Aav vector encoding diamine oxidase and uses thereof

Abstract

Provided are vectors (e.g., recombinant adeno-associated virus, "rAAV" vectors), methods of treating a condition (e.g., a condition associated with excess histamine or histamine-induced inflammation), pharmaceutical compositions, and other compositions and methods, in which the vector comprises a polynucleotide encoding a diamine oxidase ("DAO1"). Methods of treatment may include administration to the salivary glands (e.g., a parotid gland) of a subject.

Description

AAV VECTOR ENCODING DIAMINE OXIDASE AND USES THEREOFCROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of the priority date of U.S. Provisional Patent Application No. 63 / 607,089, filed December 6, 2023, the disclosure of which is incorporated by reference herein in its entirety.SEQUENCE LISTING

[0002] This application incorporates by reference in its entirety the Sequence Listing XML file entitled “SequenceListing.xml (71 KB), which was created on December 6, 2023, and filed electronically herewith.BACKGROUND

[0003] Histamine (2-[3H-imidazol-4-yl]ethanamine) is an important chemical mediator that causes vasodilation and increased vascular permeability, and elevations in plasma or tissue histamine levels have been noted during anaphylaxis and experimental allergic responses of the skin, nose, and airways. Histamine also acts on several physiological functions, such as cell differentiation, proliferation, hematopoiesis, and cell regeneration. Synthesis of histamine occurs through decarboxylation of the amino acid histidine by the enzyme L-histidine decarboxylase (HDC), which is expressed in neurons, parietal cells, gastric mucosal cells, mast cells, and basophils; whereas degradation of histamine is mediated by the enzyme diamine oxidase (DAO) and histamine N-methyltransferase (HNMT), which each catalyze histamine deamination.

[0004] Histamine is associated with various medical conditions, including allergic rhinitis, urticaria, anaphylaxis, and to a lesser degree, asthma. Prior research efforts have led to the development of various antihistamine compounds capable of interfering with the binding of histamine to its receptors (e.g., the Hl receptor). However, these drugs provide short-term relief and, in many cases, produce undesired side effects.BRIEF SUMMARY

[0005] In view of the shortcomings of current medical treatment options for histamine-related diseases, disorders, and conditions, there exists a need for improved methods for delivering antihistamine therapy to a subject, e.g., without the need for repeated self-administration (e.g., ofa daily pill). Self-administration can result in poor patient compliance. Moreover, pulsed administration of antihistamine therapy may only provide temporary or “episodic” relief and may not be suitable for chronic conditions. 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 illustrated by the accompanying figures.

[0006] In some aspects, the disclosure provides a vector, for example an adeno-associated virus (“AAV”) vector, adenoviral vector, or lentiviral vector, or a plasmid DNA for expression of diamine oxidase (“DAO1”; alternatively abbreviated as “DAO” herein) in cells of one or more salivary glands of a subject following in vivo administration.

[0007] In one aspect, the disclosure provides a recombinant adeno-associated viral (rAAV) vector comprising an AAV capsid and an expression cassette, the expression cassette comprising a polynucleotide encoding diamine oxidase, also known as DAO1, DAO or histaminase, operatively linked to a promoter. In some embodiments, the polynucleotide encodes a protein that is at least 95% identical to SEQ ID NOs: 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.

[0008] In another aspect, the disclosure provides a composition comprising an rAAV vector, wherein the rAAV vector comprises: (a) an AAV capsid, and (b) an expression cassette, wherein the expression cassette comprises a polynucleotide comprising a nucleotide sequence sharing at least 95% identity to a nucleotide sequence selected from SEQ ID NOs: 29-31, and wherein the polynucleotide is linked to a promoter.

[0009] In some embodiments, the promoter is a CMV promoter. In some embodiments, the promoter is a promoter of a gene encoding a human parotid secretory protein (PSP), a mucin protein, an amylase (e.g., AMY1C), a kallikrein (e.g., KLK1), a salivary sialoperoxidase, a salivary myeloperoxidase, a P-defensin, a lingual lipase, or a lysozyme protein. 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 (“poly A”) sequence. In some embodiments, the polyA sequence is a BGH polyA sequence. In some embodiments, the expression cassette comprises a Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (“WPRE”). In some embodiments, the expression cassettecomprises a Kozak sequence. In some embodiments, the expression cassette is flanked by two inverted terminal repeats (“ITRs”). In some embodiments, the ITRs are AAV2 ITRs.

[0010] In some embodiments, the expression cassette comprises a nucleotide sequence that shares at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 23.

[0011] 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).

[0012] In yet another aspect, the 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 comprising a nucleotide sequence sharing at least 95% identity to a nucleotide sequence selected from SEQ ID NOs: 29-31, and wherein the polynucleotide is linked to a promoter.

[0013] In some embodiments, the expression cassette comprises a polynucleotide sequence sharing at least 95% identity to SEQ ID NO: 23.

[0014] In some embodiments, the AAV capsid is AAV2. In some embodiments, the AAV capsid is AAV5. In some embodiments, the AAV capsid is AAV9.

[0015] In one aspect, the disclosure provides a pharmaceutical composition comprising a vector or plasmid as described herein (e.g., an rAAV vector) or a protein described herein (e.g., a polypeptide comprising DAO1 or a functional fragment or variant thereof), and a pharmaceutically acceptable carrier.

[0016] In some embodiments, the pharmaceutical composition comprises an rAAV vector (e.g., configured to express DAO1 or a functional fragment or variant thereof), in an amount of about 1 x 105to about 1 x 1016genome copies per milliliter of the rAAV vector. In some embodiments, the pharmaceutical composition comprises about 1 x 1012to about 6.2 x 1012genome copies per milliliter of the rAAV vector. In some embodiments, the pharmaceutical composition comprises a plasmid vector configured to express DAO1 or a functional fragment or variant thereof, in an amount of about 1 x 105to about 1 x 1016genome copies per milliliter of the plasmid vector. Insome embodiments, the pharmaceutical composition comprises about 1 x 1012to about 6.2 x 1012genome copies per milliliter of the plasmid vector.

[0017] In some embodiments, a pharmaceutical composition may comprise an rAAV vector (or any other vector described herein, such as a plasmid configured to express DAO1) in an amount or at a concentration sufficient to 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 pg / mL of DAO1, or a concentration within a range defined by any pair of the foregoing values, in saliva of a subject. Expression may be measured, e.g., 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 of time, such as after 1, 2, 3, 4, or 5 days.

[0018] In some embodiments, a pharmaceutical composition may comprise 0.25, 0.50, 0.75, 1.00, 1.25, 1.50, 1.75, 2.00, 2.25, 2.50, 2.75, 3.00, 3.25, 3.50, 3.75, 4.00, 4.25, 4.50, 4.75, 5.00, 5.25, 5.50, 5.75, or 6.00 pg of a polypeptide comprising DAO1 (or a functional variant thereof), or an amount within a range defined by any pair of the foregoing values. In some embodiments, the pharmaceutical composition may further comprise one or more excipients, carriers, and / or diluents suitable for administration to an oral cavity or a surface thereof, or to a salivary gland (e.g., a parotid gland) of a human or animal subject.

[0019] In some embodiments, the pharmaceutical composition is formulated for administration of the vector or plasmid into cells of a salivary gland. In some embodiments, the pharmaceutical composition is formulated for administration onto an oral cavity surface (e.g., as a gel capable of being absorbed by a mucosal surface within the oral cavity such that the vector or plasmid enters cells of one or more salivary glands). In some embodiments, the oral cavity surface is a surface located on top of, or adjacent to, a salivary gland. In some embodiments, the pharmaceutical composition is formulated for use, or adaptable for use, in the treatment of a disease, disorder, or condition associated with excess histamine or histamine-induced inflammation.

[0020] In some aspects, the disclosure provides a method for treating a histamine-related disease, disorder, or condition in a subject in need thereof, the method comprising administering an effective amount of a pharmaceutical composition comprising a vector or plasmid described herein to the subject for the vector or plasmid to transfect or transduce cells of a salivary gland in the subject. In some embodiments, the pharmaceutical composition is administered into an oral cavity or on a surface thereof, of the subject. In some embodiments, the pharmaceuticalcomposition is injected into a salivary gland of the subject. In some embodiments the injection is a direct injection. In other embodiments, the injection is a multiple tine injection. Other methods of administering a pharmaceutical composition comprising a vector or plasmid as described herein to the subject include, but are not limited to: a “gene gun” delivery means (e.g., gold particles coated with the vector propelled through the subject’s inner cheek via pressurized helium); application of an oral patch comprising a sustained-release pellet comprising the composition; a pharmaceutical system comprising a permeabilizer and the vector; subcutaneous placement of an implant comprising an erodible substance, such as polylactic acid (“PLA”), and the vector; by oral spray; by a temperature-shifting gel spray, or by direct topical application.

[0021] In some embodiments, the pharmaceutical composition is delivered to a main salivary gland (e.g., to a parotid gland, a sublingual gland, and / or a submandibular gland) of the subject. In some embodiments, cells within the salivary gland are transduced by the rAAV vector. In some embodiments, acinar cells within a salivary gland are transduced or transfected by the vector or plasmid. In some embodiments, myoepithelial cells within a salivary gland are transduced or transfected by the vector or plasmid. In some embodiments, stem cells within a salivary gland are transduced or transfected by the vector or plasmid. In some embodiments, the transduced or transfected cells within the salivary gland express an effective amount of DAO1 into the subject’s saliva. In some embodiments, the pharmaceutical composition is delivered to one or more minor salivary glands (e.g., located throughout the mouth).

[0022] In some embodiments, about 1 x 109to about 1 x IO10, about 1 x IO10to about 1 x 1011, about 1 x 1011to about 1 x 1012, about 1 x 1012to about 1 x 1013, or about 1 x 1013to about 1 x 1015genome copies of the rAAV vector are administered.

[0023] In some embodiments, the condition is a condition associated with increased histamine production and / or increased histamine signaling. In some embodiments, the condition is an inflammatory condition. In some embodiments, the condition is an autoimmune condition. In some embodiments, the condition is an allergic condition. In some embodiments, the condition is an allergic reaction to a therapeutic agent. In some embodiments, the condition is an allergic reaction to a microbial infection. In some embodiments, the condition is an allergen-induced anaphylaxis or a system allergic response (comprising, e.g., urticaria, respiratory distress, edema,pruritus, or any combination thereof) from food allergies, for example a tree nut or a shellfish allergy.

[0024] In some embodiments, the administration results in expression of DAO1 in cells of the salivary gland and / or an excretory duct thereof. In some embodiments, the administration results in secretion of DAO 1 into the subject’s saliva. In some embodiments, secretion of DAO 1 into the subject’s saliva is stimulated by administration of an electrical stimulus, mechanical stimulus, ultrasound stimulus, and / or a drug. In some embodiments, the drug that stimulates secretion of DAO1 into the saliva 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 mucoprotective agent (e.g., diquafosol, rebamipide, or ecabet). In some embodiments, the drug that stimulates secretion of DAO 1 into the saliva is administered orally. In some embodiments, secretion of DAO1 into the saliva is stimulated by a drug, such as a cholinergic agonist or nAChR agonist, administered into the nasal cavity. In some aspects, DAO1, or an rAAV vector, plasmid, or any other construct configured to express DAO1 described herein, may be administered to a subject as a co-therapy in combination with a tear-increasing stimulus and / or drug (e.g., those described in this paragraph). In some aspects, such co-therapies may follow any protocol, or use any components or parameters, described in International Patent Application Pub. No. WO 2022 / 235786, the entire contents of which is incorporated herein.

[0025] In some embodiments, the administration results in an improvement of one or more symptoms of the histamine-associated condition, disease, or disorder.

[0026] 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 the 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 the administration.

[0027] In some embodiments, the method further comprises administering one or more additional therapeutic agents. In some embodiments, the additional therapeutic agent is an agentthat increases tear production. In some embodiments, the additional therapeutic agent that increases tear production is a cholinergic agonist. In some embodiments, the subject is human.

[0028] In some embodiments, the disclosure provides DAO1, an rAAV or other vector configured to express DAO1, a composition, or a pharmaceutical composition described herein for use in a method of treating a condition in a subject in need thereof comprising administering an effective amount of the pharmaceutical composition to a salivary gland, or an oral cavity or a surface thereof, of the subject.

[0029] In some embodiments, the disclosure provides DAO1, an rAAV or other vector configured to express DAO1, a composition, or a pharmaceutical composition described herein for use in the manufacture of a medicament for treating a condition in a subject in need thereof.

[0030] In some embodiments, the disclosure provides a kit comprising DAO1, an rAAV or other vector configured to express DAO1, or a 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 a salivary gland, or to an oral cavity or a surface thereof, of the subject.

[0031] In some embodiments, the disclosure provides a kit comprising DAO1, an rAAV or other vector configured to express DAO1, or a 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 a salivary gland, or to an oral cavity or a surface thereof, of the subject.

[0032] In some embodiments, the disclosure provides a kit comprising DAO1, an rAAV or other vector configured to express DAO1, or a composition described herein, and a pharmaceutically acceptable carrier, and instructions for use in treating an autoimmune condition in a subject. In some embodiments, the instructions comprise administering the pharmaceutical composition to a salivary gland, or to an oral cavity or a surface thereof, of the subject.

[0033] In some embodiments, the disclosure provides a kit comprising DAO1, an rAAV or other vector configured to express DAO1, or a composition described herein, and a pharmaceutically acceptable carrier, and instructions for use in treating an allergy condition in a subject. In someembodiments, the instructions comprise administering the pharmaceutical composition to a salivary gland, or to an oral cavity or a surface thereof, of the subject.BRIEF DESCRIPTION OF THE DRAWINGS

[0034] FIG. 1A shows a vector map of an rAAV expression cassette with inverted terminal repeats (“ITRs”), a promoter, and DAO1 polynucleotide elements.

[0035] FIG. IB provides a schematic depicting the elements positioned between the ITRs of the rAAV expression cassette, including from 5' to 3': a cytomegalovirus (“CMV”) enhancer / promoter, an N-terminal secretion signal, a nucleotide sequence encoding DAO1, a woodchuck hepatitis virus post-translational regulatory element (“WPRE”), and polyadenylation (“pA”) signal.

[0036] FIG. 2 provides images obtained by confocal fluorescence microscopy of 293T cells transfected with an AAV plasmid encoding DAO1 at 20X magnification (top row) or 63X magnification (middle row) as compared to non-transfected control 293T cells at 20X magnification (bottom row). The left-most column shows detection of fluorescent signal for an AF488-labeled secondary antibody used to detect labeling with a primary anti -D AO 1 antibody, the middle column shows fluorescence signal for nuclear staining using DAPI, and the right-most column shows an overlay of the AF488 and DAPI fluorescent signals.

[0037] FIG. 3A shows an image of a Western blot of whole cell lysates harvested from 293 T cells at 48 hours following transient transfection with AAV.DAO1 (lane labeled “AAV-DAO1”) as compared to control non-transfected 293T cells (lane labeled “293T”), with detection of DAO1 using a rabbit anti -D AO 1 antibody. An arrow indicates the protein band corresponding to the DAO1 polypeptide.

[0038] FIG. 3B shows an image of a Western blot of recombinant human DAO1 polypeptide with detection of DAO 1 using a mouse anti -his antibody.

[0039] FIGs. 4A-4B show data generated using a colorimetric functional activity assay to measure DAO1 substrate cleavage. DAO1 cleaves a first substrate to yield H2O2 (hydrogen peroxide) that reacts with a second substrate to signal that is detected using fluorescence spectroscopy (excitation wavelength of 535 nm and emission wavelength of 587 nm). FIG. 4A shows a calibration curve generated by measuring fluorescence signal of the second substrate as a function of concentration of H2O2. FIG. 4B shows a graph quantifying H2O2 generated by activityof DA01 present in media or cell lysate of 293T cells transiently transfected with AAV plasmid encoding DA01 as compared to activity of media of non-transfected control cells.

[0040] FIG. 5 shows a schematic diagram depicting the elements between the ITRs of an AAV plasmid. The plasmid encodes EGFP linked to a secretion signal at its N-terminus (“secEGFP”) under control of a CMV promoter. The woodchuck hepatitis virus post-translational regulatory element (WPRE) serves to increase transgene expression and is proximal to the bovine growth hormone poly adenylation (pA) signal.

[0041] FIGs. 6A-6K are images of lacrimal tissue stained with anti-eGFP antibody. Lacrimal glands were dosed with rAAV vectors containing an expression cassette with eGFP transgene by intralacrimal injection. Lacrimal tissue was stained with anti-eGFP antibody to assess eGFP expression. Black arrows indicate staining showing eGFP expression.

[0042] FIG. 7 provides images of porcine lacrimal gland that received an injection of AAV- secEGFP (AAV2 or AAV9 serotype) and was harvested on Day 103 and fixed in paraffin. IHC of 5 pM sections was performed using anti-GFP antibody and DAPI (nuclear) counterstain. Images were captured using a confocal microscope at 100X magnification. Negative control animals received no injection.

[0043] FIG. 8 provides an image of porcine lacrimal gland that received an injection of AAV9- secEGFP and was harvested on Day 103 and fixed in paraffin. IHC of 5 pM sections was performed using anti-GFP antibody and DAPI (nuclear) counterstain. In addition to lacrimal gland acinar cells, ductile epithelial cells appear to be transduced by AAV9 (white arrows).

[0044] FIG. 9 provides an image of porcine lacrimal gland that received an injection of AAV was harvested at Day 103 and fixed in paraffin. H&E staining of 5 pM paraffin sections at lOOx reveal no inflammatory infiltrate, macro, or micro abnormalities.

[0045] FIG. 10 provides a schematic of a treatment schedule for administering AAV encoding a model protein via injection into the lacrimal gland of pigs in combination with administration of OC-01 (Varenicline) by nasal spray. Time points for tear collection and termination of the study are indicated.

[0046] FIG. 11 shows a calibration curve generated by measuring fluorescence signal of the second substrate as a function of concentration of histamine. This calibration curve was used to evaluate the histamine degradation kinetics of recombinant porcine diamine oxidase (FIGs. 12-13) and histamine degradation by conditioned medium obtained from non-transfected control 293T cells and 293T cells transfected with a plasmid engineered to express DA01 (FIGs. 14-15).

[0047] FIGs. 12-13 provide graphs showing the histamine degradation kinetics of recombinant porcine diamine oxidase at 4 (FIG. 12) and 24 hours (FIG. 13).

[0048] FIGs. 14-15 provide graphs showing histamine degradation by conditioned medium obtained from non-transfected control 293T cells and 293T cells transfected with a plasmid engineered to express DAO1, at 4 (FIG. 14) and 24 hours (FIG. 15).

[0049] FIG. 16 shows the 10% SDS-PAGE gel utilized to assess the purity of the AAV9-DAO test article in Example 6.

[0050] FIG. 17 is a graph showing parotid gland expression levels of DAO following injection with the AAV9-DAO test article in Example 7.DETAILED DESCRIPTION

[0051] The present disclosure provides methods of treating a histamine-associated condition, disease, or disorder in a subject in need thereof. Such methods comprise, e.g., transduction or transfection of cells of one or more salivary glands (sublingual, submandibular, and / or parotid) with a construct (vector or plasmid) encoding a diamine oxidase gene product, followed by the expression of diamine oxidase (DAO1) and subsequent secretion of DAO1 from a salivary gland of the subject, which may then be swallowed by the subject, thereby entering the esophagus and digestive system. Some of the DAO1 will be inactivated or degraded due to digestive processes, however a portion remains active and is thus capable of providing a therapeutic intervention. In healthy subjects, the primary function of DAO 1 is to break down excess histamine in the body. DAO1 is scarcely present in the blood circulation of humans, although it has been shown to increase substantially, e.g., 1000-fold, in pregnant women during the course of pregnancy, suggesting a protective mechanism against adverse histamine. It has also been shown that in subjects with histamine intolerance and / or experiencing ocular conjunctivitis, there is a disequilibrium between accumulated histamine and histamine degradation. Following a variety of noxious stimuli, excess histamine can be released from basophil granules and mast cells, thus creating tissue edema and starting a cascade of events leading to inflammation and allergic response.

[0052] Administration of DAO 1 according to the present methods may be used to treat various histamine-associated conditions and diseases, including histamine intolerance syndrome (HITS),eosinophilic esophagitis, inflammatory bowel disease, and migraines associated with excessive histamine (e.g., migraines induced by the menstrual cycle whereby alterations in estrogen levels increase histamine and histamine signaling, or food-triggered migraines due to direct ingestion of histamine contained in food stuffs (e.g., tuna) or in alcoholic beverages).

[0053] Estrogen stimulates mast cell and basophil degranulation, resulting in increased histamine release and histamine local levels, as mast cells and basophils contain the highest levels of histamine with respect to cell types in humans. Moreover, estrogen downregulates DAO expression. Administration of a DA01 polypeptide according to the present methods may be used to treat adverse conditions associated with elevated estrogen levels in a subject.

