor to produce a response as compared to the response at a given receptor for acetylcholine (ACh). Generally, an nAChR agonist is a full agonist when binding to a given receptor induces a response equal to or higher than that for ACh. An nAChR agonist is a partial agonist when binding to the receptor induces a lower response as compared to the response produced from ACh. 【0148】 An nAChR agonist response, which can determine receptor activation, can be generated, for example, using an appropriate cell-based assay. Cells that express a specific nAChR receptor subtype and are designed to generate a current response when bound and activated by an nAChR agonist can be used to characterize the agonist profile of a compound and the amount of receptor activation so determined. Examples of general protocols are described below. 【0149】 Cells expressing a specific human nAChR subtype are first exposed to ACh. ACh binds to and activates the receptor, thereby inducing a current. The concentration of ACh is selected to elicit a maximal response of the receptor (e.g., 1280 micromolar concentration of ACh). This current is recorded as the ACh response and serves as 100% of the nAChR agonist response against which the responses to other nAChR agonists are compared. After washing, the cells are exposed to nAChR agonists at various concentrations (e.g., 0.1, 0.3, 1, 3, 10, 30, 100, and 300 micromolar concentrations). The currents induced by exposure to the nAChR agonists are measured and recorded for each nAChR concentration. This nAChR agonist response data is then normalized against the maximal ACh-induced current and plotted as a function of the logarithm of the nAChR agonist concentration. The nAChR agonist response is then calculated as a percentage of the ACh response. 【0150】 In some embodiments, a method of determining the relative agonist activity of an nAChR agonist includes conditions under which the ACh response is induced from an ACh solution at one or more millimolar concentrations. 【0151】 nAChR agonists that induce a response equal to or higher than the maximum ACh response determined by the same receptor type are full agonists. In some embodiments, an nAChR agonist that induces a response of less than 100% of the ACh response may still be characterized as a full agonist, taking into account experimental variability. For example, variability between test or measurement methods, and statistical error, may account for differences in response results. In some embodiments, an nAChR agonist that induces 80% - 120% of the ACh response is considered a full agonist. In some embodiments, an nAChR agonist that induces 99% or more of the ACh response is considered a full agonist. In some embodiments, an nAChR agonist that induces 95% or more of the ACh response is considered a full agonist. In some embodiments, an nAChR agonist that induces 90% or more of the ACh response is considered a full agonist. In some embodiments, an nAChR agonist that induces 85% or more of the ACh response is considered a full agonist. In some embodiments, an nAChR agonist that induces 80% or more of the ACh response is considered a full agonist. 【0152】 Taking into account experimental variability, when an nAChR agonist induces less than 100% of the ACh response, the agonist is generally considered a partial agonist. In some embodiments, an nAChR agonist that induces less than 95% of the ACh response is considered a partial agonist. In some embodiments, an nAChR agonist that induces less than 90% of the ACh response is considered a partial agonist. In some embodiments, an nAChR agonist that induces less than 85% of the ACh response is considered a partial agonist. In some embodiments, an nAChR agonist that induces less than 80% of the ACh response is considered a partial agonist. 【0153】 In some embodiments, an nAChR agonist that induces 5% - 95% of the ACh response is considered a partial agonist. In some embodiments, an nAChR agonist that induces 5% - 9 nAChR agonists that induce 0% are considered partial agonists. In some embodiments, nAChR agonists that induce 5% - 85% of the ACh response are considered partial agonists. In some embodiments, nAChR agonists that induce 5% - 80% of the ACh response are considered partial agonists. 【0154】 In some embodiments, nAChR agonists that induce 10% - 95% of the ACh response are considered partial agonists. In some embodiments, nAChR agonists that induce 10% - 90% of the ACh response are considered partial agonists. In some embodiments, nAChR agonists that induce 10% - 85% of the ACh response are considered partial agonists. In some embodiments, nAChR agonists that induce 10% - 80% of the ACh response are considered partial agonists. 【0155】 nAChR agonists that produce low levels of electrical activity at relatively high concentrations of the agonist may be described as weak partial agonists. In some embodiments, nAChR agonists that induce 30% or less of the ACh response are considered weak partial agonists. In some embodiments, nAChR agonists that induce 25% or less of the ACh response are considered weak partial agonists. In some embodiments, nAChR agonists that induce 20% or less of the ACh response are considered weak partial agonists. In some embodiments, the relatively high concentration of the nAChR agonist is at least 100 micromolar concentration. In some embodiments, the relatively high concentration of the nAChR agonist is at least 200 micromolar concentration. In some embodiments, the relatively high concentration of the nAChR agonist is at least 300 micromolar concentration or more. For example, a 300 micromolar concentration of nAChR agonist that induces 25% of the maximum Ach-induced current is considered a weak partial agonist. 【0156】 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. 【0157】 In some embodiments, the nAChR agonist, or a pharmaceutically acceptable salt thereof, is an agonist of at least one nAChR subtype selected from alpha3beta4, alpha3alpha5beta4, alpha4beta2, and alpha4alpha6beta2. In some embodiments, the nAChR agonist, or a pharmaceutically acceptable salt thereof, is an agonist of at least two nAChR subtypes selected from alpha3beta4, alpha3alpha5beta4, alpha4beta2, and alpha4alpha6beta2. In some embodiments, the nAChR agonist, or a pharmaceutically acceptable salt thereof, is an agonist of at least three nAChR subtypes selected from alpha3beta4, alpha3alpha5beta4, alpha4beta2, and alpha4alpha6beta2. In some embodiments, the nAChR agonist, or a pharmaceutically acceptable salt thereof, is an agonist of the nAChR subtype alpha3beta4. In some embodiments, the nAChR agonist, or a pharmaceutically acceptable salt thereof, is an agonist of the nAChR subtype alpha3alpha5beta4. In some embodiments, the nAChR agonist, or a pharmaceutically acceptable salt thereof, is an agonist of the nAChR subtype alpha4beta2. In some embodiments, the nAChR agonist, or a pharmaceutically acceptable salt thereof, is an agonist of the nAChR subtype alpha4alpha6beta2. In some embodiments, the nAChR agonist, or a pharmaceutically acceptable salt thereof, is an agonist of the nAChR subtype alpha7. In some embodiments, the nAChR agonist, or a pharmaceutically acceptable salt thereof, is not an agonist of the nAChR subtype alpha7. In some embodiments, the nAChR agonist, or its pharmaceutically Permissible salts are not full agonists of the nAChR subtype alpha7. In some embodiments, the nAChR agonist is a full agonist of the above-described subtype. In some embodiments, the nAChR agonist is a partial agonist of the above-described subtype. In some embodiments, the nAChR agonist is a weak partial agonist of the above-described subtype. 【0158】 The term "alpha7" or "α7" nAChR refers to a homomeric alpha7 subtype in which the pentameric subunit of the nAChR is composed entirely of alpha7 subunits. Thus, an nAChR agonist that binds to and activates nAChR alpha7 is an agonist that binds to and activates the nAChR homomeric alpha7 receptor. In some embodiments described herein, the nAChR agonist is not an alpha7 receptor agonist. 【0159】 In some embodiments, the nAChR agonist, or a pharmaceutically acceptable salt thereof, selectively binds to at least one of the nAChR subtypes selected from alpha3beta4, alpha3alpha5beta4, alpha4beta2, and alpha4alpha6beta2. As used herein, "selectively binds to" or "is selective for" means that the compound has a higher affinity and / or a lower half maximal effective concentration ("EC50") for the nAChR subtype, for at least one reference nAChR subtype. Selectivity may be associated with an affinity difference of at least 5-fold, at least 10-fold, at least 20-fold, or at least 50-fold in the EC50 value. In some embodiments, the nAChR agonist, or a pharmaceutically acceptable salt thereof, selectively binds to the nAChR subtype alpha3beta4. In some embodiments, the nAChR agonist, or a pharmaceutically acceptable salt thereof, selectively binds to the nAChR subtype alpha3alpha5beta4. In some embodiments, the nAChR agonist, or a pharmaceutically acceptable salt thereof, selectively binds to the nAChR subtype alpha4beta2. In some embodiments, the nAChR agonist, or a pharmaceutically acceptable salt thereof, selectively binds to the nAChR subtype alpha4alpha6beta2. In some embodiments, the nAChR agonist, or a pharmaceutically acceptable salt thereof, selectively binds to the nAChR subtype alpha7. In some embodiments, the nAChR agonist, or a pharmaceutically acceptable salt thereof, does not selectively bind to the nAChR subtype alpha7. 【0160】 The 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. 【0161】 Varenicline is characterized as a full agonist of the nAChR subtype alpha7 and a partial agonist of the subtypes alpha3beta4, alpha4beta2, alpha6beta2, alpha3alpha5beta4, and alpha4alpha6beta2. 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. Patent No. 9,504,644, U.S. Patent No. 9,504,645, U.S. Patent No. 9,532,944, U.S. Patent No. 9,597,284, and U.S. Patent No. 10,456,386. 【0162】 Compound 1 as referred to herein refers to the following structure. 【Chemical formula】 【0163】 An alternative structural representation of Compound 1 is shown here. 【Chemical formula】 【0164】 Compound 1 may also be referred to by its chemical name. For example, Compound 1 may also be referred to as (R)-5-((E)-2-pyrrolidin-3-ylvinyl)pyrimidine, or simpanicline, 5-{(E)-2-[(3R)-pyrrolidin-3-yl]vinyl}pyrimidine and its variations including (R,E)-5-((2-pyrrolidin-3-yl)vinyl)pyrimidine. 【0165】 Compound 1 is a full agonist of the nAChR subtypes alpha4beta2, alpha3beta4, alpha3alpha5beta4, and alpha4alpha6beta2. Compound 1 is a full agonist of the nAChR subtypes alpha4beta2 and alpha3beta4. 【0166】 Compound 1 is a partial agonist of subtype alpha3beta2. 【0167】 Compound 1 is a weak partial agonist of subtype alpha7. In one example, 300 micromolar concentration of Compound 1 citrate induced only 25% of the maximal ACh-induced current. 【0168】 In some embodiments, the nAChR agonist may be Compound 1, or a pharmaceutically acceptable salt thereof. Pharmaceutically acceptable salts of Compound 1 include galactarate (e.g., hemigalactarate dihydrate) and citrate (e.g., monocitrate). Patent-related information about 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. 【0169】 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 hemigalactarate dihydrate. In some embodiments, the nAChR agonist is (R)-5-((E)-2-pyrrolidin-3-ylvinyl)pyrimidine monocitrate. 【0170】 Mode of Administration In some embodiments, the present disclosure relates to administering rAAV vectors to the eye of a subject or to the lacrimal gland of the eye of the subject A method comprising administering a vector, wherein the rAAV vector comprises an expression cassette comprising a polynucleotide encoding DAO1 operably linked to an AAV capsid and a promoter. In other aspects, the methods according to the present disclosure may comprise administering to the eye of a subject or to the lacrimal gland of the eye of a subject a polypeptide comprising DAO1 or a fragment thereof, or an alternative vector (e.g., a plasmid-based expression system) configured to express DAO1 or a fragment thereof. 【0171】 As noted above, the lacrimal gland functional unit consists of the main and accessory lacrimal glands, the ocular surface, and the interconnecting innervation. For each eye, the main lacrimal gland is located superiorly and cranially in the orbit within the lacrimal fossa of the frontal bone. The accessory glands, known as the glands of Wolfring and Krause, are located within the eyelids. There are approximately 2 - 5 glands of Wolfring and approximately 40 glands of Krause in the upper eyelid. There are approximately 6 - 8 glands of Krause in the lower eyelid. The specific location and anatomical structure of the lacrimal gland functional unit are well known (Conrady et al. J Ophthalmol.;2016:7542929(2016)). Together, the lacrimal glands secrete a tear film onto the ocular surface through the lacrimal ducts. The lacrimal glands also express and secrete proteins and products necessary for corneal regeneration and for promoting transparency into the tear film, such as transforming growth factor-β and retinol (Conrady et al.J Ophthalmol.;2016:7542929(2016);Pan et al.Optom Vis Sci.;95:27 - 31(2018)). In addition to secreting tears into the eye, the lacrimal ducts drain the lacrimal fluid into the nasal cavity. 【0172】 Administration of DAO1, or a vector (e.g., rAAV), to the lacrimal gland may be achieved by topical administration to the ocular surface, direct injection into the lacrimal gland, and / or topical 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. The lacrimal gland may be accessed surgically or by manipulation of the eyelid. Manipulation of the eyelid provides access to the tissue for topical administration (e.g., by irrigation of the tissue with a pharmaceutical composition containing a viral vector). Direct injection into the lacrimal gland may be performed by penetrating the skin overlying the lacrimal gland (Figure 2A) or by manipulating the eyelid to access the lacrimal gland (Figure 2B). Administration of a viral vector to the lacrimal gland may be achieved by transconjunctival injection. In some embodiments, rAAV is administered to the lacrimal gland by direct injection as depicted in Figure 2A. In some embodiments, rAAV is administered to the lacrimal gland by manipulating the eyelid as depicted in Figure 2B. 【0173】 In some embodiments, cells in the eye, lacrimal gland, and / or nasolacrimal duct are transduced with an rAAV vector. 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, the transduced cells in the eye, lacrimal gland, and / or nasolacrimal duct express a therapeutically effective amount of DAO1 enzyme in the tear film of the subject and optionally on the ocular surface. In some embodiments, a therapeutically effective amount of DAO1 enzyme is secreted into the nasal cavity of the subject. In some embodiments, a therapeutically effective amount of DAO1 enzyme is secreted onto the ocular surface of the subject. 【0174】 Delivery of rAAV vectors to the eye and / or lacrimal gland to express a transgene in the tear film has been demonstrated in vivo. In one example, rAAV vectors encoding a luciferase transgene with serotypes AAV2, AAV4, AAV5, AAV 5w8, AAV x5, AAV 9, AAV12, and bovine AAV (BAAV) were directly injected into the major lacrimal gland of mice. Each of AAV9, AAV 5w8, AAV5, and AAV2 was able to transduce the lacrimal ducts and acinar cells of the lacrimal gland (Rocha et al., supra). 【0175】 In some embodiments, the rAAV vector is administered to the lacrimal gland of a subject. In some embodiments, the lacrimal gland is the major lacrimal gland. In some embodiments, the lacrimal gland is either the Wolfring gland or the Krause gland of the subject. 【0176】 The compositions and rAAV vectors of the present disclosure may be administered to the lacrimal gland of a subject by any suitable method. For example, the subject compositions may be administered by direct injection into the major or accessory lacrimal gland. 【0177】 Access to the lacrimal gland in a human subject can be achieved, for example, by manually lifting the upper eyelid to expose the palpebral lobe of the lacrimal gland and delivering a therapeutic agent using a syringe with, for example, a 30G needle. 【0178】 The viral vectors of the present disclosure are generally delivered to a subject as a pharmaceutical composition. The pharmaceutical composition includes a pharmaceutically acceptable solvent (e.g., water, etc.) and one or more excipients. In some embodiments, the pharmaceutical composition includes a buffer at approximately neutral pH (pH 5, 6, 7, 8, or 9). In some embodiments, the pharmaceutical composition includes phosphate buffered saline (e.g., PBS at a pH of about 7). The pharmaceutical composition may include a pharmaceutically acceptable salt. The concentration of the salt may be selected to ensure that the pharmaceutical composition is isotonic, or nearly isotonic, with the target tissue. 【0179】 In some embodiments, the pharmaceutical composition of the present disclosure comprises about 1×10 8 genomic copies per milliliter (GC / mL) of a viral vector (e.g., an rAAV vector), about 5×10 8 GC / mL, about 1×10 9 GC / mL, about 5×10 9 GC / mL, about 1×10 10 GC / mL, about 5×10 10 GC / mL, about 1×10 11 GC / mL, about 5×10 11 GC / mL, about 1×10 12 GC / mL, about 5×10 12 GC / mL, about 5×10 13 GC / mL, or about 1×10 14 GC / mL. In some embodiments, the pharmaceutical composition of the present disclosure comprises about 1×10 8 genomic copies per milliliter (GC / mL) of a viral vector (e.g., an rAAV vector), from about 5×10 8 GC / mL to about 1×10 9 GC / mL, from about 1×10 9 GC / mL to about 5×10 9 GC / mL, from about 5×10 9 GC / mL to about 1×10 10 GC / mL, from about 1×10 10 GC / mL to about 5×10 10 GC / mL, from about 5×10 10 GC / mL to about 1×10 11 GC / mL, from about 1×10 11 GC / mL to about 5×10 11 GC / mL, from about 5×10 11 GC / mL to about 1×10 12 GC / mL, from about 1×10 12 GC / mL to about 5×10 12 GC / mL, from about 5×10 12 GC / mL to about 5×10 13 GC / mL, or from about 5×10 13 GC / mL to about 1×10 14 GC / mL. In some embodiments, the pharmaceutical composition of the present disclosure comprises from about 5×10 8 GC / mL to about 5×10 9GC / mL, about 5×10 9 GC / mL to about 5×10 10 GC / mL, about 5×10 10 GC / mL to about 5×10 11 GC / mL, about 5×10 11 GC / mL to about 5×10 12 GC / mL, or about 5×10 12 GC / mL to about 1×10 14 GC / mL of a viral vector (e.g., rAAV vector). In yet further embodiments, the pharmaceutical composition of the present disclosure is 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 a viral vector (e.g., rAAV vector). 