[0054] Administration of DA01 according to the present methods may also be used as a prophylactic intervention for allergen-induced anaphylaxis and systemic allergic responses (urticaria, respiratory distress, edema, pruritus, etc.) from food allergies (e.g., tree nut or shellfish allergies). In some aspects, prophylactic administration may be applied to pediatric patients with known and / or unknown allergic reactions to food or excessive histamine in food (e.g., tuna). Additionally, allergic airway disease may be diminished through constitutive and accumulating systemic levels of DA01 breaking down excess histamine rendering it non-functional.

[0055] As disclosed herein, AAV-based delivery of transgenes to the salivary glands provides a method to treat histamine-associated disorders. rAAV vectors have distinct advantages for transgene delivery. rAAV vectors may be maintained for a long period of time in transduced cell, allowing potential long-term expression of the transgene product. In addition, rAAV vectors may be less immunogenic as compared to other viral delivery vectors, such as adenovirus.

[0056] AAV serotypes used in AAV-based delivery of transgenes include AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, and AAV9 (Lebherz et al. J Gene Med.; 10(4): 375-382 (2008)). AAV serotypes used in AAV-based delivery of a transgene to the lacrimal gland include AAV2, AAV4, AAV5, AAV5w8, AAV x5, AAV 9, AAV 12, and BAAV (Rocha et al., Invest Ophthalmol Vis Sci. 52.956'1-95'12 (2011)).

[0057] In some embodiments, an rAAV vector is provided for the expression of DA01. In some embodiments, cells of the at least one salivary gland are transduced with the rAAV vector of the disclosure. The salivary glands produce and excrete saliva as well as various proteins contained therein, into the oral cavity of mammals. The main salivary glands include the parotid, submandibular, and sublingual glands. These glands are comprised of acinar cells, ductal cells,and / or myoepithelial cells. The main salivary glands secrete saliva as well as mucins into the oral cavity.Expression Cassettes

[0058] The rAAV vectors of the disclosure comprise 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, e.g., promoters, regulatory elements (e.g., one or more promoters), translation initiation sequences, coding sequences, and termination sequences (FIGs. 1A and IB).

[0059] Additionally, the DAO1 protein encoded by the polynucleotide sequence in the expression cassette may include a signal peptide (e.g., an IgK leader sequence) to induce secretion out of a cell following expression.

[0060] In mammals, histamine is metabolized by oxidative deamination via DAO1 (also called histaminase, amiloride-binding protein, amine oxidase copper containing 1, AOC1) (see, e.g., Elmore, et al (2002) J Biol Inorg Chem 7:565; Schwelberger, et al (2018) Inflamm Res 67:245; Finney et al (2014) Arch Biochem Biophys 546: 19). The enzyme is a homodimer that catalyzes oxidative deamination of the primary amine group of histamine.

[0061] Histamine intolerance is the build-up of excessive levels of histamine due to decreased catabolism and / or increased production. Vakal et al., Molecules 2020, 25, 1293. Histamine is released during anaphylaxis and mast cell degranulation, and it has been suggested that mast cell degranulation increases DAO1 release and DAO1 is increased in severe anaphylaxis in mastocytosis patients (Boehm et al. Allergy. 2019, 74:58). Moreover, several therapeutic agents have been shown to inhibit DAO1, including, for example, berenil, pentamidine, aminoguanidine, metformin (Vakal 2020).

[0062] Excess histamine causes a range of symptoms (see, e.g., Schnedl et al., Food Sci Biotechnol (2019) 28(6): 1779-1784). Histamine is a pro-inflammatory mediator contributing to allergic reactions (see, e.g., White MV (1990), J Allergy Clin Immunol 86: 599-605) and may contribute to Vernal Keratoconjunctivitis (VKC) (see, e.g., Abelson et al., Opthamology 1995,102(12): 1958-1963; Bonini et al., (1992), Journal of allergy and clinical immunology, 89(1), 103- 107).

[0063] Accordingly, the present disclosure provides expression cassettes comprising a polynucleotide sequence that encodes DAO1 for use in a therapy that induces breakdown of histamine (e.g., histamine present in the tear fluid). In some embodiments, the expression cassettes are used in a method of treating a condition (e.g., an ocular condition) associated with increased histamine production.DAO1 Enzyme

[0064] In some embodiments, the expression cassette of the present disclosure comprises a polynucleotide sequence encoding a DAO 1 enzyme or a functional variant thereof. As used herein, the term “DAO1 enzyme” refers to a DAO1 enzyme from any species. The term “functional variant” refers to variants having sequence substitutions, insertions, deletions, and / or N- or C- terminal truncations, where the functional variant retains one or more functions of the reference protein, e.g., a native DAO1 enzyme. DAO1 is a 200-kDa homomeric glycoprotein that catalyzes oxidative deamination of the primary amino group of histamine.

[0065] 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 nomenclature, chromosomal localization, gene products, attributes of the gene, associated markers, phenotypes, interactions, links to citations, sequence information, information regarding sequence variants, gene maps, expression reports, homologs, protein domain content, and access to external databases. As is understood by the skilled artisan, sequence information for human DAO1 isoforms known in the art may be identified by entering the appropriate accession number into the NCBI Gene Database and selecting sequence information in the desired computer- readable format (e.g., FASTA). Such sequence information may include, but is not limited to, the nucleotide sequence for 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 in the NCBI genedatabase via the accession number NM_001272072.2 and has the amino acid sequence set forth in SEQ ID NO: 2. In another example, one isoform of human DA01 is identifiable in the NCBI gene database via the accession number NM 001091 and consists of the amino acid sequence set forth by SEQ ID NO: 2.

[0066] Exemplary amino acid sequences of human DAO1 and exemplary polynucleotide sequences (mRNA and ORF) encoding human DAO1 are set forth in Table 1. In some embodiments, the DAO1 enzyme comprises or consists of an amino acid sequence set forth in Table 1 In some embodiments, the DAO1 enzyme comprises or consists of an amino acid sequence encoded by an mRNA sequence set forth in Table 1. In some embodiments, the DAO1 enzyme comprises or consists of an amino acid sequence encoded by an ORF set forth in Table 1

[0067] 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.

[0068] 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 without limitation the 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 that encodes one or more segments of SEQ ID NO: 1 (e.g., the 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 without limitation the 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 that encodes one or moresegments of SEQ ID NO: 2 (e.g., the segments spanning positions 39-125, 141-241, and / or 300- 708 of SEQ ID NO: 2).

[0069] In some embodiments, the DAO1 enzyme comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to one or more segments of SEQ ID NO: 1 or SEQ ID NO: 2, wherein each segment comprises at least 20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, or 500 contiguous amino acids of SEQ ID NO: 1 or SEQ ID NO: 2, segment comprising a length within a range defined by any pair of the foregoing values. For example, a functional DAO1 enzyme may share high sequence identity (e.g., 95%) with one or more predicted functional domains (e.g., positions 39-125, 141-241, and / or 300-727 of SEQ ID NO: 1; or 39-125, 141-241, and / or 300-708 of SEQ ID NO: 2) while having greater sequence divergence in other portions of the DAO1 enzyme. Similarly, in some embodiments, a functional DAO1 enzyme may have at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a segment spanning from position 20 to the final position of either SEQ ID NO: 1 or SEQ ID NO: 2 (e.g., omitting a predicted N-terminal signal peptide shared by both sequences).

[0070] 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.

[0071] 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, theDA01 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.Polynucleotides Encoding a DAO1 Enzyme

[0072] In some embodiments, the expression cassette comprises a polynucleotide encoding a DAO1 enzyme described herein (e.g., a human DAO1 enzyme). In some embodiments, the polynucleotide comprises an ORF encoding a DAO1 enzyme described herein (e.g., a human DAO1 enzyme).

[0073] In some embodiments, the expression cassette provides increased expression of DAO 1 in the at least one salivary gland. In some embodiments, expression of DAO 1 may be increased 5%, 10%, 15%, 20%, or 25% compared to expression of DAO 1 in an untreated subject. As used herein, “subject” means any mammal, including mice, rabbits, non-human primates (NHP) and humans. In some embodiments, the subject is a human or NHP. Moreover, “individual” or “patient” may be used interchangeably with “subject.” In some embodiments, expression of DAO1 may be increased by at least 1.5-fold, 2-fold, 3-fold, 4-fold, or 5-fold compared to expression of DAO 1 in an untreated subject. In some embodiments, DAO1 may be expressed at any detectable level in the treated salivary gland or in the oral cavity of a treated subject, whereas DAO1 may not be expressed, or expressed at undetectable levels, in a salivary gland or an oral cavity of an untreated subject. Put another way, the salivary gland to which an rAAV vector described herein is administered may express DAO1 in higher abundance than in a salivary gland that has only endogenous (ie., native) expression of DAO 1 or a salivary gland that have a lower or impaired secretion of endogenous (z.e., native) DAO1.

[0074] The term “functional variant” refers to variants having sequence substitutions, insertions, deletions, and / or N- or C-terminal truncations, where the functional variant retains one or more functions of the reference protein, c.g, native DAO1.

[0075] In some embodiments, the expression cassette comprises a polynucleotide encoding a protein that is human DAO 1 or a functional variant thereof. In some embodiments, the expression cassette comprises a polynucleotide comprising a nucleotide sequence encoding an amino acidsequence identified in Table 1. In some embodiments, the expression cassette comprises a polynucleotide comprising a nucleotide sequence identified in Table 1.

[0076] In some embodiments, the expression cassette comprises a polynucleotide comprising a nucleotide sequence encoding a protein having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the expression cassette comprises a polynucleotide comprising a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the expression cassette comprises a polynucleotide comprising a nucleotide sequence encoding a protein having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 2. In some embodiments, the expression cassette comprises a polynucleotide comprising a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 2.

[0077] 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.

[0078] In some embodiments, the expression cassette comprises a polynucleotide encoding DAO1 comprising a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 30. In some embodiments, the expression cassette comprises a polynucleotide encoding DAO1 comprising or consisting of the nucleotide sequence 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 that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 31. In some embodiments, the expression cassette comprises a polynucleotide comprising or consistingof the nucleotide sequence set forth in SEQ ID NO: 31. In some embodiments, the protein is human DA01 or a functional variant thereof.Codon Optimization

[0079] In some embodiments, the expression cassette comprises a polynucleotide encoding a DAO1 enzyme, wherein the polynucleotide comprises or consists of a nucleotide sequence codon- optimized for expression in a target cell. In some embodiments, the target cell is a mammalian cell. In some embodiments, the target cell is a human cell, a murine cell, or a non-human primate (NHP) cell.

[0080] A codon-optimized nucleotide sequence, e.g., a codon-optimized nucleotide sequence encoding a DAO 1 enzyme, typically is a sequence comprising at least one synonymous nucleobase substitution with respect to a reference sequence (e.g., a wild-type ORF encoding a DAO1 enzyme). A codon-optimized nucleotide sequence can be partially or completely different in sequence from the reference sequence. For example, a reference sequence encoding poly serine uniformly encoded by TCT codons can be sequence-optimized by having 100% of its nucleobases substituted (for each codon, T in position 1 replaced by A, C in position 2 replaced by G, and T in position 3 replaced by C) to yield a sequence encoding polyserine which would be uniformly encoded by AGC codons. The percentage of sequence identity obtained from a global pairwise alignment between the reference polyserine nucleic acid sequence and the sequence-optimized polyserine nucleic acid sequence would be 0%. However, the protein products from both sequences would be 100% identical.

[0081] Codon optimization methods are known in the art and can be useful to achieve one or more desired results, e.g., to increase expression of a synthetic gene in a target cell. In some embodiments, an expression cassette comprises a nucleotide sequence that is sequence-optimized relative to a reference sequence using a method of sequence optimization. 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).

[0082] In some embodiments, an expression cassette of the 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 comprises a codon optimization algorithm described in US 7,561,972; US 7,561,973; US 8,126,653; and US 8,401,798, each of which isincorporated herein by reference. In some embodiments, the polynucleotide sequence is sequence- optimized based on codon usage bias in a host cell (e.g., mammalian cell, e.g., human cell, murine cell, 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.

[0083] In some embodiments, the disclosure provides an expression cassette comprising a polynucleotide comprising or consisting of a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 29. In some embodiments, the polynucleotide comprises or consists of SEQ ID NO: 29.Table 1: Human DAO1 Amino Acid and Polynucleotide SequencesExpression Cassette Elements

[0084] In some embodiments, the expression cassette of the present disclosure comprises a promoter. The term “promoter” as used herein refers to a DNA sequence that directs the binding of RNA polymerase and thereby promotes RNA synthesis, z.e., a minimal sequence sufficient to direct transcription. Promoters and corresponding protein or polypeptide expression may be ubiquitous, meaning strongly active in a wide range of cells, tissues and species or cell-type specific, tissue-specific, or species specific. Promoters may be “constitutive,” meaning continually active, or “inducible,” meaning the promoter can be activated or deactivated by the presence or absence of biotic or abiotic factors. Also included in the nucleic acid constructs or vectors of the invention are enhancer sequences that may or may not be contiguous with the promoter sequence.Enhancer sequences influence promoter-dependent gene expression and may be located in the 5' or 3' regions of the native gene.

[0085] Any suitable promoter region or promoter sequence therein can be used in the subject polynucleotide cassettes, so long as the promoter region promotes expression of a polynucleotide sequence encoding a DAO1 enzyme in the at least one salivary gland of a subject. In some embodiments, the promoter promotes expression of the gene in a mammalian oral cavity or in a mammalian salivary gland. In some embodiments, the expression cassette comprises a cell-type specific promoter. The promoter may specifically promote transcription in the cells of the salivary gland and / or the cells of excretory duct associated therewith.

[0086] In some embodiments, the promoter is a CAG promoter. In some embodiments, the promoter comprises a nucleotide sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity with the nucleotide sequence set forth in SEQ ID NO: 5. In some embodiments, the promoter comprises SEQ ID NO: 5.

[0087] In some embodiments, the promoter is a CMV promoter. In some embodiments, the promoter comprises a nucleotide sequence having at least 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 21. In some embodiments, the promoter comprises or consists of SEQ ID NO: 21.

[0088] In some embodiments, the promoter is an endogenous promoter of a human parotid secretory protein (PSP), amylase (AMY1C), kallikrein (KLK1), salivary sialoperoxidase, salivary myeloperoxidase, P-defensin, lingual lipase, or lysozyme protein, or a variant comprising a truncated or mutant form of any such promoter.

[0089] The expression cassette may contain a polyadenylation (polyA) sequence. In some embodiments, the expression cassettes described herein comprise a transcription termination signal. Elements directing the efficient termination and polyadenylation of the heterologous nucleic acid transcripts increases heterologous gene expression. Transcription termination signals are generally found downstream of the polyadenylation signal. In some embodiments, vectors comprise a polyadenylation sequence 3' of a polynucleotide encoding a polypeptide to be expressed. The term “polyA site” or “polyA sequence” as used herein denotes a DNA sequence which directs both the termination and polyadenylation of the nascent RNA transcript by RNA polymerase II. Polyadenylation sequences can promote mRNA stability by addition of a polyA tail to the 3' end of the coding sequence and thus, contribute to increased translational efficiency. Cleavage and polyadenylation is directed by a poly(A) sequence in the RNA. The core poly(A) sequence for mammalian pre-mRNAs has two recognition elements flanking a cleavage-polyadenylation site. Typically, an almost invariant AAUAAA hexamer lies 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 coupled 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, ATT AAA, AGTAAA). In particular embodiments, the poly(A) sequence is an SV40 polyA sequence, a bovine growth hormone polyA sequence (BGH polyA), a rabbit P-globin polyA sequence (rPgpA), variants thereof, or another suitable heterologous or endogenous polyA sequence known in the art. In some embodiments, the expression cassette described herein comprises a polyA sequence. In some embodiments, the polyA sequence is a 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 with SEQ ID NO: 18. In some embodiments, the BGH polyA sequence comprises or consists of SEQ ID NO: 18.

[0090] Optionally the rAAV vectors of the disclosure comprise the Woodchuck Post- transcriptional Regulatory Element (WPRE). In some embodiments, the rAAV vector comprises a WPRE comprising SEQ ID NO: 22.Recombinant AAV Vector

[0091] In some embodiments of the present disclosure, the subject expression cassettes are used to deliver DAO1 or a functional variant thereof to at least one salivary gland of a subject, e.g., to treat a histamine-associated disorder. Accordingly, in some embodiments of the disclosure, the composition that provides for the expression of DAO 1 or a functional variant thereof in at least one salivary gland of a subject is a gene delivery vector, wherein the gene delivery vector comprises an expression cassette described herein.

[0092] In some embodiments, the gene delivery vector is an rAAV vector. In such embodiments, the expression cassette is flanked on the 5’ and 3’ ends by functional AAV inverted terminal repeat (ITR) sequences. By “functional AAV ITR sequences” is meant that the ITR sequences function as intended for the rescue, replication and packaging of the AAV vector. Hence, 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 the insertion, deletion or substitution of nucleotides 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 AAV 1. In some embodiments, the AAV ITR is derived from AAV 10. Certain rAAV vectors have the wild-type REP and CAP genes deleted in whole or part, but retain functional flanking ITR sequences. In some embodiments, the AAV ITR is one identified in Table 2.Table 2. Exemplary ITR Sequences

[0093] In such embodiments, the rAAV vector comprises an AAV capsid, derived from any adeno-associated virus serotype known in the art, or prospectively discovered, including without limitation, 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 comprises an AAV capsid derived from AAV2. In some embodiments, the rAAV vector comprises an AAV capsid derived from AAV3. In some embodiments, the rAAV vector comprises an AAV capsid derived from AAV4. In some embodiments, the rAAV vector comprises an AAV capsid derived from AAV5. In some embodiments, the rAAV vector comprises an AAV capsid derived from AAV6. In some embodiments, the rAAV vector comprises an AAV capsid derived from AAV7. In some embodiments, the rAAV vectorcomprises an AAV capsid derived from AAV8. In some embodiments, the rAAV vector comprises an AAV capsid derived from AAV9. In some embodiments, the rAAV vector comprises an AAV capsid derived from AAV10. AAV capsids of particular interest include AAV2, AAV5, AAV8, and AAV9. However, as with the ITRs, the capsid need not have a wildtype nucleotide sequence, but rather may be altered by the insertion, deletion or substitution of nucleotides in the VP1, VP2 or VP3 sequence, so long as the capsid is able to transduce cells of a salivary gland (e.g., a human salivary gland). Put another way, 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 ITRs 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 / l, rAAV2 / 3, rAAV2 / 4, rAAV2 / 5, rAAV2 / 6, rAAV2 / 7, rAAV2 / 8, rAAV2 / 9, etc. In some embodiments, the rAAV is rAAV2 / l. 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.

[0094] In some embodiments, the rAAV is replication defective, in that the rAAV vector cannot independently further replicate and package its genome. For example, when salivary glands are transduced with rAAV vectors, the gene is expressed in the transduced salivary gland, however, due to the fact that the transduced salivary glands lack AAV rep and cap genes and accessory function genes, the rAAV is not able to replicate.

[0095] rAAV vectors of the present disclosure encapsulating the expression cassettes as described herein, can be produced using helper-free production. rAAVs are replication-deficient viruses and normally require components from a live helper virus, such as adenovirus, in a host cell for packaging of infectious rAAV vectors. rAAV helper-free production systems allow the production of infectious rAAV vectors without the use of a live helper virus. In the helper-free system, a host packaging cell line is co-transfected with three plasmids. A first plasmid contains adenovirus gene products (i.e. E2A, E4, and VA RNA genes) needed for the packaging of rAAV vectors. A second plasmid contains the required AAV genes (i.e., REP and CAP genes). A third plasmid contains the polynucleotide sequence encoding DAO1 or a functional variant thereof and a promoter flanked by ITRs. A host packaging cell line can be, for example, AAV-293 host cells.Suitable host cells contain additional components required for packaging infectious rAAV vectors that are not supplied by the plasmids. In some embodiments, the CAP genes can encode, for example, AAV capsid proteins as described herein. In some embodiments, the promoter is a promoter sequence as described herein. In some embodiments, the promoter sequence is a CAG sequence. In some embodiments, the encoded enzyme is DA01. Exemplary amino acid and nucleotide sequences for the AAV capsid proteins are identified in Table 3.

[0096] In some embodiments, the AAV serotype used to infect the salivary 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 at least 95%, 96%, 97%, 98%, 99%, or 100% similar to SEQ ID NO: 6. In some embodiments, the AAV capsid protein shares at least 95%, 98%, or 100% identity to the AAV2 VP1 protein (SEQ ID NO: 6).

[0097] 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 at least 95%, 96%, 97%, 98%, 99%, or 100% similar to SEQ ID NO: 7. In some embodiments, the polynucleotide sequence encoding the AAV2 VP1 protein shares at least 95%, 98%, or 100% identity with SEQ ID NO: 7.

[0098] In some embodiments, the AAV capsid protein comprises SEQ ID NO: 8. In some embodiments, the AAV capsid protein comprises a sequence at least 95%, 96%, 97%, 98%, 99%, or 100% similar to SEQ ID NO: 8. In some embodiments, the AAV capsid protein shares at least 95%, 98%, or 100% identity to the AAV2 VP3 protein (SEQ ID NO: 8).