【0180】 In some embodiments, the pharmaceutical composition of the present disclosure is about 1×10 12 GC / mL to about 6.2×10 12 GC / mL of a viral vector (e.g., rAAV vector). In some embodiments, the pharmaceutical composition of the present disclosure is about 1×10 12 GC / mL or about 6.2×10 12 GC / mL of a viral vector (e.g., rAAV vector). 【0181】 In some embodiments, the pharmaceutical composition of the present disclosure is about 10 μL, about 20 μL, about 30 μ It is administered in a total volume of L, 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 composition of the present disclosure is 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 in total volume. 【0182】 Genome copies per milliliter can be determined by quantitative polymerase change reaction (qPCR) using a standard curve generated with a reference sample having a known concentration of the viral polynucleotide genome. For AAV, the reference sample used is often the transfer plasmid used in the production of the rAAV vector, although other reference samples may be used. 【0183】 Alternatively, or additionally, the concentration of the viral vector can be determined by measuring the titer of the vector in a cell line. The viral titer is typically expressed as viral particles (vp) per unit volume (e.g., vp / mL). In some embodiments, the pharmaceutical composition of the present disclosure is about 1×10 8 viral particles / milliliter (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 11vp / 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 of a viral vector (e.g., rAAV vector). In some embodiments, the pharmaceutical composition of the present disclosure is about 1×10 8 viral particles per milliliter (vp / mL) to about 5×10 8 vp / mL, about 5×10 8 vp / mL to 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 to 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 to 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 to about 1×10 12 vp / mL, about 1×10 12 vp / mL to about 5×10 12 vp / mL, about 5×10 12 vp / mL to about 5×10 13 vp / mL, or about 5×10 13 vp / mL to about 1×10 14 vp / mL of a viral vector (e.g., rAAV vector). 【0184】 A variety of tests are available to evaluate eye symptoms in a subject before, during, and after treatment with any of the methods or compositions disclosed herein. In some embodiments of the disclosure, effective treatment of a subject can be indicated by one or more of the tests, which can be, for example, a) an eye dryness score test on a visual analog scale, b) a Schirmer test, c) a corneal fluorescein staining test, and d) an ocular surface disease index test. Tests for evaluating signs and symptoms of eye symptoms may be performed under standardized or reproducible conditions to obtain a test score for the subject. The conditions include exposing the subject to an artificially created environment that imposes an adverse burden on the subject, or an environment where the environment (temperature, humidity, air flow) is monitored and carefully controlled. 【0185】 Evaluation of effectiveness The effectiveness of the methods described herein can be evaluated using any suitable method known in the art. 【0186】 In some embodiments, the methods described herein result in one or more symptoms of eye symptoms being reduced as compared to the symptoms of eye symptoms prior to administration of the rAAV vector. As used herein, "symptom" includes any diagnostic criteria or symptoms associated with a given eye symptom, 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 flushing. 【0187】 In some embodiments, symptoms (e.g., itching, swelling, tearing, and flushing) can be reduced after administration of the compositions and rAAV of the present disclosure. In some embodiments, one or more symptoms of the ocular symptoms are reduced as compared to the symptoms of the ocular symptoms in untreated control subjects. In some embodiments, one or more symptoms of the ocular symptoms are reduced as compared to the symptoms of the ocular symptoms in the contralateral eye. By "contralateral eye" is meant the eye of the subject that is opposite the eye treated with the composition according to the present disclosure. The contralateral eye can be used as a control as long as the subject has a bilateral disease or, in the case of a model animal, is subjected to an experimental protocol that leads to treatment in both eyes. 【0188】 The effectiveness of the treatment methods described herein may be evaluated by assessing the symptoms of the ocular symptoms treated according to the methods described herein on a clinical scale. Examples of clinical scales that may be used to evaluate the effectiveness of the treatment methods described herein include, but are not limited to, the conjunctival itching grading scale (Table 4), the conjunctival flushing assessment grading scale (Table 5), and the corneal staining grading scale (Figure 3). 【0189】 As used herein, the term "statistically significant" refers to a method of analysis selected by one of ordinary skill in the art based on the study design and the type of data generated to evaluate differences observed between two or more groups, where the analysis determines whether the differences are not due to chance or randomness. 【0190】 Conjunctival allergen challenge (CAC) Conjunctival allergen challenge consists of an initial visit - allergen titration visit - in which increasing allergen concentrations of a specific allergen that induced a positive skin test reaction are instilled bilaterally at 10 - minute intervals until a positive reaction is induced. After the challenge, eye / conjunctival pruritus is evaluated by the subject and eye / conjunctival hyperemia is evaluated by a trained clinician; both evaluations use a standardized rating scale ranging from 0 (none) to 4 (severe). A positive CAC reaction is defined as moderate - to - severe hyperemia in two of the three vascular beds (conjunctiva, ciliary body, episclera) of each eye within 10 minutes of allergen instillation and moderate - to - severe pruritus in both eyes. Subjects who are not positive in the test are excluded from the study. During subsequent visits, allergen confirmation, the patient is challenged with the final titer of the allergen used at the first visit. If a moderate - to - severe ocular allergic response is reproduced at two of the three time points after three CACs, the diagnosis is confirmed and the subject is enrolled. 【0191】 Conjunctival Pruritus Grading Scale In some embodiments, treatment of eye symptoms 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 pruritus grading scale shown in Table 4. 【Table 4】 【0192】 In some embodiments disclosed herein, an effective treatment is indicated by a statistically significant decrease in the subject's score on the conjunctival pruritus grading scale, and a statistically significant decrease in the subject's score on the conjunctival pruritus grading scale is determined after administration to the subject of the first dose of the rAAV vector described herein, or optionally one or more subsequent doses, and the subject's score on the conjunctival pruritus grading scale is a) the subject's score on the conjunctival pruritus grading scale prior to administration of the first dose of the rAAV vector; b) the subject's score on the conjunctival pruritus grading scale of a subject administered a control; c) the subject's score on the conjunctival pruritus grading scale of a subject administered a comparative compound; or d) compared to the contralateral eye. 【0193】 In some embodiments disclosed herein, the subject's score on the conjunctival pruritus grading scale is compared to the subject's corneal score prior to administration of the rAAV vector according to the methods described herein. In some embodiments disclosed herein, the subject's score on the conjunctival pruritus grading scale is compared to the subject's score on the conjunctival pruritus grading scale of a subject administered a control. In some embodiments disclosed herein, the subject's score on the conjunctival pruritus grading scale is compared to the subject's score on the conjunctival pruritus grading scale of a subject administered a comparative compound. In some embodiments disclosed herein, the subject's score on the conjunctival pruritus grading scale is compared to the contralateral eye. 【0194】 In some embodiments, a statistically significant decrease in the subject's eye / conjunctival pruritus score is observed in subjects administered the rAAV vector described herein compared to a control. In some embodiments described herein, the statistically significant decrease in the subject's eye / conjunctival pruritus 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, the subject's eye / conjunctival A statistically significant decrease in the pruritus score was observed in subjects administered the rAAV vectors described herein compared to the control, and the statistically significant decrease in the subject's eye / conjunctiva pruritus score is at least 5%, 10%, 15%, 20%, 25%, 50%, 75%, 80%, 90%, 95%, 100%, 125%, 150%, 200%, 250%, 300%, or 350%. 【0195】 In some embodiments described herein, a statistically significant decrease in the subject's eye / conjunctiva pruritus 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 the subject's eye / conjunctiva pruritus score is characterized by a p-value of 0.05 or less. In some embodiments described herein, a statistically significant decrease in the subject's eye / conjunctiva pruritus score is characterized by a p-value of 0.01 or less. 【0196】 In some embodiments described herein, a statistically significant decrease in the subject's eye / conjunctiva pruritus score occurs 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 after treatment with any of the disclosed methods or compositions. 【0197】 In some embodiments, the decrease in the subject's eye / conjunctiva pruritus 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 after treatment with any of the disclosed methods or compositions. 【0198】 In some embodiments described herein, a statistically significant decrease in the subject's eye / conjunctiva pruritus score is based on the subject's eye / conjunctiva pruritus score determined after administration of the first dose of the rAAV vector. 【0199】 In some embodiments described herein, a statistically significant decrease is based on the eye / conjunctiva pruritus score of a subject determined after treatment with either a method or a composition disclosed herein. 【0200】 Conjunctival injection grading scale In some embodiments, treatment of an eye condition according to the methods described herein results in an improvement of 1 point, 2 points, 3 points, or 4 points on the conjunctival injection grading scale shown in Table 5. 【Table 5】 【0201】 In some embodiments disclosed herein, an effective treatment is indicated by a statistically significant decrease in the score of a subject on the conjunctival injection grading scale, and a statistically significant decrease in the score of a subject on the conjunctival injection grading scale is determined after administration to the subject of the first dose of the rAAV vector described herein, or optionally one or more subsequent doses, and the score of the subject on the conjunctival injection grading scale is a) the score of the subject on the conjunctival injection grading scale prior to administration of the first dose of the rAAV vector; b) the score of the subject administered a control on the conjunctival injection grading scale; c) the score of the subject administered a comparative compound on the conjunctival injection grading scale; or d) compared to the contralateral eye. 【0202】 In some embodiments disclosed herein, the eye / conjunctiva injection score of a subject is compared to the eye / conjunctiva injection score of the subject prior to administration of the first dose of the rAAV vector and the first dose of a treatment that increases tear production. 【0203】 In some embodiments disclosed herein, the eye / conjunctiva pruritus score of a subject is compared to the eye / conjunctiva injection score of a subject administered a control. 【0204】 In some embodiments disclosed herein, the target eye / conjunctiva pruritus score is compared to the target eye / conjunctiva injection score of a subject administered a comparative compound. 【0205】 In some embodiments disclosed herein, the target eye / conjunctiva injection score is compared to the eye / conjunctiva pruritus score in the contralateral eye. 【0206】 In some embodiments, a statistically significant decrease in the target eye / conjunctiva injection score is observed in a subject administered an rAAV vector described herein as compared to a control. In some embodiments disclosed herein, a statistically significant decrease in the target eye / conjunctiva injection 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 decrease in the target eye / conjunctiva injection score is observed in a subject administered an rAAV vector described herein as compared to a control, and a statistically significant decrease in the target eye / conjunctiva injection score is at least 5%, 10%, 15%, 20%, 25%, 50%, 75%, 80%, 90%, 95%, 100%, 125%, 150%, 200%, 250%, 300%, or 350%. 【0207】 In some embodiments disclosed herein, a statistically significant decrease in the target eye / conjunctiva injection 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 disclosed herein, a statistically significant decrease in the target eye / conjunctiva injection score is characterized by a p-value of 0.05 or less. In some embodiments disclosed herein, a statistically significant decrease in the target eye / conjunctiva injection score is characterized by a p-value of 0.01 or less. 【0208】 In some embodiments described herein, a statistically significant decrease in the subject's eye / conjunctival hyperemia score occurs 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 after treatment with either the disclosed method or composition. 【0209】 In some embodiments, the decrease in the subject's eye / conjunctival hyperemia 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 after treatment with either the disclosed method or composition. 【0210】 In some embodiments described herein, a statistically significant decrease in the subject's eye / conjunctival hyperemia score is based on the subject's eye / conjunctival hyperemia score determined after administration of the first dose of the rAAV vector. 【0211】 In some embodiments described herein, the statistically significant decrease is based on the subject's eye / conjunctival hyperemia score determined after treatment with either the disclosed method or composition. 【0212】 In some embodiments disclosed herein, the subject's score on the conjunctival hyperemia assessment grading scale is compared to the subject's corneal score prior to administration of the rAAV vector according to the method described herein. In some embodiments disclosed herein, the subject's score on the conjunctival hyperemia assessment grading scale is compared to the score of a subject administered a control on the conjunctival hyperemia assessment grading scale. In some embodiments disclosed herein, the subject's score on the conjunctival hyperemia assessment grading scale is compared to the score of a subject administered a comparative compound on the conjunctival hyperemia assessment grading scale. In some embodiments disclosed herein, the subject's score on the conjunctival hyperemia assessment grading scale is compared to the contralateral eye. 【0213】 In some embodiments, the treatment of eye symptoms 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 greater than about 95% in at least one compartment of the corneal staining grading scale. 【0214】 Corneal surface changes are associated with the eye symptoms and eye discomfort described herein, in addition to inadequate tear flow and excessive dryness. Corneal surface changes may include disruption of the mucin coating that protects the surface epithelial cells, and / or damage to the epithelial cell wall. and / or damage to the epithelial cell wall. 【0215】 Corneal staining test Corneal surface changes are associated with the eye symptoms and eye discomfort described herein, in addition to inadequate tear flow and excessive dryness. Corneal surface changes may include disruption of the mucin coating that protects the surface epithelial cells, and / or damage to the epithelial cell wall. 【0216】 Tests of corneal staining, including fluorescein staining, lissamine green staining, and rose bengal staining, are diagnostic tests for determining the health of the corneal surface and can indicate areas of damage on the corneal surface. Any suitable method of corneal staining known in the art, for example, the method described in Begley et al., The Ocular Surface 17(2019)208-220, may be used to evaluate the effectiveness of the treatment methods described herein. A normal corneal surface does not absorb water-soluble dyes that have penetrated into the tear film. However, damaged epithelial cells allow the water-soluble dye to diffuse into the surface cells. The dye that stains the damaged epithelial cells can be visualized on the corneal surface to indicate damage on the corneal surface. 【0217】 To perform a corneal staining test, a staining dye is applied to one or both eyes. The dye penetrates and stains the compartments between the surface cells. Inactivated cells and strands (filaments) of inactivated surface tissue can be visualized in this test. The test operator then uses a corneal surface scoring system developed to assess the severity of the observed damage. This scoring system is useful for the treatment of eye symptoms over time. Figure 3 depicts the NEI / Industry Workshop Scale used in the scoring system. Other equivalent standardized scoring systems may be used. The test operator grades the compartments of the damaged corneal surface and calculates a corneal score that reflects the severity of the damage to the corneal surface. 【0218】 The test operator may use the corneal score to evaluate the effectiveness of a particular treatment in a subject. The test may be performed multiple times to monitor any changes in the severity of the subject's eye surface over a period of time. Generally, a larger number indicates greater damage to the corneal surface compared to a smaller number that indicates a lower level of damage to the corneal surface. A decrease in the corneal score over time is evidence of a decrease in the damage to the corneal surface. A decrease in the corneal score generally indicates an improvement in the subject's symptoms. A decrease in the corneal score over time is also evidence that the treatment is effective in the treatment of dry eye disease, increasing tear production, or improving eye discomfort. 