[0099] 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 at least 95%, 96%, 97%, 98%, 99%, or 100% similar to SEQ ID NO: 9. In some embodiments, the polynucleotide sequence encoding the AAV2 VP3 protein shares at least 95%, 98%, or 100% identity with SEQ ID NO: 9.

[0100] In some embodiments, the AAV serotype used to infect the salivary 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 at least 95%, 96%, 97%, 98%, 99%, or 100% similar to SEQ ID NO: 10. In some embodiments, the AAV capsid protein shares at least 95%, 98%, or 100% identity to the AAV5 capsid protein (SEQ ID NO: 10).

[0101] In some embodiments, the polynucleotide sequence encoding the AAV5 capsid protein comprises SEQ ID NO: 11. In some embodiments, the polynucleotide sequence encoding theAAV capsid protein comprises a sequence at least 95%, 96%, 97%, 98%, 99%, or 100% similar to SEQ ID NO: 11. In some embodiments, the polynucleotide sequence encoding the AAV5 capsid protein shares at least 95%, 98%, or 100% identity with SEQ ID NO: 11.

[0102] In some embodiments, the AAV serotype used to infect the salivary gland is AAV8. In some embodiments, the capsid protein comprises SEQ ID NO: 12. In some embodiments, the AAV capsid protein comprises a sequence at least 95%, 96%, 97%, 98%, 99%, or 100% similar to SEQ ID NO: 12. In some embodiments, the AAV capsid protein shares at least 95%, 98%, or 100% identity to the AAV8 capsid protein (SEQ ID NO: 12).

[0103] In some embodiments, the polynucleotide encoding the AAV8 capsid protein comprises SEQ ID NO: 13. In some embodiments, the polynucleotide sequence encoding the AAV capsid protein comprises a sequence at least 95%, 96%, 97%, 98%, 99%, or 100% similar to SEQ ID NO: 13. In some embodiments, the polynucleotide sequence encoding the AAV8 capsid protein shares at least 95%, 98%, or 100% identity with SEQ ID NO: 13.

[0104] In some embodiments, the AAV serotype used to infect the salivary 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 at least 95%, 96%, 97%, 98%, 99%, or 100% similar to SEQ ID NO: 14. In some embodiments, the AAV capsid protein shares at least 95%, 98%, or 100% identity to the AAV9 capsid protein (SEQ ID NO: 14).

[0105] In some embodiments, the polynucleotide sequence encoding the AAV9 capsid protein comprises SEQ ID NO: 15. In some embodiments, the polynucleotide sequence encoding the AAV capsid protein comprises a sequence at least 95%, 96%, 97%, 98%, 99%, or 100% similar to SEQ ID NO: 15. In some embodiments, the polynucleotide sequence encoding the AAV9 capsid protein shares at least 95%, 98%, or 100% identity with SEQ ID NO: 15.Table 3. AAV Capsid SequencesExemplary rAA Vs

[0106] In some embodiments, the rAAV comprises an AAV capsid. In some embodiments, an 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 the expression cassette, wherein the expression cassette comprises a polynucleotide operatively linked to a promoter, wherein the polynucleotide comprises a nucleotide sequence encoding DAO1. In some embodiments, the promoter is any 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 a AAV2 3'ITR. In some embodiments, the expression cassette comprises a nucleotide sequence comprising from 5' to 3': a 5'ITR (e.g., an AAV2 5'ITR), a promoter (e.g., a CMV promoter), a 5 'untranslated region (5'UTR), a polynucleotide sequence comprising a nucleotide sequence encoding a DAO1 enzyme, a polyA sequence, and a 3'ITR (e.g., an AAV2 3'ITR). In some embodiments, the expression cassette comprises a nucleotide sequence comprising from 5' to 3': a 5'ITR (e.g., an AAV2 5'ITR), an enhancer (e.g., a CMV enhancer), a promoter (e.g., a CMV promoter), a 5'UTR, a polynucleotide sequence comprising a nucleotide sequence encoding a DAO1 enzyme, a WPRE sequence, a polyA sequence, and a 3'ITR (e.g., an AAV2 3'ITR). In some embodiments, the 5'UTR comprises a Kozak sequence positioned immediately upstream of and adjacent to an initiation codon in the nucleotide sequence encoding the DAO1 enzyme.

[0107] In some embodiments, the rAAV comprises an AAV capsid and an expression cassette comprising a polynucleotide operatively linked to a promoter, wherein the polynucleotide comprises a nucleotide sequence encoding a human DAO1 enzyme described herein. In some embodiments, the rAAV comprises an AAV capsid and an expression cassette comprising a polynucleotide operatively linked to a CAG promoter or a CMV promoter, wherein the polynucleotide comprises a nucleotide sequence encoding a DAO1 enzyme described herein.

[0108] In some embodiments, the rAAV comprises an AAV capsid and an expression cassette comprising a polynucleotide operatively linked to a promoter (e.g., a CAG promoter or CMV promoter), wherein the polynucleotide comprises a nucleotide sequence encoding a DAO1 enzyme, wherein the DAO1 enzyme comprises or consists of an amino acid sequence that shares at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 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 DA01 enzyme comprises or consists of the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the rAAV comprises an AAV capsid and an expression cassette comprising a polynucleotide operatively linked to a promoter (e.g., a CAG promoter), wherein the polynucleotide comprises a nucleotide sequence encoding a DA01 enzyme, wherein the nucleotide sequence comprises a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 30. In some embodiments, the nucleotide sequence comprises SEQ ID NO: 30.

[0109] In some embodiments, the rAAV comprises an AAV capsid and an expression cassette comprising a polynucleotide operatively linked to a promoter (e.g., a CAG promoter or CMV promoter), wherein the polynucleotide comprises a nucleotide sequence encoding a DAO1 enzyme, wherein the DAO1 enzyme comprises or consists of an amino acid sequence that shares at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity 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, the rAAV comprises an AAV capsid and an expression cassette comprising a polynucleotide operatively linked to a promoter (e.g., a CAG promoter or CMV promoter), wherein the polynucleotide comprises a nucleotide encoding a DAO1 enzyme, wherein the nucleotide sequence comprises a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 31. In some embodiments, the nucleotide sequence comprises SEQ ID NO: 31. In some embodiments, the rAAV comprises an AAV capsid and an expression cassette comprising a polynucleotide operatively linked to a promoter (e.g., a CAG promoter or CMV promoter), wherein the polynucleotide comprises a nucleotide encoding a DAO1 enzyme, wherein the nucleotide sequence comprises a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99% identity to SEQ ID NO: 29. In some embodiments, the nucleotide sequence comprises SEQ ID NO: 29.

[0110] In some embodiments, the rAAV comprises an AAV capsid and an expression cassette comprising or consisting of a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 23. In some embodiments, the expression cassette comprises or consists of SEQ ID NO: 23.[OHl] In some embodiments, the rAAV comprises an AAV2 capsid and an expression cassette comprising a polynucleotide operatively linked to a promoter, wherein the polynucleotide comprises a nucleotide sequence encoding a human DAO1 enzyme described herein. In some embodiments, the rAAV comprises an AAV2 capsid and an expression cassette comprising a polynucleotide operatively linked to a CAG promoter or a CMV promoter, wherein the polynucleotide comprises a nucleotide sequence encoding a DAO1 enzyme described herein.

[0112] In some embodiments, the rAAV comprises an AAV2 capsid and an expression cassette comprising a polynucleotide operatively linked to a promoter (e.g., a CAG promoter or CMV promoter), wherein the polynucleotide comprises a nucleotide sequence encoding a DAO1 enzyme, wherein the DAO1 enzyme comprises or consists of an amino acid sequence that shares at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity 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, the rAAV comprises an AAV2 capsid and an expression cassette comprising a polynucleotide operatively linked to a promoter (e.g., a CAG promoter or CMV promoter), wherein the polynucleotide comprises a nucleotide encoding a DAO1 enzyme, wherein the nucleotide sequence comprises a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 30. In some embodiments, the nucleotide sequence comprises SEQ ID NO: 30.

[0113] In some embodiments, the rAAV comprises an AAV2 capsid and an expression cassette comprising a polynucleotide operatively linked to a promoter (e.g., a CAG promoter or CMV promoter), wherein the polynucleotide comprises a nucleotide sequence encoding a DAO1 enzyme, wherein the DAO1 enzyme comprises or consists of an amino acid sequence that sharesat 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 AAV2 capsid and an expression cassette comprising a polynucleotide operatively linked to a promoter (e.g., a CAG promoter or CMV promoter), wherein the polynucleotide comprises a nucleotide encoding a DAO1 enzyme, wherein the nucleotide sequence comprises a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 31. In some embodiments, the nucleotide sequence comprises SEQ ID NO: 31. In some embodiments, the rAAV comprises an AAV2 capsid and an expression cassette comprising a polynucleotide operatively linked to a promoter (e.g., a CAG promoter or CMV promoter), wherein the polynucleotide comprises a nucleotide encoding a DAO1 enzyme, wherein the nucleotide sequence comprises a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 29. In some embodiments, the nucleotide sequence comprises SEQ ID NO: 29.

[0114] In some embodiments, the rAAV comprises an AAV2 capsid and an expression cassette comprising or consisting of a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 23. In some embodiments, the rAAV comprises an AAV2 capsid and an expression cassette comprising or consisting of SEQ ID NO: 23.

[0115] In some embodiments, the rAAV comprises an AAV5 capsid and an expression cassette comprising a polynucleotide operatively linked to a promoter, wherein the polynucleotide comprises a nucleotide sequence encoding a human DAO1 enzyme described herein. In some embodiments, the rAAV comprises an AAV5 capsid and an expression cassette comprising a polynucleotide operatively linked to a CAG promoter, wherein the polynucleotide comprises a nucleotide sequence encoding a DAO1 enzyme described herein.

[0116] In some embodiments, the rAAV comprises an AAV5 capsid and an expression cassette comprising a polynucleotide operatively linked to a promoter (e.g., a CAG promoter or CMVpromoter), wherein the polynucleotide comprises a nucleotide sequence encoding a DAO1 enzyme, wherein the DAO1 enzyme comprises or consists of an amino acid sequence that shares at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity 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 AAV5 capsid and an expression cassette comprising a polynucleotide operatively linked to a promoter (e.g., a CAG promoter or CMV promoter), wherein the polynucleotide comprises a nucleotide encoding a DAO1 enzyme, wherein the nucleotide sequence comprises a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 30. In some embodiments, the nucleotide sequence comprises SEQ ID NO: 30.

[0117] In some embodiments, the rAAV comprises an AAV5 capsid and an expression cassette comprising a polynucleotide operatively linked to a promoter (e.g., a CAG promoter or CMV promoter), wherein the polynucleotide comprises a nucleotide sequence encoding a DAO1 enzyme, wherein the DAO1 enzyme comprises an amino acid sequence that shares at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity 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 AAV5 capsid and an expression cassette comprising a polynucleotide operatively linked to a promoter (e.g., a CAG promoter or CMV promoter), wherein the polynucleotide comprises a nucleotide encoding a DAO1 enzyme, wherein the nucleotide sequence comprises a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 31. In some embodiments, the nucleotide sequence comprises SEQ ID NO: 31. In some embodiments, the rAAV comprises an AAV5 capsid and an expression cassette comprising a polynucleotide operatively linked to a promoter (e.g., a CAG promoter or CMV promoter), wherein the polynucleotide comprises a nucleotide encoding a DAO1 enzyme, wherein the nucleotide sequence comprises a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least93%, 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.

[0118] In some embodiments, the rAAV comprises an AAV5 capsid and an expression cassette comprising or consisting of a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 23. In some embodiments, the rAAV comprises an AAV5 capsid and an expression cassette comprising or consisting of SEQ ID NO: 23

[0119] In some embodiments, the rAAV comprises an AAV9 capsid and an expression cassette comprising a polynucleotide operatively linked to a promoter, wherein the polynucleotide comprises a nucleotide sequence encoding a human DAO1 enzyme described herein. In some embodiments, the rAAV comprises an AAV9 capsid and an expression cassette comprising a polynucleotide operatively linked to a CAG promoter, wherein the polynucleotide comprises a nucleotide sequence encoding a DAO1 enzyme described herein.

[0120] In some embodiments, the rAAV comprises an AAV9 capsid and an expression cassette comprising a polynucleotide operatively linked to a promoter (e.g., a CAG promoter or CMV promoter), wherein the polynucleotide comprises a nucleotide sequence encoding a DAO1 enzyme, wherein the DAO1 enzyme comprises an amino acid sequence that shares at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity 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 AAV9 capsid and an expression cassette comprising a polynucleotide operatively linked to a promoter (e.g., a CAG promoter or CMV promoter), wherein the polynucleotide comprises a nucleotide encoding a DAO1 enzyme, wherein the nucleotide sequence comprises a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 30. In some embodiments, the nucleotide sequence comprises SEQ ID NO: 30.

[0121] In some embodiments, the rAAV comprises an AAV9 capsid and an expression cassette comprising a polynucleotide operatively linked to a promoter (e.g., a CAG promoter or CMV promoter), wherein the polynucleotide comprises a nucleotide sequence encoding a DAO1enzyme, wherein the DA01 enzyme comprises or consists of an amino acid sequence that shares at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity 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 AAV9 capsid and an expression cassette comprising a polynucleotide operatively linked to a promoter (e.g., a CAG promoter or CMV promoter), wherein the polynucleotide comprises a nucleotide encoding a DAO1 enzyme, wherein the nucleotide sequence comprises a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 31. In some embodiments, the nucleotide sequence comprises SEQ ID NO: 31. In some embodiments, the rAAV comprises an AAV9 capsid and an expression cassette comprising a polynucleotide operatively linked to a promoter (e.g., a CAG promoter or CMV promoter), wherein the polynucleotide comprises a nucleotide encoding a DAO1 enzyme, wherein the nucleotide sequence comprises a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 29. In some embodiments, the nucleotide sequence comprises SEQ ID NO: 29.

[0122] In some embodiments, the rAAV comprises an AAV9 capsid and an expression cassette comprising or consisting of a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 23. In some embodiments, the rAAV comprises an AAV9 capsid and an expression cassette comprising or consisting of SEQ ID NO: 23.METHODS OF USE

[0123] Methods and compositions described herein can be used to treat histamine-associated conditions and reduce the associated symptoms of such conditions. The terms “treatment”, “treating” and the like are used herein to generally mean obtaining a desired pharmacologic and / or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof, e.g., reducing the likelihood that the disease or symptom thereofoccurs in the subject, and / or may be therapeutic in terms of a partial or complete cure for a disease and / or adverse effect attributable to the disease. “Treatment” as used herein covers any treatment of a disease in a mammal, and includes, without limitation: (a) inhibiting progress of the disease; (b) alleviating, reducing, or reducing an increase in one or more symptoms of the disease; (c) alleviating, reducing, or reducing an increase in one or more signs of the disease; (d) causing regression of the disease. The therapeutic agent may be administered before, during or after the onset of disease or injury. The treatment of ongoing disease, where the treatment stabilizes or reduces the undesirable clinical symptoms of the patient, is of particular interest. Such treatment is desirably performed prior to complete loss of function in the affected tissues. The subject therapy will desirably be administered during the symptomatic stage of the disease, and in some cases after the symptomatic stage of the disease.

[0124] As used herein, “administer,” “administering,” “administration” and the like refers to providing a substance (e.g., an rAAV vector, or a polypeptide comprising DAO1, or a functional variant thereof) to a subject in a manner that is pharmacologically useful (e.g., to treat a disease, disorder, or condition in the subject).

[0125] In some embodiments, the condition treated in accordance with the methods described herein associated with increased histamine production and / or increased histamine signaling. In some embodiments, the condition is an inflammatory condition. In some embodiments, the condition is an autoimmune reaction. In some embodiments, the condition is an autoimmune condition. In some embodiments, the condition is an allergic reaction. In some embodiments, the condition is an allergic reaction to a therapeutic agent. In some embodiments, the condition is an allergic condition. In some embodiments, the condition is vernal keratoconjunctivitis. In some embodiments, the condition is atopic keratoconjunctivitis. In some embodiments, the condition is seasonal or perennial allergic conjunctivitis. In some embodiments, the condition is histamine intolerance syndrome (HITS), eosinophilic esophagitis, mast cell activation syndrome, inflammatory bowel disease, or migraines associated with excessive histamine (e.g., migraines induced by the menstrual cycle whereby alterations in estrogen levels increases histamine and histamine signaling).

[0126] In some embodiments, the disclosure provides a method of treating a subject with an histamine-associated disease or disorder, comprising administering to the subject an rAAV vector comprising a nucleotide encoding human DAO1 described herein, wherein expression of the human DAO1 is increased in the subject compared to expression of the human DAO1 in anuntreated subject. Similarly, methods of treatment comprising the administration of a polypeptide comprising DA01 or a functional variant thereof, as well as the administration of alternative vectors (e.g., plasmids) configured to express DA01 or a functional variant thereof, are also described herein in various embodiments.

[0127] In some embodiments, the disclosure provides rAAV vectors for use in a method of treating an ocular condition in a subject in need thereof, the method comprising administering a recombinant adeno-associated virus (rAAV) vector, the rAAV vector comprising an AAV capsid and an expression cassette comprising a polynucleotide encoding human DAO1 operatively linked to a promoter, to at least one salivary gland (or to the oral cavity or a surface thereof) of the subject. In some embodiments, the rAAV vector for use is administered to a main salivary gland of a subject (e.g., a parotid gland). In some embodiments, the rAAV vector for use comprises an expression cassette encoding human DAO1.

[0128] In some embodiments, the disclosure provides rAAV vector for use, or adaptable for use, to treat a subject with an ocular disease, disorder, or condition. In some embodiments, the rAAV vector for use or adaptable for use comprise an AAV capsid and an expression cassette comprising a polynucleotide encoding human DAO1 (or a functional variant thereof) operatively linked to a promoter.

[0129] In some embodiments, the disclosure provides methods of treating a histamine- associated condition in a subject in need thereof, comprising administering a recombinant adeno- associated virus (rAAV) vector described herein, a plasmid configured to express DAO1 or a functional variant thereof, or a polypeptide comprising DAO1 or a functional variant thereof, to at least one salivary gland of the subject. In some embodiments, the rAAV (or any other vector) being administered, may comprise a nucleotide encoding a DAO1 that shares 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 the DAO1 is administered directly, the polypeptide comprising the 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.

[0130] In some embodiments, the disclosure provides rAAV vectors for use in a method of treating a histamine-associated condition in a subject in need thereof, the method comprising administering a recombinant adeno-associated virus (rAAV) vector comprising an expressioncassette comprising the nucleic acid sequence of SEQ ID NO: 23 to at least one salivary gland of the subject.

[0131] In some embodiments, expression of the human DAO 1 is increased 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% compared to expression of human DAO1 in an untreated subject. In some embodiments, 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 expression of human DAO1 in an untreated subject.

[0132] In some embodiments, expression of the human DAO1 is increased for about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 1 year compared to expression of the human DAO1 in an untreated subject.

[0133] As used herein, “approximately” or “about,” as applied to one or more values of interest, refers to a value that is similar to a stated 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 in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).

[0134] In some embodiments, a subject is administered an rAAV or other vector comprising a nucleotide encoding human DAO1 enzyme described herein, wherein expression of the human DAO1 enzyme is increased in the subject compared to expression of human DAO1 in an untreated subject.

[0135] In some embodiments, expression of the human DAO1 enzyme is increased 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% compared to expression of the human DAO1 enzyme in an untreated subject. In some embodiments, expression of the human DAO1 enzyme may be increased at least 1.5-fold, 2-fold, 3-fold, 4-fold, or 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 25-fold, 50-fold, 100-fold, 200- fold, 300-fold, 400-fold, 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, or 1000-fold compared to expression of the human DAO1 enzyme in an untreated subject. The aforementioned percentage and fold expression level changes may be determined, e.g., by evaluating plasma levels of DAO 1 before and after treatment with the rAAV vectors described herein. The pre-treatment time pointmay be, e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days before treatment, or within a range bound by any pair of the foregoing time points.

[0136] In some embodiments, expression of the human DAO1 enzyme is increased for about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 1 year compared to expression of the human DAO1 enzyme in an untreated subject.

[0137] In some embodiments, the disclosure provides a method of treating a subject with an histamine-associated disease or disorder, comprising administering to the subject DAO1, or an rAAV (or other vector) comprising a nucleotide encoding a human DAO1 enzyme, wherein expression of human DAO1 enzyme is increased in the subject compared to expression of human DAO1 enzyme in an untreated subject.

[0138] In some embodiments, the disclosure provides a method for treating a condition associated with histamine intolerance in a subject in need thereof, the method comprising administering to the subject an effective amount of DAO 1, or an effective amount of an rAAV (or other vector) described herein. In some embodiments, the condition is associated with an overproduction of histamine in one or more tissues of the subject. In some embodiments, the condition is associated with an overproduction of histamine in one or both eyes of the subject. In some embodiments, the condition is associated with an accumulation of histamine in one or more tissues of the subject. In some embodiments, the condition is associated with an accumulation of histamine in one or both eyes of the subject.