【0219】 In some embodiments disclosed herein, an effective treatment is indicated by a statistically significant decrease in the subject's corneal score, and a statistically significant decrease in the subject's corneal score is determined after administration of a first dose of the composition described herein, or optionally one or more subsequent doses, to the subject, and the subject's corneal score is: a) the subject's corneal score prior to administration of a first dose of the rAAV vector and a first dose of a treatment that increases tear production; b) the corneal score of a subject administered a control; c) the corneal score of a subject administered a comparative compound; or d) compared to the contralateral eye. 【0220】 In some embodiments disclosed herein, the corneal score of a subject is compared to the corneal score of the subject prior to administration of a pharmaceutical composition according to the methods described herein. In some embodiments disclosed herein, the corneal score of a subject is compared to the corneal score of a subject administered a control. In some embodiments disclosed herein, the corneal score of a subject is compared to the corneal score of a subject administered a comparative compound. In some embodiments disclosed herein, the corneal score of a subject is compared to the contralateral eye. 【0221】 In some embodiments disclosed herein, the corneal score of a subject may be used to measure a definition of corneal healing defined as less than 0.5 mm of fluorescein staining or no fluorescein staining (Bonini et al., Ophthalmology. 125:1332-1343 (2018)). 【0222】 In some embodiments, a statistically significant decrease in the corneal healing score of a subject is observed in a subject administered an rAAV vector described herein compared to a control. 【0223】 In some embodiments described herein, a statistically significant decrease in the corneal healing score of a subject 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 a subject is observed in a subject administered an rAAV vector described herein compared to a control, and the statistically significant decrease in the OSDI score of the subject is at least 5%, 10%, 15%, 20%, 25%, 50%, 75%, 80%, 90%, 95%, 100%, 125%, 150%, 200%, 250%, 300%, or 350%. 【0224】 In some embodiments described herein, a statistically significant decrease in the corneal staining score of a subject is at least 5%, 10%, 15%, 20%, 25%, 50%, 75%, 80%, 90%, 95%, 100%, 125%, 150%, 200%, 250%, 300%, or 350%. 【0225】 In some embodiments described herein, a statistically significant decrease in the corneal score of a subject is characterized by a p-value of 0.05 or less, 0.01 or less, 0.005 or less, or 0.001 or less. 【0226】 In some embodiments described herein, a statistically significant decrease in the corneal score of a subject 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 after treatment according to the methods or compositions disclosed herein. 【0227】 In some embodiments, the decrease in the corneal score of 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 after treatment with any of the methods or compositions disclosed herein. 【0228】 In some embodiments described herein, the statistically significant decrease is based on the corneal score of a subject determined after administration of one or more subsequent doses of the rAAV vector and optionally the first or one or more subsequent doses of a treatment that increases tear production. 【0229】 Dry eye score test The dry eye score test on a visual analog scale may be used to evaluate eye symptoms, tearing level, or eye discomfort in a subject. The test involves the use of a visual analog scale and includes a 100 mm horizontal line where one endpoint at 0 is labeled "no discomfort" and the other endpoint at 100 is labeled "maximum discomfort". Figure 3 shows an example of a visual analog scale (not shown at actual scale). The subject is asked to rate the eye symptoms resulting from eye dryness by placing a vertical mark on the horizontal line to indicate the level of discomfort. The dry eye score is then obtained by identifying where the subject's response lies on the 100 mm scale. 【0230】 The dry eye score measured in mm can be used to evaluate the severity of the subject's eye symptoms and the effectiveness of a particular treatment. This test has the advantage of being administered to the subject as frequently as every 5 minutes, which allows the test administrator to closely monitor changes in the subject's symptoms over time. A larger number indicates a greater discomfort regarding the eye symptoms compared to a smaller number which indicates a relatively lower level of discomfort. A decrease in the dry eye score is evidence that the treatment is effective in treating eye symptoms, increasing tear production, or improving eye discomfort. A reduction in the dry eye score over time is evidence of a reduction or alleviation in the eye symptoms and generally indicates an improvement in the subject's symptoms. 【0231】 In some embodiments disclosed herein, an effective treatment is indicated by a statistically significant decrease in the subject's dry eye score, and a statistically significant decrease in the subject's dry eye score is determined after administration to the subject of a first dose of the rAAV vector, or optionally one or more subsequent doses, and a first dose of a treatment that increases tear production, or optionally one or more subsequent doses, where the subject's dry eye score is compared to a) the subject's dry eye score prior to administration of the first dose of the rAAV vector and the first dose of the treatment that increases tear production; b) the dry eye score of a subject administered a control; c) the dry eye score of a subject administered a comparative compound; or d) the dry eye score in the contralateral eye. 【0232】 In some embodiments disclosed herein, the subject's dry eye score is compared to the subject's dry eye score prior to administration of the first dose of the rAAV vector and the first dose of the treatment that increases tear production. 【0233】 In some embodiments disclosed herein, the subject's dry eye score is compared to the dry eye score of a subject administered a control. 【0234】 In some embodiments disclosed herein, the subject's dry eye score is compared to the dry eye score of a subject administered a comparative compound. 【0235】 In some embodiments disclosed herein, the subject's dry eye score is compared to the dry eye score in the contralateral eye. 【0236】 In some embodiments, a statistically significant decrease in the subject's dry eye score is observed in subjects administered the rAAV vectors described herein compared to the control. In some embodiments of the embodiments described herein, the statistically significant decrease in the subject's dry eye 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 decrease in the subject's dry eye score is observed in subjects administered the rAAV vectors described herein compared to the control, and the statistically significant decrease in the subject's dry eye score is at least 5%, 10%, 15%, 20%, 25%, 50%, 75%, 80%, 90%, 95%, 100%, 125%, 150%, 200%, 250%, 300%, or 350%. 【0237】 In some embodiments of the embodiments described herein, the statistically significant decrease in the subject's dry eye 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. In some embodiments, the statistically significant decrease in the subject's dry eye score is observed in subjects administered the rAAV vectors described herein compared to the control, and the statistically significant decrease in the subject's dry eye 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. 【0238】 In some embodiments described herein, a statistically significant decrease in the subject's dry eye score is 3 mm to 10 mm, 3 mm to 20 mm, 3 mm to 25 mm, 3 mm to 30 mm, 3 mm to 35 mm, 3 mm to 40 mm, 3 mm to 45 mm, 3 mm to 50 mm, 5 mm to 10 mm, 5 mm to 20 mm, 5 mm to 25 mm, 5 mm to 30 mm, 5 mm to 35 mm, 5 mm to 40 mm, 5 mm to 45 mm, 5 mm to 50 mm, 10 mm to 15 mm, 10 mm to 20 mm, 10 mm to 25 mm, 10 mm to 30 mm, 10 mm to 35 mm, 10 mm to 40 mm, 10 mm to 45 mm, 10 mm to 50 mm, 15 mm to 20 mm, 20 mm to 30 mm, 25 mm to 35 mm, 30 mm to 40 mm, 30 mm to 45 mm, or 30 mm to 50 mm. 【0239】 In some embodiments described herein, a statistically significant decrease in the subject's dry eye 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 the subject's dry eye score is characterized by a p-value of 0.05 or less. In some embodiments described herein, a statistically significant decrease in the subject's dry eye score is characterized by a p-value of 0.01 or less. 【0240】 In some embodiments described herein, a statistically significant decrease in the subject's dry eye 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 after treatment with any of the disclosed methods or compositions. 【0241】 In some embodiments, the decrease in the subject's dry eye 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 after treatment with any of the methods or compositions disclosed herein. 【0242】 In some embodiments described herein, the statistically significant decrease in the subject's dry eye score is based on the subject's dry eye score determined after administration of the first dose of the rAAV vector. 【0243】 In some embodiments described herein, the statistically significant decrease is based on the subject's dry eye score determined after treatment with either the method or composition disclosed herein. 【0244】 Schirmer score test Eye symptoms can affect tear volume and tear production. The Schirmer test may be used to evaluate tear production in a subject and to evaluate the severity of dry eye disease, insufficient tearing, or eye discomfort. The test measures the amount of tears produced in each eye. The test typically involves first putting an anesthetic in one or both of the subject's eyes. These droplets prevent the aqueous fluid from coming out of the eyes in response to the test strip. Next, the test practitioner places a small piece of filter paper on the inside of one or both of the lower eyelids, and the person closes their eyes. After 5 minutes, the test practitioner removes the filter paper and evaluates how far the tears have traveled on the paper. The Schirmer test may be performed on one or both eyes. may also. 【0245】 Generally, the smaller the amount of moisture on the paper, the less tears the person has produced. In healthy eyes, each piece of paper typically contains more than 10 millimeters of moisture. A Schirmer score of less than 10 millimeters of moisture may indicate one or more of the eye symptoms, abnormally low tearing, or symptoms including eye discomfort described herein. 【0246】 The Schirmer test may be used to evaluate the effectiveness of a particular treatment in a subject and may be performed multiple times to monitor any changes in the severity of the subject's symptoms over a period of time. An increase in the Schirmer score over time in a subject being treated for the eye symptoms, insufficient tearing, or eye discomfort described herein is evidence of an increase in tear volume or tear production and generally indicates an improvement in the subject's symptoms. An increase in the Schirmer score over time is evidence that the treatment is effective in treating the eye symptoms, increasing tear production, or improving eye discomfort described herein. 【0247】 In some embodiments disclosed herein, an effective treatment is indicated by a statistically significant increase in the subject's Schirmer score, and a statistically significant increase in the subject's Schirmer score is determined after treatment with any of the methods or compositions disclosed herein, where the subject's Schirmer score is: a) the subject's Schirmer score prior to administration of a first dose of the rAAV vector and a first dose of a treatment that increases tear production; b) the Schirmer score of a subject administered a control; c) the Schirmer score of a subject administered a comparative compound; or d) compared to the contralateral eye. 【0248】 In some embodiments disclosed herein, the subject's Schirmer score is compared to the subject's Schirmer score prior to administration of the pharmaceutical composition described herein. In some embodiments disclosed herein, the subject's Schirmer score is compared to the Schirmer score of a subject administered a control. In some embodiments disclosed herein, the subject's Schirmer score is compared to the Schirmer score of a subject administered a comparative compound. In some embodiments disclosed herein, the subject's Schirmer score is compared to the contralateral eye. 【0249】 In some embodiments, a statistically significant increase in the Schirmer score of a subject is observed in a subject administered an rAAV vector described herein as compared to a control. In some embodiments of the present disclosure, the statistically significant increase in the Schirmer score of the subject is at least 5%, 10%, 15%, 20%, 25%, 50%, 75%, 80%, 90%, 95%, 100%, 125%, 150%, 200%, 250%, 300%, or 350%. In some embodiments of the present disclosure, the increase in the Schirmer score of the subject is at least 100%, 200%, or 300%. In some embodiments, a statistically significant increase in the Schirmer score of a subject is observed in a subject administered an rAAV vector described herein as compared to a control, and the statistically significant increase in the Schirmer score of the subject is at least 5%, 10%, 15%, 20%, 25%, 50%, 75%, 80%, 90%, 95%, 100%, 125%, 150%, 200%, 250%, 300%, or 350%. 【0250】 In some embodiments of the present disclosure, the statistically significant increase in the Schirmer score of the subject 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. 【0251】 In some embodiments of the present disclosure, the statistically significant increase in the Schirmer score of the subject is 3 mm to 5 mm, 3 mm to 10 mm, 3 mm to 15 mm, 3 mm to 20 mm, 3 mm to 25 mm, 3 mm to 30 mm, 5 mm to 10 mm, 5 mm to 15 mm, 5 mm to 20 mm, 5 mm to 25 mm, 5 mm to 30 mm, 10 mm to 15 mm, 10 mm to 20 mm, 10 mm to 25 mm, 10 mm to 30 mm, 15 mm and 20 mm, 15 mm to 25 mm, 15 mm to 30 mm, 20 mm to 25 mm, or 20 mm to 30 mm. 【0252】 In some embodiments, a statistically significant increase in the Schirmer score of a subject is observed in a subject administered an rAAV vector described herein as compared to a control, and the statistically significant increase in the Schirmer score of the subject 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. 【0253】 In some embodiments described herein, a statistically significant decrease in the Schirmer score of a subject is characterized by a p-value of 0.05 or less, 0.01 or less, 0.005 or less, or 0.001 or less. 【0254】 In some embodiments described herein, a statistically significant decrease in the Schirmer score of a subject 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 after treatment according to the methods or compositions disclosed herein. 【0255】 In some embodiments described herein, a statistically significant decrease in the Schirmer score of a subject is based on the Schirmer score of the subject determined after treatment according to the methods or compositions disclosed herein. 【0256】 In some embodiments described herein, the statistically significant decrease is based on the Schirmer score of the subject determined after treatment according to any of the methods or compositions disclosed herein. 【0257】 Ocular Surface Disease Index Test Subjects being treated for eye symptoms and associated syndromes can provide important information used to diagnose symptoms and determine the severity of syndromes through questionnaires. A well-designed questionnaire should be able to be verified for reproducibility and consist of relevant questions that elicit responsive answers. One example of a questionnaire is the Ocular Surface Disease Index (OSDI), which is a survey of 12 questions for subjects with eye symptoms that has been shown to be a reliable and valid means for directly assessing symptom frequency. The eye symptoms evaluated include, but are not limited to, burning / stinging pain, itching, foreign body sensation, eye discomfort, eye dryness, photophobia, and pain. Most people are familiar with questionnaires, and they understand that they are an efficient way for healthcare providers to gather information. Questionnaires also reduce bias because there are no linguistic or visual cues that inadvertently influence respondents. The tester collects responses from the subject and calculates the OSDI based on the subject's answers to the questions. 【0258】 The OSDI score can be used to evaluate the severity of a subject's eye symptoms and the effectiveness of specific treatments. A larger number indicates a higher severity of eye symptoms. A reduction in the OSDI score over time is evidence of a reduction or alleviation in eye symptoms and generally indicates an improvement in the subject's symptoms. A decrease in the OSDI score is also evidence that the treatment is effective in treating eye symptoms and reducing syndromes. 【0259】 In some embodiments disclosed herein, an effective treatment is indicated by a statistically significant decrease in the OSDI score of a subject, and a statistically significant decrease in the OSDI score of the subject is determined after administration to the subject of a first dose of the rAAV vector, or optionally one or more subsequent doses, and a first dose of a treatment that increases tear production, or optionally one or more subsequent doses, wherein the OSDI score of the subject is: a) the OSDI score of the subject prior to administration of the first dose of the rAAV vector and the first dose of the treatment that increases tear production; b) the OSDI score of a subject administered a control; c) the OSDI score of a subject administered a comparator compound; or d) compared to the contralateral eye. 【0260】 In some embodiments disclosed herein, the OSDI score of a subject is compared to the OSDI score of the subject prior to treatment according to the disclosed methods described herein. In some embodiments disclosed herein, the OSDI score of a subject is compared to the OSDI score of a subject administered a control. In some embodiments disclosed herein, the OSDI score of a subject is compared to the OSDI score of a subject administered a comparator compound. In some embodiments disclosed herein, the OSDI score of a subject is compared to the OSDI score of the contralateral eye. 【0261】 In some embodiments, a statistically significant decrease in the OSDI score of a subject is observed in a subject administered the rAAV vector described herein as compared to a control. 【0262】 In some embodiments described herein, a statistically significant decrease in the OSDI score of a subject 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 a subject is observed in a subject administered an rAAV vector as described herein compared to a control, and the statistically significant decrease in the OSDI score of the subject is at least 5%, 10%, 15%, 20%, 25%, 50%, 75%, 80%, 90%, 95%, 100%, 125%, 150%, 200%, 250%, 300%, or 350%. 【0263】 In some embodiments described herein, a statistically significant decrease in the OSDI score of a subject is characterized by a p-value of 0.05 or less, 0.01 or less, 0.005 or less, or 0.001 or less. 【0264】 In some embodiments described herein, a statistically significant decrease in the OSDI score of a subject occurs 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 after treatment according to a method or composition disclosed herein. 【0265】 In some embodiments, the decrease in the OSDI score of 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 after treatment with any of the methods or compositions disclosed herein. 【0266】 In some embodiments described herein, a statistically significant decrease in the OSDI score of a subject is based on the OSDI score of the subject determined after administration of a first dose of an rAAV vector. 【0267】 In some embodiments described herein, the statistically significant decrease is based on the OSDI score of the subject determined after treatment according to the methods or compositions disclosed herein. 【0268】 Maintenance of effective treatment over time The effectiveness of the treatment methods described herein may be evaluated at any suitable time point after administration. For example, improvements in the conjunctival itching grading scale, conjunctival hyperemia evaluation grading scale, eye tearing score grading scale, and / or corneal staining grading scale may be measured about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 18 months, about 24 months, about 3 years, about 4 years, or about 5 years after administration of the rAAV vectors described herein. In some embodiments, improvements in the conjunctival itching grading scale, conjunctival hyperemia evaluation grading scale, eye tearing score grading scale, and / or corneal staining grading scale are measured at two or more time points after administration of the rAAV vectors described herein, for example, every 3 months, every 6 months, or every 12 months after administration of the rAAV vectors. 【0269】 The present disclosure may provide effective treatment over a period of time in which a statistically significant improvement in the subject's score is maintained. The term "maintained" as used in the present disclosure, when referring to the maintenance of a statistically significant improvement in the subject's score (EDS, Schirmer, corneal staining, or OSDI), refers to the statistically significant improvement not decreasing below a certain threshold over time. A statistically significant improvement may be maintained even if the subject's score changes at a later time point. Improvements after treatment according to the disclosed methods may be maintained without additional dosing or after one or more subsequent doses. 【0270】 For example, a corneal staining score of 0.5 mm or 0 mm can be defined as corneal healing and can represent a significant improvement 30 or 60 days after the start of treatment (Bonini et al., Ophthalmology. 125:1332-1343 (2018)). 【0271】 For example, an ocular dryness score that is "maintained within 10%" means that the decrease in the subject's ocular dryness score does not decrease by more than 10% during the specified time period. Further improvement in the subject's ocular dryness score is also considered to be maintenance of a statistically significant improvement (e.g., if the ocular dryness score improves by an additional 15% during the specified time period, this is considered to be "maintained within 10%"). 【0272】 In another example, if there is a statistically significant decrease (improvement) in the subject's ocular dryness 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 a benefit to the subject), it is said that the statistically significant improvement has been maintained. Alternatively, if at a later time point the subject's ocular dryness score is higher than the ocular dryness score 30 days after treatment according to the methods described herein, the subject may still be receiving a therapeutic benefit, and the score determined later may still be a statistically significant improvement compared to the pre-dose or pre-treatment baseline score. 【0273】 In another example, if there is a statistically significant decrease (improvement) in the subject's ocular dryness 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), it is said that the statistically significant improvement has been maintained. Alternatively, if at a later time point the subject's ocular dryness score is higher than the ocular dryness score 60 days after treatment according to the methods described herein, the subject may still be receiving a therapeutic benefit, and the score determined later is still a statistically significant improvement compared to before treatment according to the methods described herein. 【0274】 It is noted that what constitutes an improvement varies depending on the score being measured. For example, an improvement in the dry eye score, corneal score, and OSDI score is a decrease in the numerical value of the score. For the Schirmer test, an improvement is typically an increase in the numerical value of the Schirmer score. 【0275】 In some embodiments described herein, maintenance of a statistically significant improvement in a subject's score (e.g., EDS, Schirmer, corneal staining, or OSDI) means that the statistically significant improvement does not decrease by more than 10%, 20%, 30%, 40%, 50%, or 60%. 【0276】 In some embodiments described herein, a statistically significant improvement in a subject's score (e.g., EDS, Schirmer, corneal, 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, a statistically significant improvement in a subject's score is maintained for at least 30 days after treatment according to the methods described herein, and the statistically significant improvement does not decrease by more than 30%. In some embodiments, a statistically significant improvement in a subject's score is maintained for at least 1 month after administration of the first dose of the rAAV vector and, optionally, the first dose of a treatment that increases tear production, and the statistically significant improvement does not decrease by more than 30%. 【0277】 In some embodiments described herein, a statistically significant improvement in a subject's score (e.g., EDS, Schirmer, corneal, 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 after treatment according to the methods described herein, and the statistically significant improvement is not decreased by more than 10%, 20%, 30%, 40%, 50%, or 60% compared to the subject's score within 60 days after treatment according to the methods described herein. 【0278】 In some embodiments described herein, a statistically significant improvement in a subject's score (e.g., EDS, Schirmer, 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 after treatment according to the methods described herein, and the statistically significant improvement is not decreased by more than 20% compared to the corresponding dry eye score, corneal score, or OSDI score of the subject within 60 days after treatment according to the methods described herein. 【0279】 In some embodiments, improvements measured on the conjunctival pruritus grading scale, conjunctival hyperemia assessment grading scale, eye tearing score grading scale, and corneal staining grading scale persist for an extended period (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) after administration of the rAAV vectors provided herein. 【0280】 Timing and Method of Administration The schedule of doses administered to a subject 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 in the body. For example For example, if the patient's symptoms do not improve, at the discretion of the healthcare provider, the methods of treating the eye symptoms described herein may have the dosage adjusted or may be administered repeatedly in order to relieve, otherwise control or limit, the symptoms of the subject's eye symptoms. For example, the time period between administrations of one or more dosages may be extended, or the time period between days on which the subject is administered one or more dosages may be extended. As a non-limiting example, the administration of one or more dosages is modified to the administration of one or more dosages after measuring the symptoms of the eye symptoms. 【0281】 The term "dosage", as used herein, may refer to the dosage of the pharmaceutical composition of the present disclosure, or the dosage of a treatment that reduces symptoms in an eye symptom. 【0282】 In some embodiments described herein, the dosage of the rAAV vector is the dosage of the rAAV vector having the expression cassette. In such cases, delivery of an appropriate dosage (e.g., an effective amount) of the gene product is achieved by administering to the target site an appropriate amount / titer of the viral vector that allows expression of the effective amount of the gene product over a period of time. "Effective amount", as used herein, 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 eye symptoms described herein. The term "amount", as used herein, refers to an absolute amount (e.g., the absolute amount of a protein or rAAV particles) or a concentration (e.g., the concentration of a protein in a solution), and it will be apparent to those skilled in the art based on the context provided herein whether the amount referred to in a given instance refers to an absolute amount, a concentration, or both. 【0283】 In some embodiments, the viral vector is an rAAV vector. In some embodiments, the viral vector is administered to the lacrimal gland. In some embodiments, the rAAV is administered to the lacrimal gland by topical administration. In some embodiments, the rAAV is administered to the lacrimal gland by direct injection. In some embodiments, the dose of rAAV results in stable production of the gene product over a temporal period (e.g., about 1 day, about 2 days, about 4 days, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 9 months, about 12 months, or longer). In some embodiments, the dose of rAAV results in stable production of the gene product over about 1 week. In some embodiments, the dose of rAAV results in stable production of the gene product over about 2 weeks. In some embodiments, the dose of rAAV results in stable production of the gene product over about 3 weeks. In some embodiments, the dose of rAAV results in stable production of the gene product over about 4 weeks. In some embodiments, the dose of rAAV results in stable production of the gene product over about 1 month. In some embodiments, the dose of rAAV results in stable production of the gene product over about 2 months. In some embodiments, the dose of rAAV results in stable production of the gene product over about 3 months. In some embodiments, the dose of rAAV results in stable production of the gene product over about 4 months. In some embodiments, the dose of rAAV results in stable production of the gene product over about 5 months. In some embodiments, the dose of rAAV results in stable production of the gene product over about 6 months. In some embodiments, the dose of rAAV results in stable production of the gene product over about 9 months. In some embodiments, the dose of rAAV results in stable production of the gene product over about 12 months. 【0284】 In some embodiments, the methods described herein include administering to a subject an effective amount of the rAAV described herein, the rAAV comprising a polynucleotide encoding a gene product (e.g., DAO1). In some embodiments, the method includes delivering a first dose and one or more subsequent doses of rAAV. The one or more subsequent doses are administered after a period of time following the first dose. In some embodiments, the first dose The period of time between the subsequent dose is at least 1 day, at least 3 days, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 1 month, at least 2 months, at least 4 months, at least 6 months, at least 9 months, at least 12 months, or longer. In some embodiments, the period of time between the first dose and the next subsequent dose is 1-7 days, 1-4 weeks, 2-6 weeks, 4-8 weeks, 1-3 months, 2-4 months, 3-6 months, 4-12 months, 6-24 months. In some embodiments, the period of time 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 period of time between one or more subsequent doses is 1-7 days, 1-4 weeks, 2-6 weeks, 4-8 weeks, 1-3 months, 2-4 months, 3-6 months, 4-12 months, 6-24 months. 【0285】 In some embodiments, the methods described herein include administering to a subject an effective amount of rAAV, wherein the subject 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 includes administering one or more doses of an nAChR agonist described herein. In some embodiments, the nAChR agonist is administered via topical administration. In some embodiments, the nAChR agonist is administered as a nasal spray. 【0286】 In some embodiments, a treatment that increases tear production (e.g., a treatment that includes administering to a subject one or more doses of an nAChR agonist) is administered on a schedule that is 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 a first dose of rAAV. In some of the 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 1 year, or longer. In some of the embodiments described herein, 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. 【0287】 In some embodiments, a treatment that increases tear production (e.g., a treatment that includes administering to a subject one or more doses of an nAChR agonist) is administered to the subject 1 - 4 times per day, 1 time per day, 2 times per day, or 3 times per day after the first day of administration of rAAV. 【0288】 In some embodiments, a subject is administered a dosing cycle of the nAChR agonist described herein, and the duration between doses of the nAChR agonist is increased over time. For example, administration of a dose every 4 hours is changed to administration of a dose every 8 or 12 hours. 【0289】 In some embodiments, the treatment to increase 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 1 year. In some embodiments, to increase tear production the treatment 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. 【0290】 In some embodiments, the method comprises a first dose of rAAV and one or more subsequent doses. In some embodiments, the one or more subsequent doses are administered after a time period following the first dose. This time period between the first dose and the next subsequent dose is at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, or at least 8 hours. The time period between the first dose and the next subsequent dose is 1 - 3 hours, 2 - 4 hours, 3 - 6 hours, or 4 - 8 hours. The time period between the 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 the one or more subsequent doses is 1 - 3 hours, 2 - 4 hours, 3 - 6 hours, or 4 - 8 hours. 【0291】 In some embodiments, the period between the administration of the dose of rAAV and the administration of the dose of the treatment in an effective amount to increase tear production is less than 5 minutes, 5 - 60 minutes, 30 - 90 minutes, 1 - 3 hours, 1 - 8 hours, 1 - 12 hours, 1 - 24 hours, 8 - 12 hours, 8 - 24 hours, 12 - 24 hours, 1 - 3 days, 1 - 7 days, 1 - 14 days, 1 - 28 days, 3 - 7 days, 3 - 14 days, 3 - 28 days, 7 - 14 days, or 7 - 28 days. 【0292】 In some embodiments, the method comprises administering a first dose and one or more subsequent doses of rAAV, and a first dose and one or more subsequent doses of a treatment that increases tear production. The one or more subsequent doses are administered after a time period following the first dose. This time period between the first dose and the next subsequent dose is at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, or at least 8 hours. The time period between the first dose and the next subsequent dose is 1 to 3 hours, 2 to 4 hours, 3 to 6 hours, or 4 to 8 hours. The time period between the 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 the one or more subsequent doses is 1 to 3 hours, 2 to 4 hours, 3 to 6 hours, or 4 to 8 hours. 【0293】 In some embodiments, the treatment that increases tear production, and the rAAV are administered to a subject in need thereof in separate dosage forms. In some embodiments, the treatment that increases tear production, and the rAAV are administered to a subject in need thereof in a combined dosage form. 【0294】 Pharmaceutical Compositions and Kits In some embodiments, the present disclosure provides a pharmaceutical composition comprising the rAAV vectors described herein. In some embodiments, the pharmaceutical composition comprises the rAAV vectors described herein, and a pharmaceutically acceptable carrier, delivery agent, or excipient. In some embodiments, the present disclosure provides 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 subject. In some embodiments, the present disclosure provides 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 pharmaceutical composition comprising a pharmaceutically acceptable carrier suitable for administration to the eye of a human subject. 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. 【0295】 In some embodiments, the pharmaceutical composition comprises a polypeptide comprising DAO1 (or a fragment thereof) in an amount within a range defined by 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, or any pair of values above; and optionally may contain 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 the treatment of an eye disease, disorder, or condition. 【0296】 In some embodiments, the present disclosure provides the use of the rAAV vectors or pharmaceutical compositions described herein in the production of a medicament for the treatment of an eye disease, disorder, or condition. In some embodiments, the present disclosure provides the use of the rAAV vectors or pharmaceutical compositions herein for use in or adaptable for use in the treatment of an eye disease, disorder, or condition. 【0297】 In some embodiments, the pharmaceutically acceptable carrier includes phosphate buffered saline. In some embodiments, the pharmaceutical composition is formulated to be compatible with its intended route of administration (e.g., intraglandular). 