[0139] In some embodiments, the histamine intolerance results from an allergic condition. In some embodiments, the allergic condition is a seasonal or perennial allergic condition. In some embodiments, the histamine intolerance results from an autoimmune condition.

[0140] In some embodiments, the condition is characterized by one or more allergy symptoms (e.g., sneezing, rhinitis, conjunctivitis, shortness of breath, cough, chest tightness, hives, skin discoloration, stomach cramps, throat constriction, pain, swelling, vomiting, diarrhea, bloating), wherein the administering ameliorates or prevents the one or more symptoms. In some embodiments, the condition is characterized by one or more symptoms of the eye (e.g., itching, swelling, tearing, and redness), wherein the administering ameliorates or prevents the one or more symptoms. In some embodiments, the severity of the one or more symptoms are evaluated based upon any method known in the art for characterizing allergy-related symptoms. In some embodiments, the severity of the one or more symptoms are evaluated based upon any methodknown in the art for characterizing a pathology associated with histamine intolerance. In some embodiments the one or more symptoms are evaluated using methods described herein (e.g., conjunctival allergen challenge, corneal staining test, eye dryness score test, Schirmer score test, ocular surface disease index test).

[0141] In some embodiments, the method comprises administering a dose of DAO1, or an rAAV (or another vector configured to express DAO1) to a subject having a condition associated with histamine intolerance (e.g., an allergic condition), wherein the condition is characterized by one or more symptoms described herein (e.g., one or more symptoms of an allergy). In some embodiments, the dose is administered prior to the onset of the one or more symptoms. In some embodiments, the administering prevents the onset of the one or more symptoms. In some embodiments, the administering reduces the severity of the one or more symptoms. For example, in some embodiments, the method comprises administering a dose of the rAAV to a subject having a seasonal or perennial allergic condition characterized by one or more symptoms (e.g., rhinitis, keratoconjunctivitis, headache, fatigue, cough, sneezing), wherein the dose of the rAAV is administered prior to the season during which the allergic condition occurs, wherein the administering prevents the onset of the one or more symptoms and / or reduces the severity of the one or more symptoms. In some embodiments, the dose is administered following the onset of the one or more symptoms, wherein the administering reduces the severity of the one or more symptoms.

[0142] In some embodiments, the method comprises administering a dosing regimen of the rAAV to a subject having a condition associated with a histamine intolerance (e.g., allergic condition), wherein the condition is characterized by one or more symptoms described herein (e.g. , one or more symptoms of an allergy), wherein the dosing regimen comprises a first dose of the rAAV and at least one additional dose, wherein the first dose of the rAAV is administered to the subject prior to or subsequent to the onset of the one or more symptoms, and wherein the at least one additional dose is administered to the subject following the first dose, thereby preventing or reducing the severity of the one or more symptoms. In some embodiments, the dosing regimen comprises a frequency of dosing and / or dose amount that is selected based upon the pharmacokinetic parameters of the rAAV. In some embodiments, a clinician will administer the rAAV at a frequency and / or dose that achieves or maintains one or more desired effects. In some embodiments, the one or more desired effects is preventing one or more symptoms associated with the subject’s condition. In some embodiments, the one or more desired effects is reducing theseverity of one or more symptoms associated with the subject’s condition. In some embodiments, the severity of the one or more symptoms is measured using a method described herein or known in the art for evaluating a pathology associated with histamine intolerance. In some embodiments, the one or more desired effects is achieved immediately following administering of the first dose of the rAAV. In some embodiments, the one or more desired effects occurs at any time point following administering of the first dose of the rAAV. In some embodiments, the one or more desired effects is achieved following administration of the at least one additional dose of the rAAV. In some embodiments, the one or more desired effects is achieved at any point during the dosing regimen. In some embodiments, the one or more desired effects is achieved following administration of the first dose of the rAAV and the subject is administered at least one additional dose of the rAAV to prevent reversal of the one or more desired effects. In some embodiments, the method comprises administering a first dose of the rAAV to the subject prior to, immediately following, or during the onset of one or more symptoms and an additional dose following 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 protocol and parameters described above may be used to design regimens for treatments wherein DAO1 is administered directly or via an alternative vector system (e.g., plasmid-based expression).

[0143] In some embodiments, the disclosure provides a method for treating an inflammatory condition of the eye 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.

[0144] In some embodiments, the disclosure provides a method for treating an autoimmune condition of the eye 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.

[0145] In some embodiments, the disclosure provides a method for treating an allergic condition of the eye in a subject in need thereof, the method comprising administering to the subject one or more therapeutically effective doses of DAO 1, or an rAAV or other vector described herein.

[0146] In some embodiments, the disclosure provides a method for treating vernal keratoconjunctivitis in a subject in need thereof, the method comprising administering to thesubject one or more therapeutically effective doses of DA01, or an rAAV or other vector described herein.

[0147] In some embodiments, the disclosure provides a method for treating atopic keratoconjunctivitis 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.

[0148] In some embodiments, the disclosure provides a method for treating seasonal or perennial allergic conjunctivitis in a subject in need thereof, the method comprising administering to the subject one or more therapeutically effective doses of DAO 1, or an rAAV or other vector described herein.

[0149] In some embodiments, the disclosure provides a method for reducing a likelihood of or preventing a miscarriage in a pregnant human subject, comprising administering to the subject one or more therapeutically effective doses of a pharmaceutical composition comprising a vector or plasmid encoding a DAOl polypeptide described herein.

[0150] In some embodiments, the disclosure provides a method for increasing a likelihood of achieving a full-term pregnancy in a pregnant human subject, comprising administering to the subject one or more therapeutically effective doses of a pharmaceutical composition comprising a vector or plasmid encoding a DAO1 polypeptide described herein.

[0151] Without being bound to a theory, it is possible that breakdown of the placenta leading to biologically-aborted pregnancies is histamine-mediated. Elevated histamine during gestation increases vasoconstriction (Altura BM, et al., “Effects of vasoactive agents on isolated human umbilical arteries and veins.” Am. J. Physiol. 1972;222:345-55) the risk of pre-eclampsia (1984 Human Nutrition, Clinical Nutrition). As a routine clinical practice, some patients are administered anti-histamine during high-risk pregnancies. (BMJ Open. 2022; 12(10): e061837. Published online 2022 Oct 7. doi: 10.1136 / bmjopen-2022-061837). Additionally, histamine levels have been reported to influence morning sickness and hyperemesis gravidarum (2006 Journal of Reproductive Immunology). Histamine increases the expression of HCG hormone, which in turn leads to morning sickness and MCAS (mast cell activation syndrome). Low DAO levels have been reported to result in a 16.6x increase in miscarriage (Gahl WA, et al. “Maternal serum diamine oxidase in fetal death and low-birth-weight infants.” Br J ObstetGynaecol. 1982;89: 202-207 and Maintz L, et al. “Effects of histamine and diamine oxidase activities on pregnancy: a critical review.” Hum Reprod Update. 2008; 14: 485-495).

[0152] In some embodiments, the disclosure provides an rAAV or other vector as described herein for use in a method of treating a condition associated with histamine intolerance in a subject in need thereof, the method comprising administering to the subject an effective amount of an rAAV described herein.

[0153] some embodiments, the disclosure provides an rAAV or other vector as described herein for use in the manufacture of a medicament for treating a condition associated with histamine intolerance in a subject in need thereof.

[0154] In some embodiments, the disclosure provides an rAAV or other vector as described herein for use in a method of treating an autoimmune condition in a subject in need thereof, the method comprising administering to the subject an effective amount of the rAAV or other vector described herein.

[0155] In some embodiments, the disclosure provides an rAAV or other vector as described herein for use in the manufacture of a medicament for treating an autoimmune condition in a subject in need thereof.

[0156] In some embodiments, the disclosure provides an rAAV or other vector described herein for use in a method of treating an allergy condition in a subject in need thereof, the method comprising administering to the subject an effective amount of the rAAV or other vector as described herein.

[0157] In some embodiments, the disclosure provides an rAAV or other vector described as herein for use in the manufacture of a medicament for treating an allergy condition in a subject in need thereof.

[0158] In some embodiments, the disclosure provides an rAAV or other vector described herein for use in a method of treating a vernal keratoconjunctivitis in a subject in need thereof, the method comprising administering to the subject an effective amount of the rAAV or vector as described herein.

[0159] In some embodiments, the disclosure provides an rAAV or other vector as described herein for use in the manufacture of a medicament for treating a vernal keratoconjunctivitis in a subject in need thereof.

[0160] In some embodiments, the disclosure provides an rAAV or other vector described herein for use in a method of treating an atopic keratoconjunctivitis in a subject in need thereof, themethod comprising administering to the subject an effective amount of the rAAV or other vector as described herein.

[0161] In some embodiments, the disclosure provides an rAAV or other vector as described herein for use in the manufacture of a medicament for treating an atopic keratoconjunctivitis in a subject in need thereof.

[0162] In some embodiments, the disclosure provides an rAAV or other vector described herein for use in a method of treating a seasonal or perennial allergic conjunctivitis in a subject in need thereof, the method comprising administering to the subject an effective amount of the rAAV or other vector as described herein.

[0163] In some embodiments, the disclosure provides an rAAV or other vector as described herein for use in the manufacture of a medicament for treating a seasonal or perennial allergic conjunctivitis in a subject in need thereof.

[0164] In some embodiments, the disclosure provides an rAAV or other vector described herein for use in a method of (a) reducing a likelihood of or preventing a miscarriage and / or (b) increasing a likelihood of achieving a full-term pregnancy, in a pregnant human subject, the method comprising administering to the subject an effective amount of an rAAV or other vector as described herein.

[0165] In some embodiments, the disclosure provides an rAAV or other vector as described herein for use in the manufacture of a medicament for (a) reducing a likelihood of miscarriage and / or (b) increasing a likelihood of achieving a full-term pregnancy, in a pregnant human subject in need thereof.Combination Therapy

[0166] In some embodiments, a method of treatment described herein comprises further administering to the subject one or more additional therapeutic agents. In some embodiments, the additional therapeutic agent is an agent that increases tear production. In some embodiments, the additional therapeutic agent is another AAV-based gene therapy construct. Suitable therapeutic agents for use as a combination therapy in the present disclosure are further described in PCT / US2022 / 027653, herein incorporated by reference.

[0167] In some embodiments, the disclosure provides a method for treating a condition associated with histamine intolerance in a subject in need thereof, the method comprising administering to at least one salivary gland of a subject an effective amount of DAO 1, or aneffective amount of an rAAV or other vector described herein, wherein the patient is receiving, has received, or will subsequently receive a treatment that increases tear production and / or reestablishes tear film homeostasis. In some embodiments, the subject has decreased tear production and / or impaired tear film homeostasis. In some embodiments, the DA01, or the vector (e.g., the rAAV) is administered via injection to one or more salivary glands (e.g., to the parotid, sublingual, and / or submandibular glands). In some embodiments, the DA01, or the vector (e.g., the rAAV) is administered by topical application to a surface of the oral cavity (e.g., as a gel dosage form intended for sublingual administration resulting in transduction of the sublingual salivary gland). In some embodiments, the treatment is administered following administration of the DA01, or the vector (e.g., the rAAV) to the subject. In some embodiments, the treatment results in an increased amount of the DA01, or the vector (e.g., the rAAV) delivered to the salivary gland or saliva of the subject in a predetermined time compared to the amount of DA01 or the vector (e.g., the rAAV) delivered to the salivary gland or saliva in the absence of the subject receiving the treatment. In some embodiments, the predetermined period is about 5 minutes to about 60 minutes.

[0168] In some embodiments, the treatment that increases tear production comprises administering an effective amount of a nicotinic acetylcholine receptor (nAChR) agonist, or a pharmaceutically acceptable salt thereof. In some embodiments, the nAChR agonist is one described herein. nAChRs are a class of pentameric ligand-gated ion channels that have high affinity and selectivity for both nicotine and acetylcholine (which resembles nicotine in its protonated form) and comprise combinations of alpha and beta subunits. Examples of nAChR subtypes include, but are not limited to, alpha3beta4, alpha4beta2, alpha3alpha5beta4, and alpha4alpha6beta2. An important nAChR receptor subtype involved in instigating the nasolacrimal reflex, for example, is the alpha4beta2 subtype located on the trigeminal nerve endings in the nasal mucosa.

[0169] Administration of a nAChR agonist may desensitize the receptor. Receptor desensitization results in reduced response to an agonist even at higher agonist concentrations, which further results in diminished efficacy of the treatment. For instance, short term desensitization of the nAChR receptor to an agonist may occur over a 24-hour period after administration of the agonist. The potential for receptor desensitization may potentially limit the dosing frequency over a period of time in order to preserve an effective response to the agonist.

[0170] A nAChR agonist may be characterized as a full or partial agonist as determined by its ability to activate a given receptor to produce a response as compared to the response at thatreceptor for acetylcholine (ACh). In general, a nAChR agonist is a full agonist if it evokes a response upon binding to a given receptor that is equal or greater to that of ACh. A nAChR agonist is a partial agonist if it evokes a lower response upon binding to the receptor as compared to the response generated from ACh.

[0171] nAChR agonist response, from which receptor activation can be determined can, for example, be generated using an appropriate cell-based assay. Cells designed to express a particular nAChR receptor subtype and generate an electrical current response when bound to and activated by a nAChR agonist can be used to characterize the agonist profile of a compound and the amount of receptor activation thus determined. An example of a generic protocol is described below.

[0172] Cells that express a particular human nAChR subtype are first exposed to ACh. ACh binds and activates the receptor, thereby evoking a current. The concentration of the ACh is chosen to elicit the maximum response of the receptor (e.g., 1280 micromolar ACh). This current is recorded as the ACh response and serves as the 100% nAChR agonist response and to which responses to other nAChR agonists are compared. After washing, the cells are exposed to a nAChR agonist at various concentrations (e.g., 0.1, 0.3, 1, 3, 10, 30, 100, and 300 micromolar). The current evoked by exposure to the nAChR agonist is measured and recorded for each nAChR concentration. This nAChR agonist response data is then normalized to unity versus the maximal ACh evoked 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.

[0173] In some embodiments, the method to determine the relative agonist activity of nAChR agonist comprises conditions wherein the ACh response is evoked from a 1 or more millimolar ACh solution.

[0174] A nAChR agonist evoking a response equal to or greater than the maximum ACh response determined at the same receptor type is a full agonist. In some embodiments, a nAChR agonist evoking a response of less than 100% of the ACh response may still be characterized a full agonist, taking into account experimental variability. For example, variability between tests or measurement methods, and statistical error, may account for differences in the response results. In some embodiments, a nAChR agonist evoking 80% to 120% of the ACh response is considered a full agonist. In some embodiments, a nAChR agonist evoking 99% of the ACh response or greater is considered a full agonist. In some embodiments, a nAChR agonist evoking 95% of the ACh response or greater is considered a full agonist. In some embodiments, a nAChR agonistevoking 90% of the ACh response or greater is considered a full agonist. In some embodiments, a nAChR agonist evoking 85% of the ACh response or greater is considered a full agonist. In some embodiments, a nAChR agonist evoking 80% of the ACh response or greater is considered a full agonist.

[0175] Taking into account experimental variability, if the nAChR agonist evokes less than 100% of the ACh response, then generally the agonist is considered a partial agonist. In some embodiments, a nAChR agonist evoking less than 95% of the ACh response is considered a partial agonist. In some embodiments, a nAChR agonist evoking less than 90% of the ACh response is considered a partial agonist. In some embodiments, a nAChR agonist evoking less than 85% of the ACh response is considered a partial agonist. In some embodiments, a nAChR agonist evoking less than 80% of the ACh response is considered a partial agonist.

[0176] In some embodiments, a nAChR agonist evoking 5% to 95% of the ACh response is considered a partial agonist. In some embodiments, a nAChR agonist evoking 5% to 90% of the ACh response is considered a partial agonist. In some embodiments, a nAChR agonist evoking 5% to 85% of the ACh response is considered a partial agonist. In some embodiments, a nAChR agonist evoking 5% to 80% of the ACh response is considered a partial agonist.

[0177] In some embodiments, a nAChR agonist evoking 10% to 95% of the ACh response is considered a partial agonist. In some embodiments, a nAChR agonist evoking 10% to 90% of the ACh response is considered a partial agonist. In some embodiments, a nAChR agonist evoking 10% to 85% of the ACh response is considered a partial agonist. In some embodiments, a nAChR agonist evoking 10% to 80% of the ACh response is considered a partial agonist.

[0178] nAChR agonists that generate a low level of electrical activity at relatively high concentrations of agonist may be described as a weak partial agonist. In some embodiments, a nAChR agonist evoking 30% or less of the ACh response is considered a weak partial agonist. In some embodiments, a nAChR agonist evoking 25% or less of the ACh response is considered a weak partial agonist. In some embodiments, a nAChR agonist evoking 20% or less of the ACh response is considered a weak partial agonist. In some embodiments, the relatively high concentration of nAChR agonist is at least 100 micromolar. In some embodiments, the relatively high concentration of nAChR agonist is at least 200 micromolar. In some embodiments, the relatively high concentration of nAChR agonist is at least 300 micromolar or greater. For instance,a 300 micromolar concentration of nAChR agonist that evokes 25% of the maximal Ach-evoked current is considered a weak partial agonist.

[0179] 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.

[0180] In some embodiments, the nAChR agonist, or a pharmaceutically acceptable salt thereof, is an agonist of at least one of the 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 two of the 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 of the 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 nAChR subtype alpha3beta4. In some embodiments, the nAChR agonist, or a pharmaceutically acceptable salt thereof, is an agonist of nAChR subtype alpha3alpha5beta4. In some embodiments, the nAChR agonist, or a pharmaceutically acceptable salt thereof, is an agonist of nAChR subtype alpha4beta2. In some embodiments, the nAChR agonist, or a pharmaceutically acceptable salt thereof, is an agonist of nAChR subtype alpha4alpha6beta2. In some embodiments, the nAChR agonist, or a pharmaceutically acceptable salt thereof, is an agonist of nAChR subtype alpha?. In some embodiments, the nAChR agonist, or a pharmaceutically acceptable salt thereof, is not an agonist of nAChR subtype alpha?. In some embodiments, the nAChR agonist, or a pharmaceutically acceptable salt thereof, is not a full agonist of nAChR subtype alpha?. In some embodiments, the nAChR agonist is a full agonist of the aforementioned subtypes. In some embodiments, the nAChR agonist is a partial agonist of the aforementioned subtypes. In some embodiments, the nAChR agonist is a weak partial agonist of the aforementioned subtypes.

[0181] The terms “alpha?” or “a7” nAChR refer to the homomeric alpha? subtype, wherein the pentameric subunits of nAChR are composed entirely of alpha? subunits. Thus, a nAChR agonist that binds and activates nAChR alpha? is an agonist that binds and activates nAChR homomericalpha? receptor. In some embodiments described herein, the nAChR agonist is not an alpha? receptor agonist.

[0182] 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” or “is selective for” means that a compound has a higher affinity for the nAChR subtype and / or a lower half-maximal effective concentration (“EC50”) for that nAChR subtype for at least one reference nAChR subtype. Selectivity may be associated with at least a 5-fold affinity difference in EC50 value, at least a 10-fold affinity difference in EC50 value, at least a 20-fold affinity difference in EC50 value, or at least a 50-fold affinity difference in EC50 value. In some embodiments, the nAChR agonist, or a pharmaceutically acceptable salt thereof, selectively binds to nAChR subtype alpha3beta4. In some embodiments, the nAChR agonist, or a pharmaceutically acceptable salt thereof, selectively binds to nAChR subtype alpha3alpha5beta4. In some embodiments, the nAChR agonist, or a pharmaceutically acceptable salt thereof, selectively binds to nAChR subtype alpha4beta2. In some embodiments, the nAChR agonist, or a pharmaceutically acceptable salt thereof, selectively binds to nAChR subtype alpha4alpha6beta2. In some embodiments, the nAChR agonist, or a pharmaceutically acceptable salt thereof, selectively binds to nAChR subtype alpha?. In some embodiments, the nAChR agonist, or a pharmaceutically acceptable salt thereof, does not selectively bind to nAChR subtype alpha?.

[0183] The nAChR agonists contemplated in this 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.

[0184] Varenicline is characterized as a full agonist of the nAChR subtype alpha? and a partial agonist of subtypes alpha3beta4, alpha4beta2, alpha6beta2, alpha3alpha5beta4, and alpha4alpha6beta2. In some embodiments, the nAChR agonist is varenicline, or a pharmaceutically acceptable salt thereof. Pharmaceutically acceptable salts of varenicline include varenicline tartrate. Additional related information for varenicline may be found in, for example,U.S. Patent 9,504,644, U.S. Patent 9,504,645, U.S. Patent 9,532,944, U.S. Patent 9,597,284, andU.S. Patent 10,456,386.

[0185] Compound 1, as recited herein, refers to the structure: e structural representation of compound 1 is shown here:

[0187] Compound 1 may be also referred to by its chemical name. For instance, compound 1 is also referred to as (R)-5-((E)-2-pyrrolidin-3-ylvinyl)pyrimidine, or variations thereof including simpinicline, 5-{(E)-2-[(3R)-pyrrolidin-3-yl]vinyl}pyrimidine and (R,E)-5-((2-pyrrolidine-3- yl)vinyl)pyrimidine.