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 ocular surface. Examples of excipients, diluents, and / or carriers suitable for ocular administration that may be referred to as pharmaceutically acceptable carriers include sterile pyrogen-free water and sterile pyrogen-free buffered saline (e.g., saline buffered with phosphate (salt) or other buffers, such as HEPES, to maintain pH at appropriate physiological levels), isotonic sodium chloride solution, balanced salt solution, emulsions (e.g., oil / water emulsions), and various types of wetting agents. 【0298】 Any concentration of a vector suitable for effectively transducing cells of the eye, lacrimal gland, and / or nasolacrimal duct, such as an rAAV vector, 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 is 10 5 vector genomes / mL or more, 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; 1013 Vector genome / mL; 1.5×10 13 Vector genome / mL; 3×10 13 Vector genome / mL; 5×10 13 Vector genome / mL; 7.5×10 13 Vector genome / mL; 9×10 13 Vector genome / mL; 10 14 Vector genome / mL; 5×10 14 Vector genome / mL; 10 15 Vector genome / mL; 5×10 15 Vector genome / mL; 10 16 Vector genome / mL; or more, but typically 5×10 16 It may be formulated at a concentration of Vector genome / mL or less. In some embodiments, the rAAV vector may be formulated at a concentration within the range defined by any pair of the concentrations described in this paragraph. 【0299】 In some embodiments, the rAAV vector comprises an expression cassette configured to express the AAV capsid and DAO1 as described herein. Formulations according to the present disclosure may include such rAAV vectors at any concentration, for example, a concentration suitable for effectively transducing cells of the eye, lacrimal gland, and / or nasolacrimal duct. In some embodiments, the formulation is 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 ; 7.5×10 13 ; 1×10 14 ; 2.5×10 14 ; 5×10 14 ; 7.5×10 14 ; 1×10 15 ; 1×10 16 ; or 5×10 16 / mL of rAAV vector, or an rAAV vector at a concentration per mL within the range defined by any pair of concentrations described in this paragraph. 【0300】 In some embodiments, the present disclosure provides an alternative DAO1 vehicle, such as a plasmid configured to express DAO1. Formulations according to the present disclosure may include such a plasmid at any concentration, for example, a concentration suitable for effectively transfecting cells of the eye, lacrimal gland, and / or nasolacrimal duct. In some embodiments, the formulation is 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 ; 5×1010 ; 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 / mL of plasmid, or a plasmid at a concentration per mL within the range defined by any pair of the concentrations described in this paragraph may be included. 【0301】 Similarly, any total number of rAAV vectors suitable for providing appropriate transduction to cells of the eye, lacrimal gland, and / or nasolacrimal duct to confer a desired effect or treat a disease can be administered to a mammal or to the eye of a primate. In some embodiments, at least 10 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 ; 7.5×108 ; 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 ; 1 0 16 ; 2.5×10 16 ; 5×10 16 ; or 7.5×10 16 ; or more rAAV vectors, but typically 1×10 15 or fewer rAAv vectors are injected per target. For example, in some embodiments, about 1×10 9 to about 1×10 10 ; about 1×10 10 to about 1×10 11 ; about 1×10 11 to about 1×10 12 ; about 1×10 12 to about 1×10 13 ; or about 1×10 13 to about 1×10 15Individual genome copies are administered in one go. In some embodiments, the total number of rAAV vectors administered to the eye of the human or animal being treated may include concentrations within the range defined by any pair of the concentrations described in this paragraph. Any suitable number of rAAV vectors may be administered to the eye of a mammal or primate. In some embodiments, the method includes a single administration; in other embodiments, multiple administrations are made over time if deemed appropriate by the attending physician. 【0302】 In some embodiments, a suitable amount or concentration of the rAAV vector (or any other vector described herein) in the therapeutic formulation may be a concentration effective to cause 100 pg / mL to 50 μg / mL of DAO1 to be expressed in the tear layer of the 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) may result in the 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 (or a concentration within the range defined by any pair of the above values) of DAO1 in the tear layer of the subject. Expression may 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 1, 2, 3, 4, or 5 days later. 【0303】 The rAAV vector is 1×10 5 vector genomes or more, for example, 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, and in certain cases, 1×10 14 vector genomes, but usually contains 4×10 15 vector genomes or less, but is not limited thereto, and may be formulated at any suitable unit dosage. In some embodiments, the rAAV vector is 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 , or 1×10 15 vector genomes or more, but is not limited thereto, and is formulated at any suitable unit dosage. In some embodiments, the unit dosage is at most about 5×10 15 vector genomes, for example, 1×10 14 vector genomes or less, for example 1×10 13 , 1×10 12 , 1×10 11 , 1×10 10 , or 1×10 9 vector genomes or less, and in certain cases 1×10 8 vector genomes or less, and typically 1×10 8 vector genomes or more. In some embodiments, the unit dosage is at most about 5×10 15 vector genomes, for example, 1×10 14 vector genomes or less, for example 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 dosage is 1×10 10~1×10 11 is a vector genome. In some embodiments, the unit dosage is 1×10 10 ~3×10 12 is a vector genome. In some embodiments, the unit dosage is 1×10 9 ~3×10 13 is a vector genome. In some embodiments, the unit dosage is 1×10 8 ~3×10 14 is a vector genome. In some embodiments, the rAAV vector comprises an expression cassette configured to express an AAV capsid and DAO1, and the unit dosage is at least, at most, exactly, or about 1×10 5 、1×1 0 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 of the rAAV vector, or formulated at a concentration within the range defined by any pair of the concentrations described in this paragraph. 【0304】 In some embodiments, the present disclosure provides a vector comprising a plasmid configured to express DAO1, and the unit dosage 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 of the plasmid. 【0305】 In some embodiments, the unit dosage of the pharmaceutical composition may be measured using the multiplicity of infection (MOI). By MOI, the ratio of the vector or viral genome of the rAAV vector provided herein to the cells into which the nucleic acid can be delivered, or the multiplicity, is meant. In some embodiments, the MOI may be 1×10 6 It may be. In some embodiments, the MOI may be 1×10 5 ~1×10 7 It may be. In some embodiments, the MOI may be 1×10 4 ~1×10 8 It may be. 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 MOI. In some embodiments, the recombinant virus of the present disclosure is 1×10 8 ~3×10 14 MOI. In some embodiments, the recombinant virus of the present disclosure is at most about 1×10 1 、1×10 2 、1×10 3 、1×10 4 、1×10 5 、1×10 6 、1×10 7 、1×10 8 、1×10 9 、1×10 10 、1×10 11 、1×10 12 、1×10 13 、1×10 14 、1×1015 , 1×10 16 , 1×10 17 , and 1×10 18 MOI. 【0306】 In some embodiments, the amount of the pharmaceutical composition is about 1×10 5 to about 1×10 16 rAAV vectors, 1×10 8 to about 1×10 15 rAAV vectors, about 1×10 9 to about 1×10 14 rAAV vectors, about 1×10 10 to about 1×10 13 rAAV vectors, or about 1×10 11 to about 3×10 12 rAAV vectors. 【0307】 In the preparation of the subject rAAV composition, any host cell for producing an rAAV vector may be used, including, for example, mammalian cells (e.g., 293 cells), insect cells (e.g., SF9 cells), microorganisms, and yeast. The host cell can also be a packaging cell in which the AAV rep and cap genes are stably maintained in the host cell, or a producer cell in which the rAAV vector genome is stably maintained and packaged. Exemplary packaging and producer 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. 【0308】 In some embodiments, the present disclosure provides for the use of the rAAV vectors described herein in the production of a medicament. In some embodiments, the present disclosure provides for the use of the rAAV vectors described herein in the production of a medicament for use in the methods described herein. 【0309】 In some embodiments, the present disclosure provides a kit comprising the rAAV herein and instructions for use. In some embodiments, the kit includes the rAAV herein and a package insert containing instructions for use of the kit. In some embodiments, the kit includes the rAAV herein and a pharmaceutically acceptable carrier, or a pharmaceutical composition comprising the rAAV, and instructions for treating a disease, disorder, or symptom described herein or delaying the progression thereof in a subject in need of treatment of the disease, disorder, or symptom described herein or delaying the progression thereof. 【0310】 Exemplary embodiments The present disclosure relates to the following embodiments. 【0311】 Embodiment I-1. A recombinant AAV (rAAV) vector comprising an adeno-associated virus (AAV) capsid and an expression cassette, wherein the expression cassette comprises a polynucleotide encoding DAO1 operably linked to a promoter, the rAAV vector. 【0312】 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 NOs: 1 and 2. 【0313】 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 to 30. 【0314】 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 NOs: 29 to 30. 【0315】 Embodiment I-5. The rAAV vector according to any one of Embodiments I-1 to I-4, wherein the promoter is a CMV promoter containing the nucleotide sequence shown in SEQ ID NO: 21 or a CAG promoter containing the nucleotide sequence shown in SEQ ID NO: 5. 【0316】 Embodiment I-6. The rAAV vector according to Embodiment I-5, wherein the expression cassette contains the CMV promoter and the CMV enhancer. 【0317】 Embodiment I-7. The rAAV vector according to any one of Embodiments I-1 to I-6, wherein the expression cassette contains a polyadenylation (polyA) sequence. 【0318】 Embodiment I-8. The rAAV vector according to Embodiment I-7, wherein the polyA sequence is a BGH polyA sequence. 【0319】 Embodiment I-9. The rAAV vector according to any one of Embodiments I-1 to I-8, wherein the expression cassette contains a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE). 【0320】 Embodiment I-10. The rAAV vector according to any one of Embodiments I-1 to I-9, wherein the expression cassette contains a Kozak sequence. 【0321】 Embodiment I-11. A composition comprising an rAAV vector, wherein the rAAV vector (a) an AAV capsid, and (b) an expression cassette, wherein the expression cassette contains a polynucleotide sharing at least 95% identity with a nucleotide sequence selected from SEQ ID NOs: 29 to 31, and the polynucleotide is linked to a promoter, the expression cassette comprising the composition. 【0322】 Embodiment I-12. The rAAV vector according to any one of Embodiments I-1 to I-10 or the composition according to Embodiment I-11, wherein the expression cassette is flanked by two inverted terminal repeats (ITRs). 【0323】 Embodiment I-13. The rAAV vector or composition according to Embodiment I-12, wherein the ITR is an AAV2 ITR. 【0324】 Embodiment I-14. The rAAV vector according to any one of Embodiments I-1 to I-10 and I-12 or the composition according to Embodiment I-11 or I-12, wherein the expression cassette comprises a sequence sharing at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 23. 【0325】 Embodiment I-15. The rAAV vector according to any one of Embodiments I-1 to I-10 and I-12 to I-14 or the composition according to any one of Embodiments I-11 to I-14, wherein the AAV capsid comprises a VP3 sharing 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). 【0326】 Embodiment I-16. 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-15, wherein the AAV capsid comprises a VP3 sharing at least 95%, 98%, or 100% identity with AAV9 VP3 (SEQ ID NO: 14). 【0327】 Embodiment I-17. A composition comprising an rAAV vector, wherein the rAAV vector is (a) an AAV2, AAV5, AAV8, or AAV9 capsid, and (b) An expression cassette, wherein the expression cassette comprises a polynucleotide having at least 95% identity to a nucleotide sequence selected from SEQ ID NOs: 29 to 31, and the polynucleotide is linked to a promoter, said expression cassette The composition comprising the same. 【0328】 Embodiment I-18. A composition comprising an rAAV vector, wherein the rAAV vector (a) An AAV2, AAV5, AAV8, or AAV9 capsid, and (b) An expression cassette, wherein the expression cassette comprises a polynucleotide sharing at least 95% identity with SEQ ID NO: 23, said expression cassette The composition comprising the same. 【0329】 Embodiment I-19. The composition according to Embodiment I-17 or I-18, wherein the AAV capsid is AAV2. 【0330】 Embodiment I-20. The composition according to Embodiment I-17 or I-18, wherein the AAV capsid is AAV5. 【0331】 Embodiment I-21. The composition according to Embodiment I-17 or I-18, wherein the AAV capsid is AAV9. 【0332】 Embodiment I-22. A pharmaceutical composition comprising 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 to I-21, and a pharmaceutically acceptable carrier. 【0333】 Embodiment I-23. The pharmaceutical composition according to Embodiment I-22, wherein the composition comprises about 1 × 10 7 ~ about 1 × 10 14 genomic copies per milliliter of the rAAV vector. 【0334】 Embodiment I-24. The pharmaceutical composition according to embodiment I-22, wherein the composition contains from about 1×10 12 to about 6.2×10 12 genomic copies per milliliter of the rAAV vector. 【0335】 Embodiment I-25. The pharmaceutical composition according to any one of embodiments I-22 to I-24, wherein the composition is formulated for administration into the lacrimal gland. 【0336】 Embodiment I-26. The pharmaceutical composition according to any one of embodiments I-22 to I-25, wherein the composition is formulated for administration onto the ocular surface. 【0337】 Embodiment I-27. The pharmaceutical composition according to any one of embodiments I-22 to I-26, wherein the composition is formulated for use in the treatment of an eye disease, disorder, or condition or is adaptable for such use. 【0338】 Embodiment I-28. A method of treating a symptom in a subject in need thereof, the method comprising administering to the eye of the subject an effective amount of the pharmaceutical composition according to any one of embodiments I-22 to I-27. 【0339】 Embodiment I-29. The method according to embodiment I-28, wherein the pharmaceutical composition is delivered to the secretory glands of the eye of the subject. 【0340】 Embodiment I-30. The method according to embodiment I-28 or I-29, wherein the pharmaceutical composition is delivered to the lacrimal gland. 【0341】 Embodiment I-31. The method according to embodiment I-30, wherein the cells in the lacrimal gland are transduced by the rAAV vector. 【0342】 Embodiment I-32. The method according to embodiment I-31, wherein the transduced cells in the lacrimal gland express an effective amount of DAO1 in the tear film of the subject and optionally on the ocular surface. 【0343】 Embodiment I-33. The method according to any one of Embodiments I-28 to I-32, wherein the pharmaceutical composition is delivered to the accessory lacrimal gland. 【0344】 Embodiment I-34. About 1×10 9 to about 1×10 10 、about 1×10 10 to about 1×10 11 、about 1×10 11 to about 1×10 12 、about 1×10 12 to about 1×10 13 、or about 1×10 13 to about 1×10 15 genomic copies are administered, the method according to any one of Embodiments I-28 to I-33. 【0345】 Embodiment I-35. The method according to any one of Embodiments I-28 to I-34, wherein the symptom is associated with increased histamine production and / or increased histamine signaling. described method. 【0346】 Embodiment I-36. The method according to any one of Embodiments I-28 to I-35, wherein the symptom is an inflammatory symptom, optionally an inflammatory symptom of the eye. 【0347】 Embodiment I-37. The method according to any one of Embodiments I-28 to I-36, wherein the symptom is an autoimmune symptom. 【0348】 Embodiment I-38. The method according to any one of Embodiments I-28 to I-36, wherein the symptom is an allergic symptom. 【0349】 Embodiment I-39. The method according to Embodiment I-38, wherein the symptom is an allergic reaction to a therapeutic agent. 【0350】 Embodiment I-40. The method according to Embodiment I-38, wherein the symptom is an allergic reaction to a microbial agent. 【0351】 Embodiment I-41. The method according to any one of Embodiments I-28 to I-36, wherein the symptom includes vernal catarrh. 【0352】 Embodiment I-42. The method according to any one of Embodiments I-28 to I-36, wherein the symptom includes atopic keratoconjunctivitis. 【0353】 Embodiment I-43. The method according to any one of Embodiments I-28 to I-36, wherein the symptom includes seasonal or perennial allergic conjunctivitis. 【0354】 Embodiment I-44. The method according to any one of Embodiments I-28 to I-43, wherein the administration results in the expression of DAO1 in cells of the lacrimal gland and / or accessory lacrimal gland. 【0355】 Embodiment I-45. The method according to any one of Embodiments I-28 to I-44, wherein the administration results in the secretion of DAO1 into the tear film. 【0356】 Embodiment I-46. The method according to Embodiment I-45, wherein the secretion of DAO1 into the tear film is stimulated by a cholinergic agonist. 【0357】 Embodiment I-47. The method according to any one of Embodiments I-28 to I-46, wherein the administration results in the improvement of one or more symptoms of the symptom. 【0358】 Embodiment I-48. The method according to Embodiment I-47, wherein the symptom is selected from the group consisting of itching, swelling, tearing, and flushing. 【0359】 Embodiment I-49. The method according to any one of Embodiments I-28 to I-46, wherein the administration results in an improvement of 0.5 point, 1 point, 1.5 points, 2 points, 2.5 points, 3 points, 3.5 points, or 4 points on the conjunctival itching grading scale. 【0360】 Embodiment I-50. The method according to any one of Embodiments I-28 to I-46, wherein the administration results in an improvement of 1 point, 2 points, 3 points, or 4 points on the conjunctival hyperemia evaluation grading scale. 