[0188] Compound 1 is a full agonist of nAChR subtypes alpha4beta2, alpha3beta4, alpha3alpha5beta4, and alpha4alpha6beta2. Compound 1 is a full agonist of nAChR subtypes alpha4beta2, and alpha3beta4.

[0189] Compound l is a partial agonist of subtype alpha3beta2.

[0190] Compound 1 is a weak partial agonist of subtype alpha?. In one example, a 300 micromolar concentration of compound 1 citrate evoked only 25% of the maximal ACh-evoked current.

[0191] In some embodiments, the nAChR agonist may be compound 1, or a pharmaceutically acceptable salt thereof. Pharmaceutically acceptable salts of compound 1 include galactarate (e.g., hemi-galactarate dihydrate) and citrate (e.g., mono-citrate). Patent related information for compound 1 may be found in U.S. Patent 7,098,331, U.S. Patent 7,714,001, U.S. Patent 8,063,068, U.S. Patent 8,067,443, U.S. Patent 8,604,191, U.S. Patent 9,145,396, U.S. Patent 9,981,949, U.S. Patent 8,633,222, and PCT publication WO 2017 / 177024.

[0192] 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. Insome embodiments, the nAChR agonist is (R)-5-((E)-2-pyrrolidin-3-ylvinyl)pyrimidine monocitrate.Modes of Administration

[0193] In some embodiments, the disclosure provides methods comprising administering an rAAV vector, the rAAV vector comprising an AAV capsid and an expression cassette comprising a polynucleotide encoding DAO1 operatively linked to a promoter, to the oral cavity of the subject or to a salivary gland of the subject (e.g., the parotid glands). In other aspects, methods according to the disclosure may comprise administration of a polypeptide comprising DAO1 or a functional variant thereof, or an alternative vector configured to express DAO1 or a functional variant thereof (e.g., plasmid-based expression systems) to the oral cavity of the subject or to a salivary gland of the subject.

[0194] As noted above, the main salivary glands of a mammal are typically composed of parotid, submandibular, and sublingual glands, which are supplemented by hundreds of minor salivary glands located throughout the oral cavity and digestive tract. The parotid glands are the largest salivary glands. In humans, they are located just in front of the ears, and the saliva produced in these glands is secreted into the mouth from a duct near the upper second molar. Each parotid gland has two parts, or lobes: the superficial lobe and the deep lobe. The submandibular glands are about the size of a walnut and are located below the jaw. The saliva produced in these glands is secreted into the mouth from under the tongue. Like the parotid glands, the submandibular glands have two parts, a superficial lobe and a deep lobe. The sublingual glands are the smallest of the major salivary glands. These almond-shaped structures are located under the floor of the mouth and below either side of the tongue. There minor salivary glands are located throughout the mouth and the digestive tract. Most are found in the lining of the lips, the tongue, and the roof of the mouth, as well as inside the cheeks, nose, sinuses, and larynx.

[0195] Together, the various salivary glands secrete saliva onto the oral surface through excretory ducts. Salivary glands also express and secrete proteins and products associated with digestion and other processes.

[0196] Administration of DA01, or of a vector (e.g., an rAAV) to a salivary gland may be accomplished by topical administration to the oral cavity, direct injection into the salivary gland, and / or topical administration to the salivary gland. In some embodiments, the DA01 or the vector (e.g., the rAAV) is administered to the salivary gland by topical administration. In someembodiments, the DA01 or the vector (e.g., the rAAV) is administered to the salivary gland by direct injection. The salivary gland may be accessed surgically or by manipulation of the mouth and / or tongue of a subject. Manipulation of the mouth and / or tongue provides access to the tissue for administration topically (e.g., by lavage of the tissue with a pharmaceutical composition comprising the viral vector). Direct injection into a salivary gland may be done by penetrating the skin over the salivary gland or by manipulating the mouth and / or tongue to access the salivary gland. Administration of a viral vector to a salivary gland may be accomplished by a multipletine injection. In some embodiments, the rAAV is administered to the salivary gland by direct injection.

[0197] In some embodiments, cells within the oral cavity, the salivary gland, and / or the excretory duct of a salivary gland are transduced by the rAAV vector. Cells within the oral cavity, the salivary gland and / or the excretory duct include, without limitation, acinar cells, ductal cells, and / or myoepithelial cells. In some embodiments, the transduced cells within the oral cavity, the salivary gland, and / or the excretory duct express a therapeutically effective amount of DAO 1 enzyme into the saliva of the subject. In some embodiments, a therapeutically effective amount of DAO1 enzyme is secreted into the oral cavity of the subject. In some embodiments, a therapeutically effective amount of DAO 1 enzyme is an amount sufficient to result in a plasma level of DAO 1 generates in a desired therapeutic effect.

[0198] Delivery of rAAV vectors to the oral cavity and / or a salivary gland to express a transgene into saliva has not been demonstrated in vivo. However, delivery of rAAV vectors to the lacrimal glands has been reported. In one example, the main lacrimal glands of mice were directly injected with rAAV vectors encoding a luciferase transgene with serotypes AAV2, AAV4, AAV5, AAV 5w8, AAV x5, AAV 9, AAV12, and bovine AAV (BAAV). AAV9, AAV 5w8, AAV5, and AAV2 each are able to transduce the lacrimal ductal and acinar cells of the lacrimal gland (Rocha et al., supra).

[0199] In some embodiments, the rAAV vector is administered to one or more salivary glands of the subject. In some embodiments, the salivary gland is a main salivary gland (e.g., a parotid,submandibular, and / or sublingual gland). In some embodiments, the salivary gland is one or more of the minor salivary glands of the subject.

[0200] Compositions and rAAV vectors of the disclosure may be administered to a salivary gland of the subject by any suitable method. For example, the subject composition may be administered by direct injection to the main or minor salivary glands.

[0201] Access to the salivary glands in human subjects can be achieved, for example, by manually elevating the tongue to expose the sublingual gland and delivering the therapeutic agent using a syringe, e.g, with a 30G needle.

[0202] The viral vectors of the disclosure are generally delivered to the subject as a pharmaceutical composition. Pharmaceutical compositions comprise a pharmaceutically acceptable solvent (e.g., water, etc.) and one or more excipients. In some embodiments, the pharmaceutical compositions comprise a buffer at about neutral pH (pH 5, 6, 7, 8, or 9). In some embodiments, the pharmaceutical composition comprises phosphate buffered saline (e.g., PBS at pH of about 7). The pharmaceutical compositions may comprise a pharmaceutically acceptable salt. The concentration of the salt may be selected to ensure that the pharmaceutical composition is isotonic to, or nearly isotonic to, the target tissue.

[0203] In some embodiments, the pharmaceutical compositions of the disclosure comprise about 1 * 108genome copies per milliliter (GC / mL), about 5 * 108GC / mL, about 1 x 109GC / mL, about 5 x 109GC / mL, about 1 x 1010GC / mL, about 5 x io10GC / mL, about 1 x io11GC / mL, about 5 x io11GC / mL, about 1xio12GC / mL, about 5 x io12GC / mL, about 5 x io13GC / mL, or about 1 x io14GC / mL of the viral vector e.g., rAAV vector). In some embodiments, the pharmaceutical compositions of the disclosure comprise about 1 x 108genome copies per milliliter (GC / mL), about 5 x io8GC / mL to about 1 x io9GC / mL, about 1 x io9GC / mL to about 5 x io9GC / mL, about 5 x io9GC / mL to about 1 x io10GC / mL, about 1 x io10GC / mL to about 5 x io10GC / mL, about 5 x io10GC / mL to about 1 x 1011GC / mL, about 1 x 1011GC / mL to about 5 x io11GC / mL, about 5 x io11GC / mL to about 1 x 1012GC / mL, about 1 x 1012GC / mL to about 5 x io12GC / mL, about 5 x io12GC / mL to about 5 x io13GC / mL, or about 5 x io13GC / mL to about 1 x 1014GC / mL of the viral vector (e.g, rAAV vector). In some embodiments, the pharmaceutical compositions of the disclosure comprise about 5 x io8GC / mL to about 5 x io9GC / mL, about 5 x 109GC / mL to about 5 x io10GC / mL, about 5 x io10GC / mL to about 5 x io11GC / mL, about 5 x 1011GC / mL to about 5 x io12GC / mL, or about 5 x io12GC / mL to about 1xio14GC / mL of the viral vector (e.g., rAAV vector). In yet further embodiments, the pharmaceutical compositionsof the disclosure comprise about 5 * 108GC / mL to about 5 * IO10GC / mL, about 5 * IO10GC / mL to about 5 x io12GC / mL, or about 5 x io12GC / mL to about 1 x io14GC / mL of the viral vector (e.g., rAAV vector).

[0204] In some embodiments, the pharmaceutical compositions of the disclosure comprise about 1 x 1012GC / mL to about 6.2 x 1012GC / mL of the viral vector (e.g., rAAV vector). In some embodiments, the pharmaceutical compositions of the disclosure comprise about 1 x io12GC / mL or about 6.2 x 1012GC / mL of the viral vector (e.g., rAAV vector).

[0205] In some embodiments, the pharmaceutical compositions of the disclosure are administered in a total volume of about 10 pL, about 20 pL, about 30 pL, about 40 pL, about 50 pL, about 60 pL, about 70 pL, about 80 pL, about 90 pL, about 100 pL, 110 pL, about 120 pL, about 130 pL, about 140 pL, about 150 pL, about 160 pL, about 170 pL, about 180 pL, about 190 pL, about 200 pL, about 250 pL, about 260 pL, about 270 pL, about 280 pL, about 290 pL, about 300 pL, about 310 pL, about 320 pL, about 330 pL, about 340 pL, about 350 pL, about 360 pL, about 370 pL, about 380 pL, about 390 pL, about 400 pL, about 450 pL, about 500 pL, about 550 pL, about 600 pL, about 650 pL, about 700 pL, about 750 pL, about 800 pL, about 850 pL, about 900 pL, about 950 pL, or about 1000 pL. In some embodiments, the pharmaceutical compositions of the disclosure are administered in a total volume of about 10 pL to about 20 pL, about 20 pL to about 30 pL, about 30 pL to about 40 pL, about 40 pL to about 50 pL, about 50 pL to about 60 pL, about 60 pL to about 70 pL, about 70 pL to about 80 pL, about 80 pL to about 90 pL, about 90 pL to about 100 pL, about 100 pL to 110 pL, 110 pL to about 120 pL, about 120 pL to about 130 pL, about 130 pL to about 140 pL, about 140 pL to about 150 pL, about 150 pL to about 160 pL, about 160 pL to about 170 pL, about 170 pL to about 180 pL, about 180 pL to about 190 pL, about 190 pL to about 200 pL, about 200 pL to about 250 pL, about 250 pL to about 300 pL, about 300 pL to about 350 pL, about 350 pL to about 400 pL, about 400 pL to about 450 pL, about 450 pL to about 500 pL, about 500 pL to about 550 pL, about 550 pL to about 600 pL, about 600 pL to about 650 pL, about 650 pL to about 700 pL, about 700 pL to about 750 pL, about 750 pL to about 800 pL, about 800 pL to about 850 pL, about 850 pL to about 900 pL, about 900 pL to about 950 pL, pr about 950 pL to about 1000 pL.

[0206] 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 polynucleotide genome of the virus. For AAV, the reference sample used isoften the transfer plasmid used in generation of the rAAV vector but other reference samples may be used.

[0207] Alternatively, or in addition, the concentration of a viral vector can be determined by measuring the titer of the vector on a cell line. Viral titer is typically expressed as viral particles (vp) per unit volume (e.g., vp / mL). In some embodiments, the pharmaceutical compositions of the disclosure comprise about 1 x 108viral particles per milliliter (vp / mL), about 5 * 108vp / mL, about 1 x 109vp / mL, about 5 * 109vp / mL, about 1 x 1010vp / mL, about 5 x 1O10vp / mL, about 1 x 1011vp / mL, about 5 x 1011vp / mL, about 1 x 1012vp / mL, about 5 x 1012vp / mL, about 5 x 1013vp / mL, or about 1 x 1014vp / mL of the viral vector (e.g., rAAV vector). In some embodiments, the pharmaceutical compositions of the disclosure comprise about 1 x 108viral particles per milliliter (vp / mL) to about 5 x 108vp / mL, about 5 x 108vp / mL to about 1 x 109vp / mL, about 1 x 109vp / mL to about 5 x 109vp / mL, about 5 x 109vp / mL to about 1 x 1O10vp / mL, about 1 x 1O10vp / mL to about 5 x 1O10vp / mL, about 5 x 1O10vp / mL to about 1 x 1011vp / mL, about 1 x 1011vp / mL to about 5 x 1011vp / mL, about 5 x 1011vp / mL to about 1 x 1012vp / mL, about 1 x 1012vp / mL to about 5 x 1012vp / mL, about 5 x 1012vp / mL to about 5 x 1013vp / mL, or about 5 x 1013vp / mL to about 1 x IQ14vp / mL of the viral vector (e.g., rAAV vector).Assessment of Efficacy

[0208] The efficacy of a method described herein can be assessed using any suitable method known in the art.

[0209] In some embodiments, the methods described herein result in one or more symptoms of the condition being reduced compared to the symptoms of the condition before administration of the rAAV vector. As used herein, “symptoms” include any of the diagnostic criteria or symptoms associated with a given condition (e.g., a condition associated with excess histamine or histamine- induced inflammation), including those described herein. Non-limiting examples of symptoms that may be alleviated by treatment in accordance with the methods described herein include, for example, a reduction in the incidence or severity of migraines experienced by the subject being treated.

[0210] In some embodiments, symptoms can be reduced following administration of the compositions and vectors (e.g., rAAV vectors) described herein. In some embodiments, one ormore symptoms of the condition are reduced compared to the symptoms of the condition in an untreated control subject.

[0211] The efficacy of a method of treatment described herein may be evaluated by assessing the symptoms of a condition treated in accordance with the methods described herein on a clinical scale. Examples of the clinical scales that may be used to evaluate the efficacy of a method of treatment described herein include, without limitation, the MIDAS Questionnaire and the HALT (Headache Attributed Lost Time) test, used to clinically assess migraine incidence and severity.

[0212] As used herein, the term “statistically significant” refers to a method of analysis selected by a person of ordinary skill in the art based upon study design and the data type generated to assess the difference observed between two or more groups, wherein the analysis determines if the difference is not random or due strictly to chance.Maintenance of Effective Treatment Over Time

[0213] The efficacy of a method of treatment described herein may be evaluated at any suitable time point after administration, for example, improvement on a clinical metric associated with the disease or condition being treated may be measured at about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 18 months, about 24 months, about 3 years, about 4 years, about 5 years, about 6 years, about 7 year, about 8 years, about 9 years, or about 10 years after the administration of an rAAV vector described herein. In some embodiments, the improvement on the clinical metric is measured at two or more time points after the administration of an rAAV vector described herein, for example, every 3 months, every 6 months or every 12 months after the administration of the rAAV vector.

[0214] The present disclosure may provide for effective treatment over a period of time where a statistically significant improvement in a subject’s score is maintained. The term “maintained” as used in the present disclosure and as it relates to the maintenance of a statistically significant improvement in a subject’s score (EDS, Schirmer, corneal staining, or OSD I) refers to the statistically significant improvement not diminishing below a certain threshold over time. A statistically significant improvement can be maintained even if, at a later point in time, thesubject’s score changes. An improvement after treatment according to the disclosed methods, can be maintained without additional dosing or after one or more subsequent doses.

[0215] In some of the embodiments described herein, the maintenance of the statistically significant improvement of the subject’s score means that the statistically significant improvement does not diminish by more than 10%, 20%, 30%, 40%, 50%, or 60%.

[0216] In some of the embodiments described herein, the statistically significant improvement in the subject’s score 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 of the embodiments described herein, the statistically significant improvement in the subject’s score is maintained for at least 30 days after treatment according to the methods described herein, wherein the statistically significant improvement does not diminish by more than 30%. In some embodiments, the statistically significant improvement in the subject’s score is maintained for at least 1 month from administration of the first dose of the rAAV vector, wherein the statistically significant improvement does not diminish by more than 30%.

[0217] In some of the embodiments described herein, the statistically significant improvement in the subject’s score 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, wherein the statistically significant improvement does not diminish 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.

[0218] In some of the embodiments described herein, the statistically significant improvement in the subject’s score 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, wherein the statistically significant improvement does not diminish by more than 20% compared to the subject’s score on the same clinical metric assessed within 60 days after treatment according to the methods described herein.

[0219] In some embodiments, the improvement measured on the clinical metric persists for a prolonged time (e.g., 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 of the rAAV vector provided herein.Dosing Timing and Method of Administration

[0220] 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 on the body. For example, wherein the patient’s condition does not improve, upon the health provider’s discretion, the method of treating an ocular condition as described herein, may be adjusted in dose or administered repeatedly in order to ameliorate or otherwise control or limit the symptoms of the subject’s ocular condition. For instance, the period of time between administrations of one or more doses is extended, or the period of time between days the subject is administered one or more doses is extended. As a non-limiting example, administration of one or more doses is modified to administration of one or more doses after measuring symptoms of the condition or disease being treated.

[0221] The term “dose”, as used herein, may refer to a dose of a pharmaceutical composition of the disclosure, or a dose of the treatment that reduces symptoms of a condition or disease.

[0222] In some of the embodiments described herein, a dose of the rAAV vector is a dose of a rAAV vector carrying an expression cassette. In such cases, delivery of an appropriate dose (e.g., effective amount) of the gene product is achieved by administering an appropriate amount / titer of the viral vector to the target site which allows expression of an effective amount of the gene product over a period of time. An “effective amount,” as used herein, refers to an amount or dose of an rAAV, treatment, or composition described herein that is sufficient to reduce the symptoms and or signs of a condition, disease, or disorder described herein. The term “amount” as used herein refers to an absolute amount (e.g., an absolute amount of protein or rAAV particles) or concentration (e.g., a concentration of protein in a solution), whether the amount referred to in a given instance refers to an absolute amount, concentration, or both, will be clear to the skilled artisan based on the context provided herein.

[0223] In some embodiments, the viral vector is an rAAV vector. In some embodiments, the viral vector is administered to a salivary gland. In some embodiments, the rAAV is administered to a salivary gland by topical administration. In some embodiments, the rAAV is administered to a salivary gland by direct injection. In some embodiments, a dose of the rAAV results in the stable production of the gene product for a period of time (e.g., about 1 day, about 2 days, about 4 days, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 9 months, about 12 months, or longer). In some embodiments, a dose of the rAAV results in the stable production of the geneproduct for about 1 week. In some embodiments, a dose of the rAAV results in the stable production of the gene product for about 2 weeks. In some embodiments, a dose of the rAAV results in the stable production of the gene product for about 3 weeks. In some embodiments, a dose of the rAAV results in the stable production of the gene product for about 4 weeks. In some embodiments, a dose of the rAAV results in the stable production of the gene product for about 1 month. In some embodiments, a dose of the rAAV results in the stable production of the gene product for about 2 months. In some embodiments, a dose of the rAAV results in the stable production of the gene product for about 3 months. In some embodiments, a dose of the rAAV results in the stable production of the gene product for about 4 months. In some embodiments, a dose of the rAAV results in the stable production of the gene product for about 5 months. In some embodiments, a dose of the rAAV results in the stable production of the gene product for about 6 months. In some embodiments, a dose of the rAAV results in the stable production of the gene product for about 9 months. In some embodiments, a dose of the rAAV results in the stable production of the gene product for about 12 months.

[0224] In some embodiments, a method described herein comprises administering an effective amount of an rAAV described herein to a subject, wherein the rAAV comprises a polynucleotide encoding a gene product (e.g., DA01). In some embodiments, the method comprises delivering a first dose and one or more subsequent doses of the rAAV. The one or more subsequent doses are administered after a period of time after the first dose. In some embodiments, this period of time between the first dose and the next subsequent dose is at least 1 day, at least 3 days, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 1 month, at least 2 months, at least 4 months, at least 6 months, at least 9 months, at least 12 months, or longer. In some embodiments, this period of time between the first dose and the next subsequent dose is between 1-7 days, between 1-4 weeks, between 2-6 weeks, between 4-8 weeks, between 1-3 months, between 2-4 months, between 3-6 months, between 4-12 months, between 6-24 months. In some embodiments, the period of time between the 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 the one or more subsequent doses isbetween 1-7 days, between 1-4 weeks, between 2-6 weeks, between 4-8 weeks, between 1-3 months, between 2-4 months, between 3-6 months, between 4-12 months, between 6-24 months.

[0225] In some embodiments, a method described herein comprises administering an effective amount of the rAAV to a subject, 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 comprises administering one or more doses of a nAChR agonist described herein. In some embodiments, the nAChR agonist is administered via local administration. In some embodiments, the nAChR agonist is administered as an intranasal spray.

[0226] In some embodiments, the treatment that increases tear production (e.g., a treatment comprising administering to the subject one or more doses of an nAChR agonist) is administered on a separate schedule from the administration of the rAAV. In some embodiments, the treatment that increases tear production is administered throughout the period between the multiple doses of the rAAV. In some embodiments, the treatment that increases tear production is administered after the first dose of the 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 one 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.