【0361】 Embodiment I-51. The method according to any one of Embodiments I-28 to I-46, 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 greater than about 95% in the Schirmer test. 【0362】 Embodiment I-52. The method according to any one of Embodiments I-28 to I-46, 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 greater than about 95% in at least one compartment of the corneal staining grading scale. 【0363】 Embodiment I-53. The method according to any one of Embodiments I-47 to I-52, wherein the improvement is measured 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. 【0364】 Embodiment I-54. The 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 the administration. 【0365】 Embodiment I-55. The method according to any one of Embodiments I-28 to I-54, further comprising administering one or more additional therapeutic agents to the subject. 【0366】 Embodiment I-56. The method according to Embodiment I-55, wherein the one or more additional therapeutic agents increase tear production. 【0367】 Embodiment I-57. The method according to Embodiment I-56, wherein the one or more additional therapeutic agents that increase tear production are administered via local nasal administration. 【0368】 Embodiment I-58. The method according to Embodiment I-57, wherein the local nasal administration is performed via a nasal spray. 【0369】 Embodiment I-59. The method according to any one of Embodiments I-56 to I-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. 【0370】 Embodiment I-60. The method according to Embodiment I-59, wherein the nAChR agonist is a full agonist of an nAChR subtype selected from alpha4beta2, alpha3beta4, alpha3alpha5beta4, alpha4alpha6beta2, and combinations thereof. 【0371】 Embodiment I-61. The method according to Embodiment I-59 or I-60, wherein the nAChR agonist is varenicline or a pharmaceutically acceptable salt thereof. 【0372】 Embodiment I-62. The method according to Embodiment I-59 or I-60, wherein the nAChR agonist is (R)-5-((E)-2-pyrrolidin-3-ylvinyl)pyrimidine or a pharmaceutically acceptable salt thereof. 【0373】 Embodiment I-63. The method according to 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 the administration of the pharmaceutical composition. 【0374】 Embodiment I-64. The method according to any one of Embodiments I-56 to I-63, wherein the one or more additional therapeutic agents that increase tear production are administered about one week after the administration of the pharmaceutical composition. 【0375】 Embodiment I-65. The method according to any one of Embodiments I-56 to I-64, which results in the expression of DAO1 in the tear layer and / or cornea of the subject after administration of the pharmaceutical composition. 【0376】 Embodiment I-66. The method according to Embodiment I-65, wherein the expression of DAO1 in the tear layer and / or cornea is increased within a predetermined amount of time as compared to administration of the pharmaceutical composition without the one or more additional therapeutic agents that increase tear production. 【0377】 Embodiment I-67. The method according to Embodiment I-66, wherein the predetermined amount of time is about 5 minutes. 【0378】 Embodiment I-68. The method according to Embodiment I-66, wherein the predetermined amount of time is about 1 hour. 【0379】 Embodiment I-69. The method according to any one of Embodiments I-28 to I-68, wherein the subject is a human. 【0380】 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 symptom in a subject in need of treatment of the symptom, the method comprising administering to the eye of the subject an effective amount of the pharmaceutical composition, the pharmaceutical composition for said use. 【0381】 Embodiment I-71. A pharmaceutical composition according to any one of Embodiments I-22 to I-27 for use in the production of a medicament for treating a symptom in a subject in need of treatment of the symptom. 【0382】 Embodiment I-72. A kit comprising an rAAV vector described in any one of Embodiments I-1 to I-10 and I-12 to I-15, or a composition described in any one of Embodiments I-11 to I-21, and a pharmaceutically acceptable carrier, and instructions for use in treating a symptom in a subject, the treating comprising administering the pharmaceutical composition to the eye of the subject. 【0383】 Embodiment I-73. A kit comprising an rAAV vector described in any one of Embodiments I-1 to I-10 and I-12 to I-15, or a composition described in any one of Embodiments I-11 to I-21, and a pharmaceutically acceptable carrier, and instructions for use in treating a symptom associated with histamine production and / or increased histamine signaling in a subject, the treating comprising administering the pharmaceutical composition to the eye of the subject. 【0384】 Embodiment I-74. A kit comprising an rAAV vector described in any one of Embodiments I-1 to I-10 and I-12 to I-15, or a composition described in any one of Embodiments I-11 to I-21, and a pharmaceutically acceptable carrier, and instructions for use in treating an autoimmune symptom in a subject, the treating comprising administering the pharmaceutical composition to the eye of the subject. 【0385】 Embodiment I-75. A kit comprising an rAAV vector described in any one of Embodiments I-1 to I-10 and I-12 to I-15, or a composition described in any one of Embodiments I-11 to I-21, and a pharmaceutically acceptable carrier, and instructions for use in treating an allergic symptom in a subject, the treating comprising administering the pharmaceutical composition to the eye of the subject. 【0386】 Embodiment I-76. A kit according to any one of Embodiments I-72 to I-75, wherein the subject has been, is being, or will subsequently be administered one or more additional therapeutic agents that increase tear production. 【0387】 Kit according to embodiment I-76, wherein said one or more additional therapeutic agents for increasing tear production comprise a nicotinic acetylcholine receptor (nAChR) agonist, or a pharmaceutically acceptable salt thereof. 【0388】 Kit according to embodiment I-76 or I-77, wherein said one or more additional therapeutic agents for increasing tear production comprise varenicline, or a pharmaceutically acceptable salt thereof. 【0389】 Kit according to embodiment I-76 or I-77, wherein said one or more additional therapeutic agents for increasing tear production comprise (R)-5-((E)-2-pyrrolidin-3-ylvinyl)pyrimidine, or a pharmaceutically acceptable salt thereof. 【0390】 Kit according to any one of embodiments I-76 to I-80, wherein said subject is administered said one or more additional therapeutic agents after administration of said pharmaceutical composition. 【0391】 A pharmaceutical composition comprising: a) a polypeptide comprising a diamine oxidase ("DAO1") enzyme, or a fragment thereof, optionally wherein said 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 eyes of a human subject. 【0392】 A vector comprising: a) a polynucleotide encoding a polypeptide comprising a diamine oxidase ("DAO1") enzyme, or a fragment thereof, optionally wherein said 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 eyes of a human subject. 【0393】 Embodiment I-84. The 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; wetting agent; surfactant; tonicity agent; pH modifier; viscosity modifier; buffering agent; disaccharide, optionally sucrose or trehalose; cellulose and / or its derivatives; amino acid, optionally histidine; or any combination thereof. 【0394】 Embodiment I-85. The 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. 【0395】 Embodiment I-86. The pharmaceutical composition according to any one of Embodiments I-82 to I-85, wherein the polypeptide comprises at least 20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 30 0, 320, 340, 360, 380, 400, 420, 440, 460, 480, or 500 amino acids. 【0396】 Embodiment I-87. The pharmaceutical composition according to any one of Embodiments I-82 to I-86, wherein the formulation is a liquid formulated for application to or into the ocular surface of the eye of the human subject or for intracanalicular injection. 【0397】 Embodiment I-88. The pharmaceutical composition according to any one of Embodiments I-82 to I-87, comprising the vector, wherein the vector is present in the composition in an amount effective to express the polypeptide at 100 pg / mL to 50 μg / mL in the tear layer of the subject after administration of the composition to the subject. 【0398】 Embodiment I-89. The 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. 【0399】 Embodiment I-90. The 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 with at least 90% identity to the amino acid sequence selected from SEQ ID NOs: 1 and 2. 【0400】 Embodiment I-91. The pharmaceutical composition according to any one of Embodiments I-82 to I-90, comprising the vector, wherein the vector comprises a virus, optionally an adenovirus vector or a lentivirus 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 endocytosis receptor proteins. 【0401】 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. 【0402】 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. 【0403】 Embodiment I-94. The 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. 【0404】 Embodiment I-95. A method for treating an eye disease, disorder, or condition in a subject in need of treatment of the eye disease, disorder, or condition, the method comprising administering to the eye of the subject an effective amount of the pharmaceutical composition according to any one of Embodiments I-82 to I-94. 【0405】 Embodiment I-96. The method according to Embodiment I-95, wherein the eye disease, disorder, or condition is associated with increased histamine production and / or increased histamine signaling. 【0406】 Embodiment I-97. The method according to Embodiment I-95 or I-96, wherein the condition is an inflammatory condition, optionally an inflammatory condition of the eye. Embodiment I-98. The method according to any one of Embodiments I-95 to I-97, wherein the condition is an autoimmune condition. 【0407】 Embodiment I-99. The method according to any one of Embodiments I-95 to I-98, wherein the condition is an allergic condition. 【0408】 Embodiment I-100. The method according to any one of Embodiments I-95 to I-99, wherein the condition is an allergic reaction to a therapeutic agent. 【0409】 Embodiment I-101. The method according to any one of Embodiments I-95 to I-100, wherein the condition is an allergic reaction to a microbial agent. 【0410】 Embodiment I-102. The method according to any one of Embodiments I-95 to I-101, wherein the condition includes vernal catarrh. 【0411】 Embodiment I-103. The method according to any one of Embodiments I-95 to I-102, wherein the condition includes atopic keratoconjunctivitis. 【0412】 Embodiment I-103. The method according to any one of Embodiments I-95 to I-102, wherein the condition includes atopic keratoconjunctivitis. 【0413】 Embodiment I-104. The method according to any one of Embodiments I-95 to I-103, wherein the symptom includes seasonal or perennial allergic conjunctivitis. 【0414】 Embodiment I-105. The method according to any one of Embodiments I-95 to I-104, wherein the administration results in the expression of functional diamine oxidase in one or more cells of the lacrimal gland and / or accessory lacrimal gland of the subject. 【0415】 Embodiment I-106. The method according to any one of Embodiments I-95 to I-105, wherein the administration results in the secretion of functional diamine oxidase into the tear film layer of the subject. 【0416】 Embodiment I-107. The method according to any one of Embodiments I-95 to I-106, wherein the secretion of the functional diamine oxidase into the tear film layer is stimulated by a cholinergic agonist. 【0417】 Embodiment I-108. The method according to Embodiment I-107, wherein the administration is performed on the ocular surface of the subject, or into the ocular surface, and / or on the lacrimal gland of the subject. 【0418】 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 flushing. 【0419】 Embodiment I-110. The method according to any one of Embodiments I-95 to I-109, wherein the administration results in an improvement of 0.5 point, 1 point, 1.5 points, 2 points, 2.5 points, 3 points, 3.5 points, or 4 points on the conjunctival itching grading scale. 【0420】 Embodiment I-111. The method according to any one of Embodiments I-95 to I-110, wherein the subject is a human subject. 【0421】 Embodiment I-112. A kit comprising the pharmaceutical composition according to any one of Embodiments I-82 to I-94, and instructions for use in the treatment of a condition in a human subject, the treatment comprising administering the pharmaceutical composition to the eyes of the human subject. Instructions for use in the treatment of a condition in a human subject, the treatment comprising administering the pharmaceutical composition to the eyes of the human subject. 【0422】 Embodiment I-113. A pharmaceutical composition according to any one of Embodiments I-82 to I-94 for use in the production of a medicament for treating a condition in a human subject in need of treatment of the condition. 【Examples】 【0423】 The following specific examples should be construed as merely illustrative and should in no way be construed as limiting the remainder of the present disclosure. 【0424】 Example 1: In Vitro Transfection of 293T Cells with an AAV Transfer Plasmid Encoding DAO1 Studies were conducted to evaluate the expression and activity of the DAO1 polypeptide after in vitro transfection of 293T cells with the AAV transfer plasmid depicted in Figure 1A (referred to as “AAV.DAO1”). The AAV transfer plasmid contained 5’ and 3’ AAV2 ITRs (SEQ ID NOs: 16 and 17, respectively). Between the 5’ ITR and the 3’ ITR, there was a cDNA encoding the human DAO1 polypeptide and a cDNA for elements to provide an optimal expression level of the DAO1 polypeptide. A schematic depicting the construct is shown in Figure 1B. The encoded human DAO1 polypeptide has the amino acid sequence shown in SEQ ID NO: 2. The cDNA encoding the human DAO1 polypeptide was codon-optimized for improved expression in human cells, and the cDNA has the nucleotide sequence shown in SEQ ID NO: 29. The nucleotide sequence from the 5’ ITR to the 3’ ITR is shown in SEQ ID NO: 23. 【0425】 Plasmid amplification was performed by adding 10 ng of AAV.DAO1 plasmid to 50 μl of New England Biolabs (NEB) Stable Competent E. coli High Efficiency (NEB C3040) according to the producer's rapid transformation protocol. A single bacterial colony was selected and used to inoculate 5 mL of Luria Broth (LB) with kanamycin (50 μg / mL) as a positive selection for the relevant colony clones. The starting culture was incubated at 30 °C for 16 hours and then used to inoculate 250 mL of LB / kanamycin at a 1 / 500 dilution. This culture was incubated at 30 °C for 16 hours and then centrifuged at 4,000 × g for 20 minutes to pellet the bacteria. Plasmid DNA was purified using the Qiagen Hi-Speed Maxi Kit (Qiagen 12662) as per the producer's protocol. The plasmid was verified by restriction enzyme digestion and agarose gel electrophoresis based on fragment size. 【0426】 293T cells were plated in 6-well plates at 6.5×10 5 cells / well and transfected with 2.5 μg of AAV.DAO1 plasmid DNA using Lipofectamine 3000 as per the producer's protocol. Control cells were transfected with an empty vector. The cells were grown and then plated on 35 mm glass-bottom tissue culture plates at 5.5×10 5 . 【0427】 Immunofluorescence Immunofluorescence imaging was performed to detect DAO1 expression in transfected cells. 【0428】 293T cells were plated at 5.5×10 5 on poly-L-lysine (VWR 103701-192)-coated 33 mm glass-bottom TC dishes (MatTek P35G-0-10-C), and PANC-1 cells were plated at 4×10 5 . The plates 12 to 24 hours after winging, 3.75 μl of Lipofectamine per well 3000 and 2.5 μg of pAAV-DAO plasmid were used to transfect cells according to the Lipofectamine 3000 (ThermoFisher L300015) transfection protocol. 24 hours after transfection, the medium was aspirated and the cells were washed twice with pre-warmed PBS (37 °C). 500 μl of 4% paraformaldehyde was added and the cells were incubated at room temperature for 10 minutes. The cells were washed twice with PBS. 500 μl 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 against diamine oxidase and a fluorescent-tagged secondary antibody. The cells were counterstained with DAPI and then imaged on a fluorescence microscope. Specifically, the primary antibody was diluted in the blocking buffer and then 200 μl of the diluted mixture was added to the central glass part of the 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 three times with PBS for 15 minutes each. The fluorescently labeled secondary antibody and DAPI were diluted 1:400 in PBS, added to the glass part of the dish and incubated at room temperature for 1 hour (protected from light). The cells were then washed three more times with PBS for 15 minutes each. The cells were then imaged on a Keyence BZ-X series microscope. 【0429】 As shown in Figures 4A - 4B, the expression of DAO1 protein (indicated by bright punctate signals) was measured in 293T cells transfected with the AAV transfer plasmid, but not in control cells. As shown in Figures 4C - 4D, the expression of DAO1 protein was further detected in PANC-1 cells transiently transfected with the AAV.DAO plasmid. 【0430】 Western blot Western blot was performed to detect DAO1 expression into the conditioned media from AAV.DAO1 transfected cells compared to control cells. Detection of DAO1 was performed using rabbit anti-DAO1 antibody and labeled anti-rabbit secondary antibody. For conditioned media collection, the cell media was aspirated 24 hours after transfection and washed twice with 2 mL of pre-warmed Opti-MEM (ThermoFisher 31985-070). 1.4 mL of Opti-MEM was then added and conditioned media was collected at 2, 4, and 6 hour intervals. Immediately after collection, 1X protease inhibitor was added, the conditioned media was placed on ice for 5 minutes, and centrifuged at 4°C, 12,000×g for 5 minutes to remove cell debris. The conditioned media supernatant was then concentrated 40-fold using an Amicon Ultra-2 Centrifugal Filter Unit (Millipore UFC201024) as per the manufacturer's protocol and stored at -20°C. 