[0227] In some embodiments, the treatment that increases tear production (e.g., a treatment comprising administering to the subject one or more doses of an nAChR agonist) is administered to the subject one to four times daily after the first day of administration of the rAAV, once a day after the first day of administration of the rAAV, twice a day after the first day of administration of the rAAV, or three times a day after the first day of administration of the rAAV.

[0228] In some embodiments, the subject is administered a dosing cycle of an nAChR agonist described herein, wherein the duration between doses of the nAChR agonist is increased over time.For instance, administration of a dose every 4 hours is modified to administration of a dose every 8 or 12 hours.

[0229] In some embodiments, the treatment that increases tear production is administered for at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, or at least one year. In some embodiments, the treatment that increases tear production is administered for 2-52 weeks, 2-40 weeks, 2-36 weeks, 2-24 weeks, 2-12 weeks, 2-8 weeks, 4-52 weeks, 4-40 weeks, 4- 36 weeks, 4-24 weeks, 4-12 weeks, 4-8 weeks, 5-52 weeks, 5-40 weeks, 5-36 weeks, 5-24 weeks, 5-12 weeks, 5-8 weeks, 6-52 weeks, 6-40 weeks, 6-36 weeks, 6-24 weeks, 6-12 weeks, or 6-8 weeks.

[0230] In some embodiments, the method comprises a first dose and one or more subsequent doses of the rAAV. In some embodiments, the one or more subsequent doses are administered after a period of time after the first dose. This period of time between the first dose and the next subsequent dose is at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, or at least 8 hours. The period of time between the first dose and the next subsequent dose is between 1-3 hours, 2-4 hours, 3-6 hours, or 4-8 hours. The period of time 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 period of time between the one or more subsequent doses is between 1-3 hours, 2-4 hours, 3-6 hours, or 4- 8 hours.

[0231] In some embodiments, the time period between administration of a dose of a rAAV, and a dose of an effective amount of a treatment that increases tear production is less than 5 minutes, between 5-60 minutes, between 30-90 minutes, between 1-3 hours, between 1-8 hours, between 1-12 hours, between 1-24 hours, between 8-12 hours, between 8-24 hours, between 12-24 hours, between 1-3 days, between 1-7 days, between 1-14 days, between 1-28 days, between 3-7 days, between 3-14 days, between 3-28 days, between 7-14 days, or between 7-28 days.

[0232] In some embodiments, the method comprises administering a first dose and one or more subsequent doses of the rAAV, and a first dose and one or more subsequent doses of the treatment that increases tear production. The one or more subsequent doses are administered after a period of time after the first dose. This period of time between the first dose and the next subsequent dose is at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, or at least 8 hours. The period of time between the first dose and the nextsubsequent dose is between 1-3 hours, 2-4 hours, 3-6 hours, or 4-8 hours. The period of time 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 period of time between the one or more subsequent doses is between 1-3 hours, 2-4 hours, 3-6 hours, or 4- 8 hours.

[0233] In some embodiments, the treatment that increases tear production, and the rAAV, are administered to the subject in need thereof in separate dosage forms. In some embodiments, the treatment that increases tear production, and the rAAV, are administered to the subject in need thereof in a combined dosage form.Pharmaceutical Compositions and Kits

[0234] In some embodiments, the disclosure provides a pharmaceutical composition comprising an rAAV vector described herein. In some embodiments, the pharmaceutical composition comprises an rAAV vector described herein, and a pharmaceutically acceptable carrier, delivery agent, or excipient. In some embodiments, the disclosure provides a pharmaceutical composition comprising a polypeptide comprising a DAO1 enzyme, or a functional variant thereof, optionally wherein the polypeptide has an amino acid sequence having at least 90% identity to an amino acid sequence selected from SEQ ID NO: 1 and 2; and b) a pharmaceutically-acceptable carrier suitable for administration to a salivary gland, or to an oral cavity or a surface thereof, of a human subject. In some embodiments, the disclosure provides a pharmaceutical composition comprising a) a vector comprising a polynucleotide that encodes a polypeptide comprising a DAO1 enzyme, or a functional variant thereof, optionally wherein the polypeptide has an amino acid sequence having at least 90% identity to an amino acid sequence selected from SEQ ID NO: 1 and 2; and b) a pharmaceutically-acceptable carrier suitable for administration to a salivary gland, or an oral cavity or surface thereof, 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.

[0235] In some embodiments, a pharmaceutical composition may comprise 0.25, 0.50, 0.75, 1.00, 1.25, 1.50, 1.75, 2.00, 2.25, 2.50, 2.75, 3.00, 3.25, 3.50, 3.75, 4.00, 4.25, 4.50, 4.75, 5.00, 5.25, 5.50, 5.75, or 6.00 pg of a polypeptide comprising DAO1 (or a functional variant thereof), or an amount within a range defined by any pair of the foregoing values; and optionally one ormore 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 NO: 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 a disease, disorder, or condition (e.g., associated with excess histamine or histamine-induced inflammation).

[0236] In some embodiments, the disclosure provides use of an rAAV vector or pharmaceutical composition described herein, in the manufacture of a medicament for treatment of a disease, disorder, or condition (e.g., associated with excess histamine or histamine-induced inflammation). In some embodiments, the disclosure provides use of an rAAV vector or pharmaceutical composition herein for use, or adaptable for use, in the treatment of any such disease, disorder, or condition.

[0237] In some embodiments, the pharmaceutically acceptable carrier comprises phosphate buffered saline. In some embodiments, the pharmaceutical composition is formulated to be compatible with its intended route of administration . In some embodiments, the pharmaceutical composition is formulated for administration into a salivary gland (e.g., via direct injection). In some embodiments, the pharmaceutical composition is formulated for administration onto aa surface of an oral cavity of the subject (e.g., a gel administered sublingually). Examples of excipients, diluents, and / or carriers suitable for administration to the salivary gland or oral cavity, which can be referred to as pharmaceutically-acceptable carriers, include sterile, pyrogen-free water and sterile, pyrogen-free, buffered saline (e.g., saline buffered using phosphate 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.

[0238] Any concentration of the vector, such as the rAAV vector, that is suitable to effectively transduce cells of a salivary gland, and / or the excretory duct of a salivary gland, can be prepared for contacting cells of the salivary gland or the excretory duct in vitro or in vivo. For example, the rAAV vector may be formulated at a concentration of 105vector genomes per mL or more, for example 5xl05vector genomes per mL; 106vector genomes per mL; 5xl06vector genomes per mL; 107vector genomes per mL; 5xl07vector genomes per mL; 108vector genomes per ml; 5* 108vector genomes per mL; 109vector genomes per mL; 5* 109vector genomes per mL, 1010vectorgenomes per mL, 5><1O10vector genomes per mL; 1011vector genomes per mL; 5xlOnvector genomes per mL; 1012vector genomes per mL; 5*1012vector genomes per mL; 1013vector genomes per mL; 1.5* 1013vector genomes per mL; 3*1013vector genomes per mL; 5*1013vector genomes per mL; 7.5* 1013vector genomes per mL; 9*1013vector genomes per mL; 1014vector genomes per mL; 5*1014vector genomes per mL; 1015vector genomes per mL; 5*1015vector genomes per mL; 1016vector genomes per mL; or more, but typically not more than 5* 1016vector genomes per mL. In some aspects, the rAAV vector may be formulated at a concentration within a range defined by any pair of concentrations described in this paragraph.

[0239] In some embodiments, the rAAV vector comprises an AAV capsid and an expression cassette configured to express DAO1, as described herein. Formulations according to the disclosure may comprise any concentration of such rAAV vectors, e.g., at a concentration suitable to effectively transduce cells of the oral cavity, the salivary glands, and / or the excretory duct of one or more salivary glands. In some embodiments, a formulation may comprise 105; 2.5xl05; 5xl05; 107; 2.5xl07; 5xl07; 7.5xl07; 108; 2.5xl08; 5xl08; 7.5xl08; 109; 2.5xl09; 5xl09; 7.5xl09; IO10; 2.5xlO10; 5xlO10; 7.5xlO10; 1011; 2.5xlOn; 5xl0n; 7.5xlOn; 1012; 2.5xl012; 5xl012; 7.5xl012; 1013; 2.5xl013; 5xl013; 7.5xl013; IxlO14; 2.5xl014; 5xl014; 7.5xl014; IxlO15; IxlO16; or 5xl016rAAV vectors per mL, or a concentration of rAAV vectors per mL within a range defined by any pair of concentrations described in this paragraph.

[0240] In some embodiments the disclosure provides alternative DAO1 vehicles, such as plasmids configured to express DAO1. Formulations according to the disclosure may comprise any concentration of such plasmids, e.g., at a concentration suitable to effectively transfect cells of the oral cavity, the salivary glands, and / or the excretory duct of one or more salivary glands. In some embodiments, a formulation may comprise 105; 2.5xl05; 5xl05; 106; 2.5xl06; 5xl06; 7.5X106;107; 2.5X107; 5X107; 7.5X107; 108; 2.5xl08; 5xl08; 7.5xl08; 109; 2.5xl09; 5xl09; 7.5xl09; IO10; 2.5xlO10; 5xlO10; 7.5xlO10; 1011; 2.5xlOn; 5xl0n; 7.5xlOn; 1012; 2.5xl012; 5xl012; 7.5xl012; 1013; 2.5xl013; 5xl013; 7.5xl013; 1014; 2.5xl014; 5xl014; 7.5xl014; 1015; 2.5xl015; 5xio15; 7.5xl015; 1016; 2.5xl016; 5xl016; or 7.5xl016plasmids per mL, or a concentration of plasmids per mL within a range defined by any pair of concentrations described in this paragraph.

[0241] Similarly, any total number of rAAV vectors suitable to provide appropriate transduction of cells of the oral cavity, the salivary glands, and / or the excretory duct of one or more salivary glands to confer the desired effect or treat the disease can be administered to the mammal or to the primate’s oral cavity. In some embodiments, at least 105; 2.5xl05; 5xl05; 7.5xl05; 106; 2.5xl06;5xl06; 7.5xl06; 107; 2.5xl07; 5xl07; 7.5xl07; 108; 2.5xl08; 5xlO8; 7.5xl08; 109; 2.5xl09; 5xl09; 7.5xl09; IO10; 2.5xlO10; 5xlO10; 7.5xlO10; IO11; 2.5xlOn; 5xlOn; 7.5xlOn; 1012; 2.5xl012; 5xl012; 7.5xl012; 1013; 2.5xl013; 5xlO13; 7.5xl013; 1014, 2.5xl014; 5xl014; 7.5xl014; IO15; 2.5xl015; 5xlO15; or 7.5xl015; 1016; 2.5xl016; 5xl016; or 7.5xlO16rAAV vectors, or more, are injected. For example, in some embodiments, about 1 x 109to about 1 x IO10, about 1 x IO10to about 1 x 1011, about 1 x 1011to about 1 x 1012, about 1 x 1012to about 1 x 1013, or about 1 x 1013to about 1 x 1015genome copies of the rAAV vector are administered. In some aspects, the total number of rAAV vectors administered to the human subject or animal being treated may comprise a concentration within a range defined by any pair of concentrations described in this paragraph. Any suitable number of administrations of the rAAV vectors to the mammal or the primate salivary gland can be made. In some embodiments, the methods comprise a single administration; in other embodiments, multiple administrations are made over time as deemed appropriate by an attending clinician.

[0242] In some embodiments, a suitable amount or concentration of rAAV vector (or any other vector described herein) in a therapeutic formulation may be a concentration effective to express from 100 pg / mL to 50 pg / mL of DAO 1 in saliva of a subject subsequent to 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 be one that results 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 pg / mL of DAO1 (or a concentration within a range defined by any pair of the foregoing values) in saliva of a subject. Expression may be measured, e.g., 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 of time, such as after 1, 2, 3, 4, or 5 days.

[0243] The rAAV vector may be formulated into any suitable unit dosage, including, without limitation, IxlO5vector genomes or more, for example, IxlO6, lx 107, 1X108’1X109, IxlO10, IxlO11, IxlO12, IxlO13, IxlO14, or IxlO15vector genomes or more, in certain instances, IxlO14vector genomes, but usually no more than 4xl015vector genomes. In some embodiments, the rAAV vector is formulated into any suitable unit dosage, including, without limitation, IxlO5, IxlO6, IxlO7, IxlO8, IxlO9, IxlO10, IxlO11, IxlO12, IxlO13, IxlO14, or IxlO15vector genomes or more. In some embodiments, the unit dosage is at most about 5x 1015vector genomes, e.g., 1 x 1014vector genomes or less, for example 1 x 1013, 1 x 1012, 1 x 1011, 1 x 1010, or 1 x 109vector genomes orless, in certain instances IxlO8vector genomes or less, and typically no less than IxlO8vector genomes. In some embodiments, the unit dosage is at most about 5xl015vector genomes, e.g., IxlO14vector genomes or less, for example IxlO13, IxlO12, IxlO11, IxlO10, IxlO9, IxlO8, or IxlO7vector genomes or less. In some embodiments, the unit dosage is IxlO10to IxlO11vector genomes. In some embodiments, the unit dosage is IxlO10to 3xl012vector genomes. In some embodiments, the unit dosage is 1 x 109to 3 x 1013vector genomes. In some embodiments, the unit dosage is 1 x 108to 3 x 1014vector genomes. In some embodiments, the rAAV vector comprises an AAV capsid and an expression cassette configured to express DA01 and a unit dosage is formulated at a concentration of at least, at most, exactly, or about IxlO5, IxlO6, IxlO7, IxlO8, IxlO9, IxlO10, IxlO11, IxlO12, IxlO13, IxlO14, IxlO15, or IxlO16of the rAAV vector, or at a concentration within a range defined by any pair of concentrations described in this paragraph.

[0244] In some embodiments, the disclosure provides a vector comprising a plasmid configured to express DAO1, and a unit dosage is formulated at a concentration of at least, at most, exactly, or about IxlO7, IxlO8, IxlO9, IxlO10, IxlO11, IxlO12, IxlO13, IxlO14, or IxlO15of the plasmid.

[0245] In some embodiments, the unit dosage of pharmaceutical composition may be measured using multiplicity of infection (MOI). By MOI it is meant the ratio, or multiple, of vector or viral genomes of the rAAV vectors provided herein to the cells to which the nucleic acid may be delivered. In some embodiments, the MOI, measured as viral genomes, may be IxlO6. In some embodiments, the MOI may be 1 x 105to IxlO7. In some embodiments, the MOI may be 1 x 104to IxlO8. In some embodiments, recombinant viruses of the disclosure are at least about IxlO1, IxlO2, IxlO3, IxlO4, IxlO5, IxlO6, IxlO7, IxlO8, IxlO9, IxlO10, IxlO11, IxlO12, IxlO13, IxlO14, IxlO15, IxlO16, IxlO17, and IxlO18MOI. In some embodiments, recombinant viruses of this disclosure are 1 x 108to 3 x 1014MOI. In some embodiments, recombinant viruses of the disclosure are at most about IxlO1, IxlO2, IxlO3, IxlO4, IxlO5, IxlO6, IxlO7, IxlO8, IxlO9, IxlO10, IxlO11, 1 x 1012, 1 x 1013, 1 x 1014, 1 x 1015, 1 x 1016, 1 x 1017, and IxlO18MOI.

[0246] In some embodiments, the amount of pharmaceutical composition comprises about IxlO5to about IxlO16rAAV vectors, IxlO8to about IxlO15rAAV vectors, about IxlO9to about IxlO14rAAV vectors, about IxlO10to about IxlO13rAAV vectors, or about IxlO11to about 3xl012rAAV vectors.

[0247] In preparing the subject rAAV compositions, any host cells for producing rAAV vectors may be employed, including, for example, mammalian cells (e.g., 293 cells), insect cells (e.g., SF9 cells), microorganisms and yeast. Host cells can also be packaging cells in which the AAV repand cap genes are stably maintained in the host cell or producer cells 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. rAAV vectors are purified and formulated using standard techniques known in the art.

[0248] In some embodiments, the disclosure provides for use of an rAAV vector described herein in the manufacture of a medicament. In some embodiments, the disclosure provides for use of an rAAV vector described herein in the manufacture of a medicament for use in a method described herein.

[0249] In some embodiments, the disclosure provides a kit comprising an rAAV herein, and instructions for use. In some embodiments, the kit comprises an rAAV herein, and a package insert containing instructions for use of the kit. In some embodiments, the kit comprises an rAAV herein, and a pharmaceutically acceptable carrier, or a pharmaceutical composition comprising the rAAV and instructions for treating or delaying progression of a disease, disorder, or condition described herein in a subject in need thereof.EXEMPLARY EMBODIMENTS

[0250] The disclosure relates to the following embodiments.

[0251] Embodiment 1-1. An rAAV vector comprising an AAV capsid and an expression cassette, the expression cassette comprising a polynucleotide encoding DAO1, operatively linked to a promoter.

[0252] Embodiment 1-2. The rAAV vector of embodiment 1-1, wherein the polynucleotide encodes an amino acid sequence having at least 95% identity to an amino acid sequence selected from SEQ ID NO: 1 and 2.

[0253] Embodiment 1-3. The rAAV vector of any one of embodiments 1-1 to 1-2, wherein the polynucleotide comprises a nucleotide sequence having at least 95% identity to a nucleotide sequence selected from SEQ ID NOs: 29-30.

[0254] Embodiment 1-4. The rAAV vector of any one of embodiments 1-1 to 1-3, wherein the polynucleotide comprises a nucleotide sequence selected from SEQ ID NOs: 29-30.

[0255] Embodiment 1-5. The rAAV vector of any one of embodiments 1-1 to 1-4, wherein the promoter comprises a) a CMV promoter comprising the nucleotide sequence set forth in SEQ ID NO: 21 or a CAG promoter comprising the nucleotide sequence set forth in SEQ ID NO: 5; or b) a promoter of a gene encoding a human parotid secretory protein (“PSP”), an amylase (optionally,AMY1C), a kallikrein (optionally, KLK1), a salivary sialoperoxidase, a salivary myeloperoxidase, a P-defensin, a lingual lipase, or a lysozyme.

[0256] Embodiment 1-6. The rAAV vector of embodiment 1-5, wherein the expression cassette comprises the CMV promoter and a CMV enhancer.

[0257] Embodiment 1-7. The rAAV vector of any one of embodiments 1-1 to 1-6, wherein the expression cassette comprises a polyA sequence.

[0258] Embodiment 1-8. The rAAV vector of embodiment 1-7, wherein the polyA sequence is a BGH polyA sequence.

[0259] Embodiment 1-9. The rAAV vector of any one of embodiments 1-1 to 1-8, wherein the expression cassette comprises a WPRE.

[0260] Embodiment I- 10. The rAAV vector of any one of embodiments 1-1 to 1-9, wherein the expression cassette comprises a Kozak sequence.

[0261] Embodiment 1-11. A composition comprising an rAAV vector, wherein the rAAV vector comprises: (a) an AAV capsid, and (b) an expression cassette, wherein the expression cassette comprises a polynucleotide comprising a nucleotide sequence sharing at least 95% identity to a nucleotide sequence selected from SEQ ID NOs: 29-31, and wherein the polynucleotide is linked to a promoter.

[0262] Embodiment 1-12. The rAAV vector of any one of embodiments 1-1 to 1-10 or the composition of embodiment 1-11, wherein the expression cassette is flanked by two ITRs.

[0263] Embodiment 1-13. The rAAV vector or the composition of embodiment 1-12, wherein the ITRs are AAV2 ITRs.

[0264] Embodiment 1-14. The rAAV vector of any one of embodiments 1-1 to I- 10 and 1-12 or the composition of embodiment 1-11 or 1-12, wherein the expression cassette comprises a sequence that shares at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 23.

[0265] Embodiment 1-15. The rAAV vector of any one of embodiments 1-1 to I- 10 and 1-12 to 1-14 or the composition of any one of embodiments 1-11 to 1-14, wherein the AAV capsid comprises a VP3 that shares at least 95%, 98%, or 100% identity with AAV2 VP3 (SEQ ID NO: 8), AAV5 VP3 (SEQ ID NO: 10), AAV8 VP3 (SEQ ID NO: 12), or AAV9 VP3 (SEQ ID NO: 14).

[0266] Embodiment 1-16. The rAAV vector of any one of embodiments 1-1 to 1-10 and 1-12 to 1-15 or the composition of any one of embodiments 1-11 to 1-15, wherein the AAV capsidcomprises a VP3 that shares at least 95%, 98%, or 100% identity with AAV9 VP3 (SEQ ID NO: 14).

[0267] Embodiment 1-17. 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 comprising a nucleotide sequence having at least 95% identity to a nucleotide sequence selected from SEQ ID NOs: 29-31, and wherein the polynucleotide is linked to a promoter.

[0268] Embodiment 1-18. 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 sharing at least 95% identity to SEQ ID NO: 23.

[0269] Embodiment 1-19. The composition of embodiment 1-17 or 1-18, wherein the AAV capsid is AAV2.

[0270] Embodiment 1-20. The composition of embodiment 1-17 or 1-18, wherein the AAV capsid is AAV5.

[0271] Embodiment 1-21. The composition of embodiment 1-17 or 1-18, wherein the AAV capsid is AAV9.

[0272] Embodiment 1-22. A pharmaceutical composition comprising the rAAV vector of any one of embodiments 1-1 to I- 10 and 1-12 to 1-15 or the composition of any one of embodiments I- 11 to 1-21, and a pharmaceutically acceptable carrier.

[0273] Embodiment 1-23. The pharmaceutical composition of embodiment 1-22, wherein the composition comprises about 1 x 109to about 1 x 1013genome copies per milliliter of the rAAV vector.

[0274] Embodiment 1-24. The pharmaceutical composition of embodiment 1-22, wherein the composition comprises about 1 x 1013to about 1 x 1014genome copies per milliliter of the rAAV vector.

[0275] [Embodiment 1-25. The pharmaceutical composition of any one of embodiments 1-22 to 1-24, wherein the composition is formulated for administration into a human salivary gland, optionally wherein the human salivary gland is a parotid gland.

[0276] Embodiment 1-26. The pharmaceutical composition of any one of embodiments 1-22 to 1-25, wherein the composition is formulated for administration by direct injection, ballisticdelivery, as a sustained release dosage form, as a subcutaneous implant, as an oral spray, as a temperature-shifting gel spray, as a direct topical application, or as a multiple-tine injection.

[0277] Embodiment 1-27. The pharmaceutical composition of any one of embodiments 1-22 to 1-26, wherein the composition is formulated for use, or adaptable for use, in the treatment of a disease, disorder, or condition associated with excess histamine or histamine-induced inflammation.

[0278] Embodiment 1-28. A method of treating a condition in a subject in need thereof, the method comprising administering an effective amount of the pharmaceutical composition of any one of embodiments 1-22 to 1-27 to a salivary gland or an oral cavity of the subject.

[0279] Embodiment 1-29. The method of embodiment 1-28, wherein the pharmaceutical composition is delivered to a salivary gland of the subject.

[0280] Embodiment 1-30. The method of embodiment 1-28 or 1-29, wherein the pharmaceutical composition is delivered to a parotid gland of the subject.

[0281] Embodiment 1-31. The method of embodiment 1-30, wherein cells within the salivary gland or the parotid gland are transduced by the rAAV vector.

[0282] Embodiment 1-32. The method of embodiment 1-31, wherein the transduced cells within the salivary gland or the parotid gland express: a) an effective amount of DAO 1 in the subject’s saliva; b) an amount of DAO 1 sufficient to increase a plasma level of DAO 1 in the subject by at least 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1,000-fold compared to a plasma level of DAO1 in the subject measured prior to administration of the pharmaceutical composition.

[0283] Embodiment 1-33. The method of embodiment 1-32, wherein the transduced cells within the salivary gland or the parotid gland express an amount of DAO 1 sufficient to increase a plasma level ofDAOl in the subject by at least 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1,000-fold compared to a plasma level ofDAOl in the subject measured within one day, week, or month, of the administration of the pharmaceutical composition

[0284] Embodiment 1-34. The method of any one of embodiments 1-28 to 1-33, wherein the pharmaceutical composition is delivered to the salivary gland or the parotid gland by direct injection.

[0285] Embodiment 1-35. The method of any one of embodiments 1-28 to 1-34, wherein about 1 x 109to about 1 x 1010, about 1 x 1010to about 1 x 1011, about 1 x 1011to about 1 x 1012, about1 x 1012to about 1 x 1013, or about 1 x 1013to about 1 x 1015genome copies of the rAAV vector are administered.

[0286] Embodiment 1-36. The method of any one of embodiments 1-28 to 1-35, wherein the condition is an ocular condition.

[0287] Embodiment 1-37. The method of any one of embodiments 1-28 to 1-36, wherein the condition is associated with increased histamine production and / or increased histamine signaling.

[0288] Embodiment 1-38. The method of any one of embodiments 1-28 to 1-37, wherein the condition is an inflammatory condition.

[0289] Embodiment 1-39. The method of any one of embodiments 1-28 to 1-38, wherein the condition is an autoimmune condition.

[0290] Embodiment 1-40. The method of any one of embodiments 1-28 to 1-39, wherein the condition is an allergic condition.

[0291] Embodiment 1-41. The method of embodiment 1-40, wherein the condition is an allergic reaction to a therapeutic agent.

[0292] Embodiment 1-42. The method of embodiment 1-40, wherein the condition is an allergic reaction to a microbial agent.

[0293] Embodiment 1-43. The method of any one of embodiments 1-28 to 1-42, wherein the condition comprises histamine intolerance syndrome (“HITS”).

[0294] Embodiment 1-44. The method of any one of embodiments 1-28 to 1-42, wherein the condition comprises eosinophilic esophagitis.

[0295] Embodiment 1-45. The method of any one of embodiments 1-28 to 1-42, wherein the condition comprises a migraine.

[0296] Embodiment 1-46. The method of any one of embodiments 1-28 to 1-53, wherein the administration results in expression of DAO 1 in the cells of a salivary gland and / or a parotid gland of the subject.

[0297] Embodiment 1-47. The method of any one of embodiments 1-28 to 1-46, wherein the administration results in secretion of DAO 1 into saliva of the subject.

[0298] Embodiment 1-48. The method of embodiment 1-47, wherein secretion of DAO1 into the tear film is stimulated by a cholinergic agonist.

[0299] Embodiment 1-49. The method of any one of embodiments 1-28 to 1-48, wherein the administration results in an improvement of one or more symptoms of the condition.

[0300] Embodiment 1-50. The method of embodiment 1-49, wherein the improvement is measured about 1 months, 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.

[0301] Embodiment 1-51. The method of any one of embodiments 1-49 to 1-50, 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, at least 5 years, at least 6 years, at least 7 years, at least 8 years, at least 9 years, or at least 10 years, after the administration.

[0302] Embodiment 1-52. The method of any one of embodiments 1-49 to 1-51, further comprising administering one or more additional therapeutic agents to the subject.

[0303] Embodiment 1-53. The method of embodiments 1-28 to 152, wherein the subject is human.

[0304] Embodiment 1-54. The pharmaceutical composition of embodiments 1-22 to 1-27 for use in a method of treating a condition in a subject in need thereof comprising administering an effective amount of the pharmaceutical composition to a salivary gland, a parotid gland, or an oral cavity or a surface thereof, of the subject

[0305] [Embodiment 1-55. The pharmaceutical composition of any one of embodiments 1-22 to 1-27 for use in the manufacture of a medicament for treating a condition in a subject in need thereof.

[0306] Embodiment 1-56. A kit comprising a pharmaceutical composition of rAAV vector of any one of embodiments I- 1 to I- 10 and 1-12 to 1-16 or the composition of any one of embodiments 1-11 or 1-17 to 1-21, and a pharmaceutically acceptable carrier, and instructions for use in treating a condition in a subject, comprising administering the pharmaceutical composition to a salivary gland, a parotid gland, or an oral cavity or a surface thereof, of the subject.

[0307] Embodiment 1-57. A kit comprising a pharmaceutical composition of rAAV vector of any one of claims 1-10 and 12-16 or the composition of any one of claims 11 or 17-21, 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, comprising administering the pharmaceutical composition to a salivary gland, a parotid gland, or an oral cavity, of the subject.

[0308] Embodiment 1-58. A kit comprising a pharmaceutical composition of rAAV vector of any one of claims 1-10 and 12-16 or the composition of any one of claims 11 or 17-21, and apharmaceutically acceptable carrier, and instructions for use in treating an autoimmune condition in a subject, comprising administering the pharmaceutical composition to a salivary gland, a parotid gland, or an oral cavity or a surface thereof, of the subject.

[0309] Embodiment 1-59. A kit comprising a pharmaceutical composition of rAAV vector of any one of claims 1-10 and 12-16 or the composition of any one of claims 11 or 17-21, and a pharmaceutically acceptable carrier, and instructions for use in treating an allergy condition in a subject, comprising administering the pharmaceutical composition to a salivary gland, a parotid gland, or an oral cavity or a surface thereof, of the subject.

[0310] Embodiment 1-60. A pharmaceutical composition, comprising a) a polypeptide comprising a diamine oxidase (“DAO1”) enzyme, or a functional variant thereof, optionally wherein the polypeptide has an amino acid sequence having at least 90% identity to an amino acid sequence selected from SEQ ID NO: 1 and 2; and b) a pharmaceutically-acceptable carrier suitable for administration to a salivary gland, a parotid gland, or an oral cavity or a surface thereof, of a human subject.

[0311] Embodiment 1-61. A pharmaceutical composition, comprising a) a vector comprising a polynucleotide that encodes a polypeptide comprising a diamine oxidase (“DAO1”) enzyme, or a functional variant thereof, optionally wherein the polypeptide has an amino acid sequence having at least 90% identity to an amino acid sequence selected from SEQ ID NO: 1 and 2; and b) a pharmaceutically-acceptable carrier suitable for administration to a salivary gland, a parotid gland, or an oral cavity or a surface thereof, of a human subject.

[0312] Embodiment 1-62. The pharmaceutical composition of embodiments 1-60 or 1-61, wherein the pharmaceutically-acceptable carrier comprises water; sterile water; pyrogen-free water; phosphate-buffered saline; HEPES-buffered saline; an isotonic sodium chloride solution; a balanced salt solution; a wetting agent; a surfactant; a tonicity agent; a pH modifier; a viscosity-modifying agent; a buffering agent; a disaccharide, optionally, sucrose or trehalose; a cellulose and / or a derivate thereof; an amino acid, optionally histidine; or any combination thereof.

[0313] Embodiment 1-63. The pharmaceutical composition of any one of embodiments 1-60 to 1-62, 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.

[0314] Embodiment 1-64. The pharmaceutical composition of any one of embodiments 1-60 to 1-63, wherein the polypeptide comprises at least 20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, or 500 amino acids.

[0315] Embodiment 1-65. The pharmaceutical composition of any one of embodiments 1-60 to 1-64, wherein the formulation is a liquid formulated for application to an ocular surface, or into an ocular surface, or for intralacrimal injection, of the eye of the human subject.

[0316] Embodiment 1-66. The pharmaceutical composition of any one of embodiments 1-61 to 1-65, comprising the vector, wherein the vector is present in the composition in an amount effective to express from 100 pg / mL to 50 pg / mL of the polypeptide in a tear film of a subject subsequent to administration of the composition to the subject.

[0317] Embodiment 1-67. The pharmaceutical composition of any one of embodiments 1-61 to 1-67, comprising the vector, wherein the polynucleotide is operably linked to a promotor.

[0318] Embodiment 1-68. The pharmaceutical composition of any one of embodiments 1-61 to 1-67, comprising the vector, wherein the vector is engineered to constitutively express the polypeptide having an amino acid sequence having at least 90% identity to an amino acid sequence selected from SEQ ID NO: 1 and 2.

[0319] Embodiment 1-69. The pharmaceutical composition of any one of embodiments 1-61 to 1-68, comprising the vector, wherein the vector comprises a virus, optionally an adenoviral vector or a lentiviral vector; a plasmid; an episome; or an artificial chromosome; and optionally comprises one or more lipids, polycations, DNA-carrier proteins, histones, pseudocapsids, chimeric proteins, or endocytosis receptor proteins.

[0320] Embodiment 1-70. The pharmaceutical composition of any one of embodiments 1-60 or 1-62 to 1-65, comprising the polypeptide, wherein the polypeptide is present in the pharmaceutical composition at a concentration of 100 pg / mL to 50 ug / mL.

[0321] Embodiment 1-71. The pharmaceutical composition of any one of embodiments 1-60, 1- 62 to 1-65, or 1-70, comprising the polypeptide, wherein the polypeptide is present in the pharmaceutical composition in an amount of 500 ng to 5 ug.

[0322] Embodiment 1-72. The pharmaceutical composition of any one of 1-60, 1-62 to 1-65, I-70, or 1-71, comprising the polypeptide, wherein the polypeptide is present in the pharmaceutical composition in a unit dose amount.

[0323] Embodiment 1-73. A method of treating a disease, disorder, or condition in a subject in need thereof, the method comprising administering an effective amount of the pharmaceutical composition of any one of embodiments 1-64 o 1-76 to a salivary gland, a parotid gland, or an oral cavity or surface thereof, of the subject.

[0324] Embodiment 1-74. The method of embodiment 1-73, wherein the disease, disorder, or condition is associated with increased histamine production and / or increased histamine signaling.

[0325] Embodiment 1-75. The method of embodiments 1-73 or 1-74, wherein the condition is an inflammatory condition.

[0326] Embodiment 1-76. The method of any one of embodiments 1-73 to 1-75, wherein the condition is an autoimmune condition.

[0327] Embodiment 1-77. The method of any one of embodiments 1-73 to 1-76, wherein the condition is an allergic condition.

[0328] Embodiment 1-78. The method of any one of embodiments 1-73 to 1-77, wherein the condition is an allergic reaction to a therapeutic agent.

[0329] Embodiment 1-79. The method of any one of embodiments 1-73 to 1-78, wherein the condition is an allergic reaction to a microbial agent.

[0330] Embodiment 1-80. The method of any one of embodiments 1-73 to 1-79, wherein the condition comprises vernal keratoconjunctivitis.

[0331] Embodiment 1-81. The method of any one of embodiments 1-73 to 1-80, wherein the condition comprises atopic keratoconjunctivitis.

[0332] Embodiment 1-82. The method of any one of embodiments 1-73 to 1-81, wherein the condition comprises seasonal or perennial allergic conjunctivitis.

[0333] Embodiment 1-83. The method of any one of embodiments 1-95 to 1-104, wherein the administration results in expression of a functional diamine oxidase in one or more cells of a main salivary gland of the subject.

[0334] Embodiment 1-84. The method of any one of embodiments 1-73 to 1-83, wherein the administration results in secretion of a functional diamine oxidase into saliva of the subject.

[0335] Embodiment 1-85. The method of embodiment 1-84, wherein secretion of the functional diamine oxidase into the saliva is stimulated by a cholinergic agonist.

[0336] Embodiment 1-86. The method of any one of embodiment 1-73 to 1-85, wherein the administration is to an oral cavity or a surface thereof, or into a salivary gland or a parotid gland of the subject.

[0337] Embodiment 1-87. The method of any one of embodiments 1-73 to 1-86, wherein the subject is a human subject.

[0338] Embodiment 1-88. A kit comprising the pharmaceutical composition of any one of embodiments 1-60 to 1-72 and instructions for use in treating a condition in a human subject, comprising administering the pharmaceutical composition to a salivary gland, a parotid gland, or an oral cavity or a surface thereof, of the human subject.

[0339] Embodiment 1-89. The pharmaceutical composition of any one of embodiments 1-60 to 1-72 for use in the manufacture of a medicament for treating a condition in a human subject in need thereof.EXAMPLES

[0340] The following specific examples are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.Example 1: In vitro transfection of 293T cells with an AAV transfer plasmid encoding DAO1

[0341] A study was performed to assess expression and activity of DAO 1 polypeptide following in vitro transfection of 293 T cells with the AAV transfer plasmid depicted in FIG. 1A (referred to as “AAV.DAO1”). The AAV transfer plasmid contained a 5' and 3' AAV2 ITR (SEQ ID NOs: 16 and 17 respectively). Between the 5' and 3' ITR was a cDNA encoding a human DAO1 polypeptide and cDNA for elements to provide an optimal expression level of DAO 1 polypeptide. A schematic depicting the construct is shown in FIG. IB. The encoded human DAO1 polypeptide has the amino acid sequence set forth in SEQ ID NO: 2. The cDNA encoding the human DAO1 polypeptide was codon optimized for improved expression in human cells and has the nucleotide sequence set forth in SEQ ID NO: 29. The nucleotide sequence from 5TTR to 3 'ITR is set forth in SEQ ID NO: 23.

[0342] Plasmid expansion was performed by adding 10 ng of AAV.DAO1 plasmid to 50 pl of New England Biolabs (NEB®) Stable Competent coli High Efficiency (NEB® C3040) following the manufacturer’s rapid transformation protocol. A single bacterial colony was selected andutilized to inoculate 5 mL Luria Broth (LB) using kanamycin (50 ug / mL) as a positive selection for relevant colony clones. The starter culture was incubated for 16 hours at 30°C, and then used to inoculate 250 mL of LB / kanamycin at a 1 / 500 dilution. This culture was incubated for 16 hours at 30°C, then centrifuged for 20 minutes at 4,000 x g to pellet the bacteria. The Qiagen Hi-Speed Maxi Kit (Qiagen® 12662) was used to purify the plasmid DNA per the manufacturer’s protocol. The plasmid was verified by restriction enzyme digestion and agarose gel electrophoresis based upon fragment sizes.

[0343] 293T cells were plated in 6 well plates at 6.5 xlO5cells per well and transfected with 2.5 pg of AAV.DA01 plasmid DNA utilizing Lipofectamine® 3000 per the manufacturer’s protocol. Control cells were transfected with an empty vector. The cells were grown out and then plated at 5.5xl05on 35 mm glass bottom tissue culture plates.Immunofluorescence

[0344] Immunofluorescence imaging was performed to detect DA01 expression in transfected cells.

[0345] 293T cells were plated at 5.5xl05and PANC-1 cells were plated at 4 xlO5on Poly-L- Lysine (VWR 103701-192) coated 33mm glass bottom TC dish (MatTek® P35G-0-10-C). Twelve to twenty-four (12-24) hours after plating the cells were transfected according to the Lipofectamine® 3000 (Thermo Fisher Scientific L300015) transfection protocol using 3.75pl Lipofectamine® 3000 and 2.5ug pAAV-DAO plasmid per well. 24 hours post transfection the media was aspirated, and the cells were washed twice with pre-warmed PBS (37°C). 500 pl of 4% paraformaldehyde was added and incubated for 10 minutes at room temperature. The cells were washed 2 times with PBS. 500pl of blocking buffer was added and the cells were incubated for 45 minutes at room temperature. Blocking buffer consisted of PBS, 0.1% Triton X and 1% bovine serum albumin (BSA). The cells were stained with a primary antibody to diamine oxidase and a fluorescently-tagged secondary antibody. The cells were counterstained with DAPI and then imaged on a fluorescent microscope. Specifically, primary antibodies were diluted in blocking buffer then 200 pl of the diluted mixture was added to the central glass portion of the dish and incubated for 2 hours at 37°C. Primary antibody concentrations were optimized experimentally, and a 1 :50 dilution was used. The cells were washed 3 times for 15 minutes with PBS. Fluorescently labeled secondary antibodies and DAPI were diluted 1 :400 in PBS, added to the glass portion of the dish, and incubated at room temperature for 1 hour (protected from light). Thecells were then washed 3 more times for 15 minutes with PBS. The cells were then imaged on a Keyence® BZ-X series microscope.

[0346] As shown in FIG. 2, expression of DAO 1 protein (as indicated by the bright and punctate signal) was measured in 293T cells transfected with the AAV transfer plasmid, but not in control cells.Western Blot

[0347] Western blot was performed to detect DAO1 expression in conditioned media from AAV.DAO1 -transfected cells as compared to control cells, with detection of DAO 1 performed using a rabbit anti-DAOl antibody and labeled anti-rabbit secondary antibody. For conditioned media collection, cell media was aspirated at 24 hours post-transfection and washed twice with 2 mL pre-warmed Opti-MEM® (Thermo Fisher Scientific 31985-070). 1.4mL of Opti-MEM® was then added and conditioned media was collected at 2, 4, and 6 hour intervals. Immediately following collection, IX protease inhibitor was added and the conditioned media was placed on ice for 5 min, centrifuged at 4°C 12,000 x g for 5 minutes to remove culture debris. The conditioned media supernatant was then concentrated 40x using an Amicon® Ultra-2 Centrifugal Filter Unit (Millipore UFC201024) per manufacturer’s protocol and stored at -20 °C.

[0348] Western blot was also performed using whole cell extracts. Whole cell extracts were prepared by aspirating cell media at 24 or 48 hours post-transfection. The cells were washed twice with 2 mL of 4°C PBS, then lysed using 300 pl RIPA buffer (Sigma R0278) containing protease / phosphatase inhibitor (Cell Signaling 5872S) diluted to IX from 100X stock. The whole cell extract was then centrifuged at 12,000 x g for 5 min at 4°C to remove any cellular debris and then supernatant was aliquoted and stored at -20°C. The collected whole cell extract (per above methodology) was quantified using a micro- bicinchoninic acid assay (BCA) protein assay kit (Thermo Fisher Scientific 23235) per manufacturer’s recommendations. Sample concentrations were normalized by dilution with double distilled water (ddELO) and Laemmli buffer. The extracts were then heated to 100°C for 10 minutes. Samples were electrophoresed on 10% SDS-PAGE gels and then transferred to a PVDF membrane using the Invitrogen iBlot® 2 Dry Blotting System. The membrane was blocked using 5% Blotting-Grade Blocker (Bio-Rad Laboratories 1706404) for 1 hour at room temperature on a rotating platform. Primary antibodies were diluted in 5% blocking buffer with 0.1% Tween 20 and incubated at 4°C overnight on a rocking platform. Optimized primary antibody concentrations were determined experimentally. The membrane waswashed in PBS with 0.05% Tween 20 for 10 minutes on a rotating platform. The washing step was repeated 4 times. HRP conjugated secondary antibodies were diluted (1 : 10,000) in 5% blocker with 0.1% Tween 20 and incubated at room temperature on a rotating platform for 1 hour. The previous washing steps were repeated with the final wash being PBS alone. The membrane was incubated for 1 minute in SuperSignal® West Pico Plus (Thermo Fisher Scientific 34577) and chemiluminescent detection was done on a BioRad ChemiDoc® MP.