【0431】 Western blot was also performed using whole cell extracts. Whole cell extracts were prepared by aspirating the cell media 24 or 48 hours after transfection. Cells were washed twice with 2 mL of 4°C PBS and then lysed using 300 μl of RIPA buffer (Sigma R0278) containing protease / phosphatase inhibitor (Cell Signaling 5872S) diluted from 100X stock to 1X. Whole cell extracts were then centrifuged at 12,000×g for 5 minutes at 4°C to remove any cell debris, and then the supernatant was aliquoted and stored at -20°C. The whole cell extracts collected (as per the methodology above) were quantified using a bicinchoninic acid assay (BCA) protein assay kit (Thermo Fisher 23235) as per the manufacturer's recommendation. Sample concentrations were normalized by dilution with double distilled water (ddH20) and Laemmli buffer. The extracts were then heated to 100°C for 10 minutes. Samples were electrophoresed on a 10% SDS-PAGE gel and then transferred to a PVDF membrane using an Invitrogen iBlot 2 Dry Blotting System. On a rotating platform The membrane was blocked with 5% Blotting-Grade Blocker (BioRad 1706404) for 1 hour at room temperature. The primary antibody was diluted in 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. The membrane was washed in PBS containing 0.05% Tween 20 for 10 minutes on a rotating platform. The washing step was repeated 4 times. The HRP-conjugated secondary antibody was diluted (1:10,000) in 5% blocker containing 0.1% Tween 20 and incubated for 1 hour at room temperature on a rotating platform. The previous washing steps were repeated along with a final wash with PBS alone. The membrane was incubated in SuperSignal West Pico Plus (Thermo Fisher 34577) for 1 minute and chemiluminescence detection was performed on a BioRad ChemiDoc MP. 【0432】 As a control, Western blots were also performed using human DAO1 recombinant protein (40 ng) and mouse anti-his antibody. Western blots of GAPDH (30 μg / well) served as a loading control. 【0433】 As shown in Figure 5A, the DAO1 polypeptide was observed in the medium obtained from transfected cells but not in the medium obtained from control cells. The DAO1 polypeptide had the same molecular weight as the human DAO1 recombinant protein used as a positive control (Figure 5B). As shown in Figures 5C-5D, DAO1 of the expected size (kDa) was detected in the whole cell extracts obtained from transfected 293T cells. Recombinant DAO1 served as a positive control (Figure 5C) and GAPDH served as a loading control (Figure 5D). 【0434】 Additionally, DAO1 expression was measured in the conditioned medium 2, 4, and 6 hours after the exchange of fresh medium. As shown in Figure 5E, significant levels of DAO1 were detected within just 2 hours of medium exchange. 【0435】 When combined, these data demonstrate the expression of DAO1 and its secretion into the cell culture medium. 【0436】 DAO1 Enzyme Activity Assay To evaluate the functional activity of the expressed DAO1 protein, an activity assay was performed. The principle of the assay is that the substrate provided in the presence of active DAO1 results in a reaction that releases hydrogen peroxide (H2O2). The H2O2 released by the cleavage of the substrate reacts with a second substrate to generate a fluorescent signal that is excited at 535 nm and emits light at 587 nm. The amount of fluorescence recorded at 587 nm indicates the amount of enzyme activity. 【0437】 The experimental approach involved plating 293T cells in a 6-well plate format and transfection with AAV.DAO. Negative control cells were not transfected. Twenty-four hours after transfection, the medium was removed and replenished with 2 mL / well of maintenance medium. After 24 hours, the conditioned medium and whole cell extract (WCE) were collected. At the time of collection, protease inhibitors were added, and the conditioned medium / WCE was placed on ice for 5 minutes and centrifuged at 4°C, 12,000 × g for 5 minutes to remove debris. The medium / WCE was aliquoted and stored at -20°C. A micro BCA assay was performed to quantify the protein level of the WCE. Standards were prepared using 10 mM H2O2 supplied with the Diamine Oxidase Activity Assay Kit (Sigma MAK351). Serial diluted standards, undiluted AAV.DAO conditioned medium, 100 ng of WCE, and control samples were added to a 96-well reaction plate. A reaction mix was prepared using DAO assay buffer, substrate, enzyme mix, and probe. The reaction plate was then Incubate at 37 °C for 90 minutes and obtain kinetic measurements at 30, 60, and 90 minutes after incubation. Measurements were obtained using a Molecular Devices ID3 plate reader at an excitation wavelength of 535 nm and an emission wavelength of 587 nm. DAO1 activity was measured in nmol H2O2 generated within 90 minutes (Experiment 1) or pmol / min / μl (Experiment 2 using kinetic measurements). 【0438】 As shown in Figure 6A, 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 media obtained from AAV.DAO1 transfected 293T cells or cell lysates combined with conditioned media and compared to the activity measured in conditioned media obtained from non-transfected 293T cells. As shown in Figure 6B, substantially higher H2O2 was generated for conditioned media obtained from AAV.DAO1 transfected 293T cells alone or in combination with cell lysates compared to conditioned media obtained from non-transfected 293T cells. As shown in Figure 6C, kinetic measurements of DAO1 activity in the conditioned media of transfected 293T cells at 30, 60, and 90 minutes demonstrate the target modulation ability and functional activity of DAO1 expressed by AAV-DAO. 【0439】 This demonstrates that the DAO1 protein present in cells and expressed in cell culture media can cleave a given substrate and also demonstrates functional enzymatic activity. 【0440】 Example 2: Intraglandular gene therapy using rAAV vectors This example shows a 9-day pilot study of a single dose of rAAV vector administered as an intraglandular injection to Dutch Belted rabbits, followed by a single dose of varenicline administered as an intranasal dose. It evaluates the efficacy and tolerability of a panel of rAAV vector embodiments administered once via injection into the lacrimal gland. Each rAAV vector composition in the panel was at two concentrations (1×1012 GC / mL and 6.2×10 12 It is tested at (). The panel of rAAV vectors includes embodiments using capsid proteins having AAV2, AAV5, AAV8, and AAV9 serotypes. The expression cassette delivered by the rAAV vector encodes an enhanced green fluorescent protein (eGFP) transgene operably linked to a CMV promoter (Figure 7). On the 9th day after injection of the rAAV vector, the animals are given an intranasal dose of varenicline. Varenicline induces tear production in the animals, and as a result, the eGFP delivered to the lacrimal gland by the rAAV vector and expressed under the control of the CMV promoter in the cells of the lacrimal gland is secreted into the tear film of the animals and onto the ocular surface. Two main objectives are achieved using this approach: (1) To test the feasibility of capsid protein serotypes AAV2, AAV5, AAV8, and AAV9 for delivering a transgene to cells in the lacrimal gland and resulting in measurable CMV promoter-driven expression of the transgene intracellularly. (2) To evaluate the feasibility of increasing the relative amount of the transgene encoded by the expression cassette in the tear film of the animals and on the ocular surface. 【0441】 Animal studies were performed by CRL scientific staff at the Charles River Laboratories (CRL) facility. 【0442】 Animal test system, housing, and in-life monitoring The animals used in this study were male Dutch Belted rabbits 4 - 5 months old and weighing 1.3 - 2.3 kg. The animals were acclimated for 10 days prior to the start of treatment. Each animal was housed individually and fed using a standard husbandry protocol that included normal environmental conditions, a food intake schedule, and veterinary care. 【0443】 rAAV vector compositions and formulations In this study, a panel of compositions comprising rAAV vectors containing an expression cassette encoding an eGFP transgene operably linked to a CMV promoter is provided for intraductal injection of the lacrimal gland under the conditions in Table 6. Each composition contains an rAAV vector having a different AAV capsid protein serotype. The compositions are labeled as OC-100a-d, each corresponding to a different AAV capsid protein serotype. Dose formulations for intraductal injection of the lacrimal gland were prepared using a clean procedure at the target concentrations described in Table 8 below by dilution with phosphate-buffered saline aqueous solution. 【Table 6】 【0444】 Intraductal injection of rAAV composition On day 1 of the study, animals were dosed via intraductal injection of the lacrimal gland. A summary of the formulation concentration, dosing volume, dosing frequency, and the number of animals and lacrimal glands for each composition tested is found in Table 7. Prior to injection, animals were anesthetized by intramuscular injection of dexmedetomidine (0.25 mg / kg), followed by isoflurane / oxygen mix through a mask to maintain anesthesia if necessary. Topical antibiotics were applied to each eye after dosing. On day 9 of the study, animals were given intranasal administration of varenicline tartrate (50 μL per nostril of 1.2 mg / mL varenicline) to induce tear production. 【Table 7】 【0445】 Biological analysis Blood was collected from the auricular vessels of all animals before dosing on day 1, and again on days 8 and 9 (approximately 1 hour after dosing following intranasal administration). Blood samples were placed on ice until the plasma was separated by centrifugation. Plasma samples were separated into 250 μL aliquots and then frozen at -80 °C for subsequent analysis. 【0446】 The Schirmer tear test was performed, and eye moisture was collected from the animals on days 8 and 9. The test strip was placed on the inner surface of the lower eyelid for about 1 minute. The paper was removed, placed in separate tubes, and frozen at -80 °C for subsequent analysis. 【0447】 In the Syneos Analytical Laboratory, plasma samples and Schirmer test strips were analyzed for eGFP concentration and eGFP mRNA concentration using validated procedures. 【0448】 Immunohistochemistry of lacrimal gland tissue On day 9, the animals were euthanized after collection of blood and eye moisture 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 the laboratory's standard operating procedures. Lacrimal gland IHC samples were stained for eGFP and subjected to microscopic evaluation. 【0449】 Results Microscopic evaluation was performed to determine the efficiency of eGFP expression in lacrimal gland tissue dosed in vivo with the rAAV composition. Isolated positive acinar cells in the IHC samples had pink to red cytoplasmic staining indicative of GFP expression (Figures 8A - 8K; exemplary staining is indicated by black arrows). Positive eGFP expression was observed for the rAAV composition (OC - 100a) containing AAV2 capsid protein at 6.2×10 12 GC / mL (Figure 8A), 1×10 12 GC / mL (Figure 8B) and both rAAV compositions containing AAV5 capsid protein at 6.2×10 12 GC / mL (Figures 8C - 8H), as well as the rAAV composition containing AAV9 capsid protein at 6.2×10 12 GC / mL (Figures 8I - 8K). 【0450】 Conclusions The results from this example indicate that rAAV vectors can be used to deliver an expression cassette to the lacrimal gland by direct injection. The results also show that rAAV vectors containing capsid proteins with at least AAV2, AAV5, or AAV9 serotypes can be used to deliver an expression cassette to cells within the lacrimal gland. Furthermore, the results demonstrate that delivery of an expression cassette containing a transgene operably linked to a CAG promoter sequence results in transgene expression in cells of the lacrimal gland. 【0451】 Example 3: Expression of the EGFP transgene in porcine lacrimal glands delivered by rAAV via intralacrimal injection The research objective was to evaluate whether the lacrimal gland could be utilized as a method to modify or enrich the tear film with a protein of interest in pigs. Subsequently, an in vivo study was conducted to test whether EGFP could be produced in the acinar cells of the lacrimal gland and then secreted into the tear film after delivery of an adenoviral vector consisting of a plasmid encoding eGFP. The approach for introducing the cDNA encoding EGFP into acinar cells was to inject an adeno-associated virus (AAV) containing the cDNA encoding secreted EGFP (secEGFP) into the lacrimal gland. To generate each of the two different serotypes (2 and 9) of AAV for secEGFP, an AAV transfer plasmid containing the essential elements for secEGFP expression between the inverted terminal repeats (ITRs) was generated. The DNA sequence between the ITRs was packaged into the produced AAV (Figure 7). 【0452】 Analysis of design and methodology Research-grade AAVs (serotypes 2 and 9) for secreted EGFP were synthesized at Sirion, 5×10 12Provided at a stock concentration. The AAV was tested in vitro by CJ Solutions using HEK 293T cells and ELISA to ensure that the produced AAV transduced the cells. At Texas A&M, eight domestic pigs were given a single intraductal injection of EGFP, with a low dose given to the right (OD; right eye) gland and a high dose given to the left (OS; left eye) gland. Six weeks after the first injection, a second injection with high doses of AAV2 and AAV9 was performed. EGFP expression was evaluated on day 35 of the study. After confirmation of the 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 gland abnormalities. (Tables 8 & 9). In the study, nasal spray dosing was administered in weeks 3 - 4 (Table 10). 【Table 8-1】 【Table 8-2】 【Table 9-1】 【Table 9-2】 【Table 10】 【0453】 After the second AAV-secEGFP injection, tears were collected from each eye via Schirmer strips on day 82. Tears were collected by placing a Schirmer tear test strip in the lower conjunctival sac and leaving it there for 2 minutes. Tear proteins were extracted from the Schirmer tear test strips and mesoscale discovery (MSD) analysis was performed to detect the presence of EGFP protein in the tears. 【0454】 The lacrimal glands were harvested on day 103 for histological diagnosis of the eyes, and samples were sent to Zyagen, Inc. (San Diego, CA) for EGFP immunohistochemical analysis (IHC). 【0455】 ELISA results showed that the produced AAV serotypes could transduce HEK 293T cells in vitro and produce secreted EGFP. EGFP expression in tear samples was confirmed by MSD analysis and IHC at some level > 400 pg / mL, 82 days after AAV transduction of eGFP (Figure 9). IHC indicated that EGFP expression was within acinar cells and higher acinar cell infectivity was observed for AAV2 compared to AAV9. Additionally, transduction of ductal epithelial cells was observed for AAV9-injected lacrimal glands (Figure 10). Hematoxylin and eosin staining of porcine lacrimal glands after repeated AAV injection showed no inflammatory infiltrates, atrophy, or edema (Figure 11). 【0456】 Porcine lacrimal glands injected with either AAV2-secEGFP or AAV9-secEGFP expressed the EGFP transgene product in ductal epithelial cells in addition to acinar cells. EGFP expressed in the lacrimal glands was found to be secreted into the tear film. Additionally, no safety signals or inflammatory infiltrates were observed in any animals after repeated injection of AAV2 or AAV9, regardless of whether a low or high dose of AAV was given first during the first injection. The results of this study demonstrate that acinar cells of the lacrimal gland are a target for gene therapy approaches to modify and / or enrich the tear film. 【0457】 Example 4: Expression of rAAV transgene in porcine lacrimal glands in combination with OC-1 nasal spray This example describes a study in pigs to evaluate the expression of rAAV encoding a model protein after a single intraglandular injection. The purpose of the study is to determine the expression of mRNA encoding the model protein (referred to as "Protein A" in the examples and figures) in the lacrimal gland and the transgene protein levels in the tears, following a single injection of AAV encoding Protein A (referred to as "AAV-Protein A" in the examples and figures). The study is further designed to confirm the expression and secretion of Protein A after intraglandular injection and to evaluate the relative amounts of proteins present on the ocular surface following stimulation of tear production with varenicline nasal spray. Varenicline ("OC-1") is the following compound: to be determined after a single injection of AAV encoding Protein A (referred to as "AAV-Protein A" in the examples and figures). The study is further designed to confirm the expression and secretion of Protein A after intraglandular injection and to evaluate the relative amounts of proteins present on the ocular surface following stimulation of tear production with varenicline nasal spray. Varenicline ("OC-1") is the following compound: 【Chemical formula】 【0458】 The AAV-Protein A plasmid encodes, from 5' to 3', the AAV2 5' ITR, CMV enhancer / promoter, intron sequence including Kozak, open reading frame encoding Protein A, woodchuck hepatitis virus post-transcriptional regulatory element, polyA sequence, and AAV2 3' ITR. 【0459】 The study parameters are detailed in Table 11. The administration and dosing are shown in Table 12. 【Table 11-1】 【Table 11-2】 【Table 12】 【0460】 The timeline for the pig study is shown in Figure 12. For all pigs (N = 14), day 0 is the day of injection. Tear fluid is collected from each eye via Schirmer strips (applied for approximately 2 minutes and then removed) on days 7, 14, 21, 28, 35, 42, 60 / 61, and 90. The Schirmer strip is immediately cut with scissors on top of a fluid or dye line. The lower part of the Schirmer strip (saturated with tear fluid) is then placed in a microcentrifuge tube and kept on ice until transferred to a freezer (-80 °C). On days 14 and 42, tear fluid collection is performed first, and then a nasal spray dose is administered. A second tear fluid collection on the same day is performed approximately 2 minutes after the administration of the nasal spray dose. For days 22 - 28, the OC-01 nasal spray is administered twice a day (with at least 6 hours between administrations) on days 22 - 27 and once on day 28 into both nostrils. The tear fluid collection on day 28 is performed 2 minutes after the administration of the nasal spray. 【0461】 On day 90, gross pathological diagnosis is performed and body weight is evaluated. Additionally, one lacrimal gland is collected from each animal for histological diagnosis of the eye by immunohistochemistry (IHC). After the lacrimal glands are harvested, they are fixed in 10% formalin for 24 - 48 hours (room temperature), then in 70% EtOH, and stored at 4 °C. Maintain the volume in the tube 5 times the volume of the tissue and submerge the tissue completely. 【0462】 The second lacrimal gland is collected, rinsed with phosphate-buffered saline, and immediately placed into RNA-Later. The sample is snap-frozen in liquid nitrogen. Small pieces of tissue approximately 0.5 - 1 cm square from the apex of the heart, kidney, and liver are collected, immediately placed in 2 mL cryotubes, and snap-frozen in liquid nitrogen. The cryotubes are then stored at -80 °C until shipment for mRNA analysis. 【0463】 Using the developed one-step duplex RT-qPCR method, mRNA analysis was performed to analyze the AAV-derived gene expression of protein A in the lacrimal glands of domestic pigs. Each lacrimal gland was homogenized and the lysate was loaded onto a QIASymphony for automated RNA extraction utilizing silica-based RNA purification, magnetic separation, and enzymatic removal of DNA. The AAV-protein A vector contains a bovine growth hormone (bGH) polyA sequence in the 3’untranslated region of the transgene. Protein A mRNA was analyzed using primers and probes targeting the bGH polyA sequence having the sequence shown in Table 13. Amplification was performed for 76 bp of the bGH sequence. The total RNA samples extracted for both bGH mRNA copy number and the Ct value of porcine endogenous Hprt1 mRNA were analyzed on a 96-well plate using the QuantStudio 7 Flex Real Time PCR system and the one-step duplex RT-qPCR method (see Table 14). Each plate included a standard curve, negative control, and quality control samples, which were prepared separately to avoid cross-contamination. Each standard curve included bGH standard DNA levels at 108, 107, 105, 104, 103, 102, 50, 25, and 0 copies per well. RT-qPCR of the RNA samples was performed at up to 100 ng per well in duplicate wells. The bGH mRNA copy number in each RT-qPCR well was interpolated from the bGH DNA standard curve (acceptance criteria: R2≧0.980). Adjustment was made using a 2-fold multiplication step to interpolate single-stranded (ss) mRNA from the double-stranded (ds) standard curve, and the average copy number of the two replicate wells was reported as the copy of ss bGH mRNA per 100 ng of the RNA sample. Additionally, each RNA sample was tested using qScript XLT One-step RT-qPCR (without reverse transcriptase) to monitor for potential vector DNA contaminants in the RNA samples. 【Table 13】 【Table 14】 【0464】 Example 5: Expression of Functional DAO1 in 293T Cells This example describes a study that evaluated the expression of the DAO1 transgene in 293T cells using a colorimetric histamine quantification assay. The purpose of the study was to confirm whether transfection would result in the expression of functional DAO1. 【0465】 Background Five established biogenic amine neurotransmitters have been identified to date, including dopamine, norepinephrine, epinephrine, histamine, and serotonin, which are formed by the decarboxylation of amino acids or the amination of ketones or aldehydes. In particular, histamine is formed by the decarboxylation of histidine and has been described for 115 years (Windaus A, Vogt W. “Synthese des Imidazolyl-athylamins.” Ber. Dtsch. Chem. Ges. 1907;40:3691-3695). The degradation of histamine by DAO1 occurs mainly extracellularly, which is most relevant to therapeutic approaches when attempting to modulate localized mast cell degranulation that results in histamine responses from histamine uptake or histamine release into the extracellular matrix and systemic circulation. One mechanism by which histamine is degraded by DAO1 is through the oxidative deamination of histamine. Oxidation is the first step in the degradation / metabolism of histamine for the ultimate release of by-products from the body in the form of urinary constituents. 【0466】 DAO1 Enzyme Activity Assay In this assay, 293T cells transfected with a plasmid engineered to express DAO1, and non-transfected 293T cells (negative control), were generated and / or cultured using standard protocols, and conditioned media from each sample was collected and concentrated using an Amicon® protein concentrator. As described below, DAO1 enzyme activity was evaluated using the Sigma-Aldrich® Histamine MAK432 Histamine Quantification Assay Kit subjected to a modified protocol. 【0467】 The MAK432 kit provides a colorimetric method for the detection of total histamine from various sources. This kit is based on the enzymatic oxidation of histamine, linked to the reduction of the formazan WST reagent included. The intensity of the color of the product measured at 450 nm is directly 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 generate a histamine standard curve. The producer's protocol was modified by excluding the use of the provided reaction enzyme, for the purpose of determining whether the DAO1 transgene is functional and able to oxidize histamine. Briefly, 25 μg / ml of histamine and phosphate buffered saline were combined with 600 μL of conditioned media (from 293T cells transfected with the 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 of 4 or 24 hours was carried out at 37 °C on a rotary shaker at 150 rpm. The producer's detection solution was then added and stopped after 35 minutes. Figure 13 1 shows an R of 0.998 2Shows the standard curve generated for histamine having values. Figures 14 - 15 demonstrate the kinetics of histamine degradation by recombinant porcine DAO1 at increasing concentrations at 4 (Figure 14) and 24 (Figure 15) hours. Figures 16 - 17 demonstrate the ability of the conditioned medium of 293T cells transfected with the DAO1 plasmid, which significantly degrades histamine after 4 (Figure 16) and 24 (Figure 17) hours of exposure, compared to the non - transfected negative control. 【0468】 As shown by the provided histamine level data, transfection with the DAO1 expression plasmid resulted in a substantial reduction in histamine levels (e.g., >50% after 4 hours and >90% after 24 hours) compared to the negative control, verifying that transfection of 293T cells with the DAO1 plasmid results in the expression of functional DAO1. **** 【0469】 Embodiments of the present invention have been shown and described herein. Those skilled in the art will understand that such embodiments are provided by way of example only. Numerous variations, modifications, and substitutions will occur to those skilled in the art without departing from the present invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in the practice of the present invention. The following claims are intended to define the scope of the present invention, and methods and structures within the scope of these claims and their equivalents are thereby intended to be covered. 【0470】 【Table 15 - 01】 【Table 15 - 02】 【Table 15 - 03】 【Table 15 - 04】 【Table 15-05】 【Table 15-06】 【Table 15-07】 【Table 15-08】 【Table 15-09】 【Table 15-10】 【Table 15-11】 【Table 15-12】 【Table 15-13】 【Table 15-14】 【Table 15-15】 【Table 15-16】 【Table 15-17】 【Table 15-18】 【Table 15-19】 【Table 15-20】 【Table 15-21】 【Table 15-22】 【Table 15-23】 【Table 15-24】 【Table 15-25】 【Table 15-26】 【Table 15-27】 【Table 15-28】 【Table 15-29】 【Table 15-30】 【Table 15-31】 【Table 15-32】 【Table 15-33】 【Table 15-34】 【Table 15-35】 【Table 15-36】 【Table 15-37】 【Table 15-38】 【Table 15-39】 【Table 15-40】 【Table 15-41】 【Table 15-42】 【Table 15-43】 【Table 15-44】 【Table 15-45】 【Table 15 - 46】 【Table 15 - 47】 【Table 15 - 48】 【Table 15 - 49】 【Table 15 - 50】 【Table 15 - 51】 【Table 15 - 52】 【Table 15 - 53】

Claims

[Claim 1] A recombinant AAV (rAAV) vector comprising an adeno-associated virus (AAV) capsid and an expression cassette, wherein the expression cassette comprises a polynucleotide encoding diamine oxidase (DAO1) operably linked to a promoter. [Claim 2] (a) The polynucleotide encodes an amino acid sequence shown in SEQ ID NO: 1 or 2, or an amino acid sequence having at least 95% identity with said amino acid sequence, (b) The polynucleotide comprises a nucleotide sequence selected from SEQ ID NOs: 29-31 or a nucleotide sequence having at least 95% identity with said nucleotide sequence, (c) The promoter is a CMV promoter containing the nucleotide sequence shown in SEQ ID NO: 21 or a CAG promoter containing the nucleotide sequence shown in SEQ ID NO: 5 The rAAV vector according to claim 1. [Claim 3] (a) The expression cassette comprises the CMV promoter and the CMV enhancer, (b) The expression cassette contains a polyadenylated (poly-A) sequence, The rAAV vector according to claim 2. [Claim 4] (a) The polyA sequence is a BGH polyA sequence, (b) The expression cassette contains a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), (c) The expression cassette contains a Kozak sequence, The rAAV vector according to claim 3. [Claim 5] A composition comprising an rAAV vector, wherein the rAAV vector is (a) AAV capsid, and (b) An expression cassette wherein the expression cassette has at least 95% identity with a nucleotide sequence selected from SEQ ID NOs: 29 to 31 The expression cassette comprises a polynucleotide containing a nucleotide sequence, and the polynucleotide is linked to a promoter. The composition comprising the above. [Claim 6] The rAAV vector according to any one of claims 1 to 4 or the composition according to claim 5, wherein the expression cassette is adjacent to each other by two inverted terminal repeats (ITRs). [Claim 7] The rAAV vector or composition according to claim 6, wherein the ITR is an AAV2 ITR. [Claim 8] The rAAV vector according to claim 1 or the composition according to claim 5, wherein the expression cassette comprises a nucleotide sequence that shares at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with respect to SEQ ID NO:

23. [Claim 9] The rAAV vector according to claim 1 or the composition according to claim 5, wherein the AAV capsid shares at least 95%, 98%, or 100% identity with AAV2 (SEQ ID NO: 8), AAV5 (SEQ ID NO: 10), AAV8 (SEQ ID NO: 12), or AAV9 (SEQ ID NO: 14). [Claim 10] The rAAV vector or composition according to claim 9, wherein the AAV capsid shares at least 95%, 98%, or 100% identity with AAV9 (SEQ ID NO: 14). [Claim 11] The composition according to claim 5, wherein the rAAV vector comprises an AAV2, AAV5, AAV8, or AAV9 capsid. [Claim 12] A composition comprising an rAAV vector, wherein the rAAV vector is (a) AAV2, AAV5, AAV8, or AAV9 capsid, and (b) Expression cassette, wherein the expression cassette includes a polynucleotide sequence that shares at least 95% identity with SEQ ID NO: 23 The composition comprising the above. [Claim 13] A pharmaceutical composition comprising the rAAV vector described in claim 1 or the composition described in claim 5 or 12, and a pharmaceutically acceptable carrier. [Claim 14] The composition contains approximately 1 × 10⁶ of the rAAV vector per milliliter. ７ ~Approx. 1×10 １４ 10¹² genome copies, or containing approximately 1 × 10¹² to approximately 6.2 × 10¹² genome copies of the rAAV vector per milliliter, The pharmaceutical composition according to claim 13. [Claim 15] The pharmaceutical composition according to claim 13, wherein the composition is formulated for administration into the lacrimal gland. [Claim 16] The pharmaceutical composition according to claim 13, wherein the composition is formulated for administration onto the surface of the eye. [Claim 17] The pharmaceutical composition according to claim 13, wherein the composition is formulated for use in the treatment of an eye disease, disorder, or symptom. [Claim 18] The pharmaceutical composition according to claim 13 for treating ocular symptoms in a subject requiring treatment of such symptoms. [Claim 19] (a) The pharmaceutical composition is delivered to the target eye or the secretory gland of the target eye, (b) The pharmaceutical composition is delivered to the lacrimal gland of the subject, The pharmaceutical composition according to claim 18. [Claim 20] The pharmaceutical composition according to claim 19, wherein the cells in the lacrimal gland are transduced by the rAAV vector. [Claim 21] (a) The transduced cells in the lacrimal gland express an effective amount of DAO1 in the tear film of the subject and on the ocular surface, (b) The pharmaceutical composition is delivered to the accessory lacrimal gland, (c) Approximately 1 × 10⁹ to approximately 1 × 10¹⁰, approximately 1 × 10¹⁰ to approximately 1 × 10¹, approximately 1 × 10¹ to approximately 1 × 10¹², approximately 1 × 10¹² to approximately 1 × 10¹³, or approximately 1 × 10¹³ to approximately 1 × 10¹⁵ genome copies of the rAAV vector are administered, or (d) The symptoms are ocular symptoms, or (e) The symptoms are associated with increased histamine production or increased histamine signaling, (f) The symptoms described above are vernal keratoconjunctivitis, atopic keratoconjunctivitis, or seasonal or perennial allergic conjunctivitis, or (g) The above symptoms are autoimmune symptoms, or (h) The symptoms are allergic symptoms, and the symptoms are an allergic reaction to the therapeutic agent, or the symptoms are an allergic reaction to a microorganism factor, (i) Administration of the composition results in the expression of DAO1 in the cells of the lacrimal gland or accessory lacrimal gland, (j) Administration of the composition results in the secretion of DAO1 into the tear film, and the secretion of DAO1 into the tear film is stimulated by a cholinergic agonist, (k) Administration of the composition results in improvement of one or more of the symptoms, the symptoms being selected from the group consisting of itching, swelling, lacrimation, and flushing, (l) Administration of the composition results in an improvement of 0.5, 1, 1.5, 2, 2.5, 3, 3.5, or 4 points on the conjunctival pruritus grading scale, or (m) Administration of the composition results in an improvement of 1, 2, 3, or 4 points on the conjunctival redness evaluation grading scale, or (n) The administration of the composition results in an improvement greater than approximately 10% to approximately 20%, approximately 20% to approximately 30%, approximately 30% to approximately 40%, approximately 40% to approximately 50%, approximately 50% to approximately 60%, approximately 60% to approximately 70%, approximately 70% to approximately 80%, approximately 80% to approximately 90%, or approximately 95% in the Schirmer test, or (o) Administration of the composition 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 about 95% in at least one compartment of the corneal staining grading scale, or (p) The improvement is measured approximately 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 9 months, or 12 months after the administration, or (q) The subject is one to be further administered one or more additional therapeutic agents, (r) The subject is a human, The pharmaceutical composition according to claim 20. [Claim 22] A pharmaceutical composition, (a) Polypeptides comprising the diamine oxidase ("DAO1") enzyme or a fragment thereof; and (b) A pharmaceutically acceptable carrier suitable for administration to the eye of human subjects The pharmaceutical composition comprising the above. [Claim 23] The pharmaceutical composition according to claim 22, wherein the polypeptide has an amino acid sequence selected from SEQ ID NOs: 1 and 2 or an amino acid sequence having at least 90% identity with said amino acid sequence. [Claim 24] (a) The pharmaceutically acceptable carrier comprises water; sterile water; pyrogen-free water; phosphate-buffered saline; HEPES-buffered saline; isotonic sodium chloride solution; equilibrium salt solution; wetting agent; surfactant; tonic agent; pH modifier; viscosity modifier; buffering agent; disaccharide; cellulose and / or derivatives thereof; amino acids; or any combination thereof, (b) The polypeptide has an amino acid sequence that has at least 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity with SEQ ID NO: 1 or SEQ ID NO: 2, or (c) The polypeptide contains 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, (d) The formulation is a liquid formulated for application to or into the ocular surface of the eye of the human subject, or for intralacrimal injection. The pharmaceutical composition according to claim 22. [Claim 25] The polypeptide is encoded by polynucleotides contained in the vector, (a) The vector is present in the composition in an amount effective to express the polypeptide in the tear film of the subject at a concentration of 100 pg / mL to 50 μg / mL after administration of the composition to the subject, or (b) The polynucleotide is operably linked to a promoter, (c) The vector is manipulated to constitutively express the polypeptide having an amino acid sequence that has at least 90% identity with the amino acid sequences selected from SEQ ID NOs: 1 and 2, or (d) The vector includes an adenovirus vector or lentiviral vector; plasmid; episome; or artificial chromosome, The pharmaceutical composition according to claim 22. [Claim 26] (a) The polypeptide is present in the pharmaceutical composition at a concentration of 100 pg / mL to 50 μg / mL, or (b) The polypeptide is present in the pharmaceutical composition in an amount of 500 ng to 5 μg, or (c) The polypeptide is present in the pharmaceutical composition in a unit dose. The pharmaceutical composition according to claim 22. [Claim 27] The pharmaceutical composition according to claim 22 for treating an eye disease in a person requiring treatment for an eye disease. [Claim 28] (a) The eye disease is associated with increased histamine production or increased histamine signaling, (b) The eye disease includes or includes inflammatory symptoms of the eye. (c) The eye disease includes autoimmune symptoms, (d) The eye disease includes allergic symptoms, (e) The eye disease includes an allergic reaction to the therapeutic agent or an allergic reaction to a microbiological factor, (f) The eye disease includes vernal keratoconjunctivitis, (g) The eye disease includes atopic keratoconjunctivitis, (h) The eye disease includes seasonal or perennial allergic conjunctivitis, (i) Administration of the pharmaceutical composition results in the expression of functional diamine oxidase in one or more cells of the target lacrimal gland or accessory lacrimal gland, (j) Administration of the pharmaceutical composition results in the secretion of functional diamine oxidase into the tear film of the subject, and the secretion of functional diamine oxidase into the tear film is stimulated by a cholinergic agonist, (k) The administration of the pharmaceutical composition is made on or within the ocular surface of the subject, or into the lacrimal gland of the subject, (l) The symptoms of the eye disease are selected from the group consisting of itching, swelling, tearing, and redness, or (m) Administration of the pharmaceutical composition results in an improvement of 0.5, 1, 1.5, 2, 2.5, 3, 3.5, or 4 points on the conjunctival pruritus grading scale, or (n) The subject is a human subject, The pharmaceutical composition according to claim 27.

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