[0349] As a control, Western blot was also performed using a human DAO1 recombinant protein (40ng) and a mouse anti-his antibody. Western blot of GAPDH (30pg / well) served as a loading control.

[0350] As shown in FIG. 3A, DAO1 polypeptide was observed in media obtained from transfected cells, but not control cells. The DAO1 polypeptide had the same molecular weight as the human DAO 1 recombinant protein that was used as a positive control (FIG. 3B). Also, DAO 1 of the expected size (kDa) was detected in whole cell extracts obtained from transfected 293 T cells, where recombinant DAO1 served as a positive control and GAPDH as a loading control.

[0351] Additionally, DAO1 expression was measured in conditioned media at 2, 4, and 6 hours after a fresh media change. A significant level of DAO1 was detected in as little as 2 hours following media change.

[0352] Together these data demonstrate expression and secretion of DAO 1 into the cell culture media.DAO1 Enzymatic Activity Assay

[0353] To assess the functional activity of the expressed DAO1 protein, an activity assay was conducted. The principle of the assay is a substrate is provided that in the presence of active DAO1 results in a reaction where hydrogen peroxide (H2O2) is released. The H2O2 released by the cleavage of a substrate reacts with a second substrate and produces a fluorescent signal that excites at 535 nm and emits at 587 nm. The amount of fluorescence recorded at 587 nm indicates the amount of enzymatic activity.

[0354] The experimental approach involved plating 293T cells in a 6-well plate format and transfecting with AAV.DAO. Negative control cells were non-transfected. Twenty-four hours post-transfection, the media was removed and replenished with 2 mL / well maintenance media. Twenty-four later the conditioned media and whole cell extract (WCE) were collected. Upon collection, protease inhibitor was added, and the conditioned media / WCE was placed on ice for 5min and centrifuged at 4°C 12,000 x g for 5 minutes to remove debris. The media / WCE was aliquoted and stored at -20°C. A micro-BCA assay was conducted to quantify protein levels of the WCE. Standards were prepared using 10 mM H2O2 supplied with the Diamine Oxidase Activity Assay Kit (Sigma MAK351). The serially diluted standards, undiluted AAV.DAO conditioned media, 100 ng WCE and control samples were added to the 96-well reaction plate. A reaction mix was prepared using DAO Assay Buffer, substrate, enzyme mix and probe. The reaction plate was then incubated at 37°C for 90 minutes and kinetic measurements were taken at 30-, 60-, and 90- minutes post-incubation. Measurements were taken at 535 nm excitation wavelength and a 587 nm emission wavelength using a Molecular Devices ID3 plate reader. DAO1 activity was measured in nmol H2O2 generated in 90 minutes (experiment 1) or pmol / min / pl (experiment 2 using kinetic measurements).

[0355] As shown in FIG. 4A, a calibration curve was generated by measuring fluorescent signal (535 nm / 587 nm) in the presence of increasing concentrations of H2O2. The assay was used to measure the activity of DAO 1 in conditioned media obtained from AAV.DAO 1 -transfected 293 T cells or cell lysates combined with the conditioned media and compared to activity measured in conditioned media obtained from non-transfected 293T cells. As shown in FIG. 4B, substantially higher H2O2 was generated for conditioned media obtained from AAV.DAO 1 -transfected 293T cells alone or combined with cell lysates as compared to conditioned mediate obtained from nontransfected 293T cells. Kinetic measurements of DAO1 activity in conditioned media of transfected 293T cells at 30, 60, and 90 minutes demonstrated target modulation capability and functional activity of DAO 1 expressed by AAV-DAO.

[0356] This demonstrates that the DAO1 protein present in the cells and expressed into the cell culture media is able to cleave the given substrate and demonstrates functional enzymatic activity.Example 2: Intra-Lacrimal Gland Gene Therapy using rAAV vector

[0357] This example shows a 9-day pilot study of single dose rAAV vectors administered as an intra-lacrimal gland injection followed by a single dose of varenicline administered as an intranasal dose to Dutch-Belted rabbits. It evaluates the effectiveness and tolerability of a panel of rAAV vector embodiments administered one time via injection to the lacrimal gland. While this study did not evaluate treatment of the salivary glands of test subjects, the data from this study is believed to further support the safety and effectiveness of rAAV gene therapy directed tomammalian glands generally, and by extension the administration and use of such constructs in connection with the salivary glands, as described elsewhere herein.

[0358] Each rAAV vector composition in the panel is tested at two concentrations (l >< 1012GC / mL and 6.2* 1012GC / mL). The panel of rAAV vectors include embodiments with capsid proteins having AAV2, AAV5, AAV8, and AAV9 serotypes. The expression cassette delivered by the rAAV vector encoded an enhanced green fluorescent protein (eGFP) transgene that is operatively linked to a CMV promoter (FIG. 5). On day 9 following injection of the rAAV vector, the animal is given an intranasal dose of varenicline. The varenicline induces tear production in the animals such that eGFP delivered to the lacrimal by the rAAV vector and expressed under control of the CMV promoter in cells of the lacrimal gland will be secreted into the tear film and onto the ocular surface of the animal. Two main objectives will be achieved using this approach:(1) Test the feasibility of capsid protein serotypes AAV2, AAV5, AAV8, and AAV9 to deliver a transgene to cells in the lacrimal gland resulting in measurable CMV promoter-driven expression of the transgene within the cells.(2) Assess the feasibility of increasing the relative amount of the transgene encoded by the by the expression cassette into the tear film and onto the ocular surface of the animal.

[0359] Animal studies were carried out in the Charles River Laboratories (CRL) facilities by CRL staff scientific personnel.Animal Test System, Husbandry, and In-life Monitoring

[0360] The animals used in this study were male Dutch-Belted rabbits between the ages of 4 to 5 months and weighed between 1.3 to 2.3 kg. Animals were acclimated for 10 days prior to the start of treatment. Each animal was housed individually and cared for using standard caregiving protocols including regular environmental conditions, feeding schedules, and veterinary care. rAA V Vector Compositions and Formulations

[0361] In this study, the panel of compositions comprising rAAV vectors containing an expression cassette encoding an eGFP transgene operatively linked to a CMV promoter are provided for intralacrimal injection under the conditions in Table 4. Each composition contains an rAAV vector with a different AAV capsid protein serotype. The compositions are labeled as OC-lOOa-d, each corresponding to a different AAV capsid protein serotype. Dose formulationsfor intralacrimal injection were prepared using clean procedures at the target concentrations described below in Table 6 by diluting with phosphate buffered saline solution.Table 4. Summary of rAAV CompositionsIntralacrimal Injection of rAA V Compositions

[0362] Animals were dosed via intralacrimal injection on day 1 of the study. A summary of the formulation concentration for each composition tested, dose volume, dose frequency, and number of animals and lacrimal glands is found in Table 5. Prior to the injection, animals were anesthetized by intramuscular injection of dexmedetomidine (0.25 mg / kg) followed by an isoflurane / oxygen mix through a mask to maintain anesthesia, if necessary. A topical antibiotic was applied to each eye after dose administration. On day 9 of the study, animals were given intranasal administration of varenicline tartrate (50 pL per nostril of 1.2 mg / mL varenicline) to induce tear production.Table 5. Summary of Experimental DesignBioanalysis

[0363] Blood was collected on Day 1 before dosing, and again on Day 8 and 9 (after the intranasal dosing at approximately 1 hour post dose) from an auricular vessel from all animals. Blood samples were placed on ice until plasma is separated by centrifugation. Plasma samples were separated into 250 pL aliquots and frozen at -80°C for subsequent analysis.

[0364] A Schirmer tear test were performed to collect eye moisture from the animals on Day 8 and 9. Test strips were placed inside the lower eyelid for approximately 1 minute. The paper was removed and placed into separate tubes and frozen at -80°C for subsequent analysis.

[0365] Plasma samples and Schirmer’s test strips were analyzed for concentration of eGFP and eGFP mRNA concentration using a validated procedure at Syneos analytical laboratories.Immunohistochemistry of Lacrimal Gland Tissue

[0366] Animals were euthanized on day 9 following 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 lab standard operating procedure. Lacrimal gland IHC samples were stained for eGFP and subject to microscopic evaluation.Results

[0367] Microscopic evaluation was performed to determine the efficiency of eGFP expression in lacrimal gland tissues dosed in vivo with the rAAV compositions. Isolated positive acinar cells in the IHC samples had pink to red cytoplasmic staining indicative of GFP expression (FIGS. 6A- 6K; exemplary staining indicated by black arrows). Positive eGFP expression was observed in for the rAAV composition containing an AAV2 capsid protein (OC-lOOa) at 6.2* 1012GC / mL (FIG.6A), the rAAV composition containing an AAV5 capsid protein at both 1 x 1012GC / mL (FIG. 6B) and 6.2x l012GC / mL (FIGS. 6C-6H), and the rAAV composition containing an AAV9 capsid protein at 6.2 1012GC / mL (FIGS. 6I-6K).Conclusion

[0368] The results from this example show 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 the AAV2, AAV5, or AAV9 serotypes can be used to deliver an expression cassette to cells within the lacrimal gland. Furthermore, the results demonstrate delivery of an expression cassette containing a transgene operatively linked to a CAG promoter sequence results in expression of the transgene in the cells of the lacrimal gland. This study also provides a proof of concept for rAAV gene therapy directed to other mammalian glands, e.g., the salivary glands as described elsewhere herein.Example 3: Expression of EGFP Transgene in Porcine Lacrimal Gland Delivered by rAAV via Intralacrimal Injection

[0369] The study objective was to assess if the lacrimal gland is able to be leveraged as a method to modify or enrich the tear film with a protein of interest in pigs. Subsequently, in vivo study was performed to test if 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. To get cDNA encoding EGFP into acinar cells, the approach was to inject the lacrimal gland with an adeno-associated virus (AAV) which contained cDNA encoding for secreted EGFP (secEGFP). To create each AAV of 2 different serotypes (2 and 9) for secEGFP, an AAV transfer plasmid was generated which contained between the inverted terminal repeats (ITRs) the essential elements for secEGFP expression. The DNA sequence between the ITRs was packaged into the AAV (FIG. 5) that was manufactured.Design Analysis and Methodology

[0370] Research grade AAVs for secreted EGFP (serotypes 2 and 9) were synthesized at Sirion and were provided at a stock concentration of 5xl012. The AAVs were in vitro tested by CJ Solutions using HEK 293T cells and ELISA to ensure that the manufactured AAVs would transduce cells. At Texas A&M, eight domestic pigs received a one-time intralacrimal glandinjection of EGFP with the right (OD; oculus dexter) gland receiving a low dose and the left (OS; oculus sinister) gland receiving a high dose. Six weeks after the first injection, a second injection with AAV2 and AAV9 high doses were performed. The study assessed EGFP expression at Day 35. Following tear EGFP level confirmation, the study was terminated 8 weeks after the second injection to assess the presence of EGFP in the lacrimal glands and assess any potential inflammation or gland abnormalities. (Tables 6 & 7). In the study, nasal spray dosing was administered between weeks 3 to 4 (Table 8)Table 6: Study plan for in vivo study of AAV2-secEGFP and AAV9-secEGFP in domestic Pigs-Table 7: Injected dose and volume of AAV.vg = viral genomesTable 8: OC-Ol nasal spray dosing was from day 21 to day 28.mcg = micrograms

[0371] Following the second AAV-secEGFP injection, tears were collected from each eye via Schirmer strips on day 82. Tears were collected by placing a Schirmer’s Tear Test strip in the lower conjunctival cul-de-sac and leaving in place for 2 minutes. Tear protein was extracted from the Schirmer’s Tear Test strip and mesoscale discovery (MSD) analysis was conducted to detect the presence of EGFP protein in the tears.

[0372] Lacrimal gland was collected for ocular histopathology on Day 103 and samples were sent to Zyagen, Inc. (San Diego, CA) for EGFP immunohistochemistry (IHC).

[0373] ELISA results showed that the AAV serotypes that were manufactured could transduce HEK 293T cells and produced secreted EGFP in vitro. EGFP expression in the tear samples was confirmed by MSD analysis 82 days after AAV transduction with eGFP some levels >400 pg / mL as well as by IHC (FIG. 7). IHC indicated that EGFP expression was within the acinar cells with greater acinar cell infectivity observed for AAV2 compared to AAV9. Additionally, transduction of ductile epithelial cells was observed for AAV9 injected lacrimal glands (FIG. 8). Hematoxylin and eosin staining of pig lacrimal gland after repeat AAV injections did not show any inflammatory infiltrate, atrophy or edema (FIG. 9).

[0374] Porcine lacrimal gland that is injected with either AAV2-secEGFP or AAV9-secEGFP expressed the EGFP transgene product in acinar cells as well as ductile epithelial cells. The EGFP that was expressed in the lacrimal gland was found to be secreted into the tear film. Additionally, no safety signals or inflammatory infiltrates were observed in any animals after repeat injections of AAV2 or AAV9 regardless if they initially received a low or high dose of AAV during the first injection. The results of this study demonstrate that the acinar cells of the lacrimal gland are a target for a gene therapy approach to modify and / or enrich the tear film.Example 4: Expression of rAAV Transgene in Porcine Lacrimal Gland in Combination with OC-1 Nasal Spray

[0375] This example describes a study in pigs to assess expression of rAAV encoding a model protein following a single intralacrimal gland injection. The objective of the study is to determine expression of mRNA encoding the model protein (referred to in the Examples and Figures as “protein A”) in the lacrimal gland and transgene protein levels in tears following a one-time injection of an AAV encoding protein A (referred to in the Examples and Figures as “AAV- protein A”) to transduce the porcine lacrimal gland. The study is further designed to confirm expression and secretion of protein A following intralacrimal gland injection and to assess the relative amount of protein present on the ocular surface upon stimulating tear production with varenicline nasal spray. Varenicline (“OC-1”) is the following compound:

[0376] The AAV-protein_A plasmid encodes from 5' to 3' an AAV2 5'ITR, a CMV enhancer / promoter, an intronic sequence comprising a Kozak, an open reading frame encoding protein A, a Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element, polyA sequence, and an AAV2 3'ITR.

[0377] The study parameters are detailed in Table 9. The administration and dosing are shown in Table 10.Table 9: Study plan for in vivo evaluation of intralacrimal gland injection of rAAV in domestic pigsTable 10: Injected dose and volume of AAVAAV-protein A stock concentrations are 5 x 1012vg / mL and are provided in 500 pl. aliquots and stored at -20°C (short-term, <2 years) or -80°C (long-term). In this study, there is only 1 dose, (l x 1011vg).

[0378] The timeline for the porcine study is shown in FIG. 10. For all pigs (N=14), day 0 is the day of injection. Tears are collected from each eye via Schirmer strips (applied for approximately 2 minutes then removed) on days 7, 14, 21, 28, 35, 42, 60 / 61, and 90. Schirmer’s Strips are immediately cut above the fluid or dye line with a pair of scissors. The bottom portion (tearsaturated) of the Schinner’s Strip is then placed in a microcentrifuge tube and kept on ice until transferred to freezer (-80°C). On days 14 and 42, tear collection occurs first and then nasal spray dose is administered. Approximately 2 minutes after administration of nasal spray dose a second day tear collection occurs. For days 22 to 28, OC-Ol nasal spray is administered twice daily (at least 6 hours between administrations) to both nares on days 22 to 27 and once on day 28. Tear collection on day 28 will occur 2 minutes after administration of nasal spray.

[0379] On day 90, gross pathology is performed and body weights are assessed. Additionally, one lacrimal gland from each animal is collected for ocular histopathology by immunohistochemistry (IHC). After harvesting the lacrimal glands, they are fixed in 10% formalin for 24 to 48 hours (at room temperature) then to 70% EtOH and store at 4°C. The volume in the tubes is maintained at 5 times that of the tissue and the tissue is fully submerged.

[0380] A second lacrimal gland is collected, rinsed with phosphate-buffered saline and immediately placed in RNA-Later. The sample is snap frozen in liquid nitrogen. ~0.5-lcm square pieces of tissue from heart apex, kidney and liver are collected and immediately placed in a 2mL cryotube and snap frozen in liquid nitrogen. Cryotubes are later stored at -80°C until shipped for mRNA analysis.

[0381] mRNA analysis is performed to analyze AAV-derived gene expression of protein_A in the lacrimal glands of domestic pigs, using a developed one-step duplex RT-qPCR method. Each lacrimal gland is homogenized and lysates loaded into a QIASymphony® for automated RNA extraction utilizing silica-based RNA purification, magnetic separation and enzymatic removal of DNA. The AAV-protein_A vectors contain the bovine growth hormone (bGH) polyA sequence at the 3’ untranslated region of the transgene. Protein A mRNA is analyzed using primers and a probe targeting the bGH poly A sequence, with sequences shown in Table 11. Amplification is for 76 bp of the bGH sequence. The extracted total RNA samples are analyzed for both bGH mRNA copy numbers and the Ct values of porcine endogenous Hprtl mRNA on 96-well plates using the QuantStudio® 7 Flex Real Time PCR system and a one-step duplex RT-qPCR method (see Table 12). Each plate includes a standard curve, negative controls and quality control samples, which are prepared separately to avoid cross-contamination. Each standard curve includes bGH standard DNA levels at 108, 107, 105, 104, 103, 102, 50, 25 and 0 copies per well. RT-qPCR of the RNA samples is performed in duplicate wells up to 100 ng per well. The bGH mRNA copy number in each RT-qPCR well is interpolated from the bGH DNA standard curve (Acceptance Criteria: R2 > 0.980). A two-fold multiplication step is used to adjust for interpolatingsingle stranded (ss) mRNA from the double stranded (ds) standard curve, and the mean copy number of the two replicate wells will be reported as copies of ss bGH mRNA per 100 ng of RNA sample. In addition, each RNA sample is tested using qScript® XLT One-step RT-qPCR (without added reverse transcriptase) to monitor the potential vector DNA contamination in the RNA samples.Table 11: primer and probe sequences for bGH sequenceTable 12: RT-qPCR assay conditionsReagent Final concentration2x qScript XLT One-Step ToughMix Master Mix* lx bGH-F3 Forward Primer 200 nM bGH-R3 Reverse Primer 800 nM bGH-P3 Probe 150 nMPorcine Hprtl-Fl Primer 200 nMPorcine Hprtl-Rl Primer 800 nMPorcine Hprtl-Pl Probe 150 nMPorcine lacrimal gland matrix RNA** 100 ng bGH Standard DNA** 0 - 108copiesNuclease-free water To final volume of 20 pL*ToughMix is replaced with One-Step RT-qPCR Master Mix, without reverse transcriptase, when analyzing RNA samples for monitoring potential vector DNA contamination.Example 5: Expression of Functional DAO1 in 293T Cells

[0382] This example describes a study which evaluated the expression of a DAO1 transgene in 293T cells using a colorimetric histamine quantification assay. The objective of the study was to confirm whether transfection resulted in the expression of functional DAO1.Background

[0383] There are five established biogenic amine neurotransmitters identified to date, which include dopamine, norepinephrine, epinephrine, histamine and serotonin formed bydecarboxylation of amino acids or an amination event of ketones or aldehydes. Histamine, in particular, is formed by the decarboxylation of histidine and described over 115 years ago (Windaus A, Vogt W. “Synthese des Imidazolyl-athylamins.” Ber. Dtsch. Chem. Ges. 1907;40:3691-3695). The degradation of histamine by DAO1 primarily occurs extracellularly, which is the most relevant from a therapeutic approach when trying to modulate a histamine response from histamine ingestion or localized mast cell degranulation resulting in histamine release into the extracellular matrix and systemic circulation. One mechanism by which histamine is degraded by DAO1 is through oxidative deamination of histamine. Oxidation is the first step in degrading / metabolizing histamine for eventual release of the byproducts from the body in the form of urine components.DAO1 Enzymatic Activity Assay

[0384] In this assay, 293T cells transfected with a plasmid engineered to express DAO1, and non-transfected 293 T cells (a negative control) were generated and / or cultured using standard protocols, and conditioned media from each sample was collected and concentrated using Amicon® protein concentrators. DAO1 enzymatic activity was evaluated using a Sigma-Aldrich® Histamine MAK432 Histamine Quantification Assay Kit, subject to a modified protocol, as explained below.

[0385] 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, which is coupled to the reduction of the included formazan WST reagent. The intensity of the product color, measured at 450 nm, is directly proportional to the histamine concentration in the sample. The MAK432 kit includes a “Reaction Enzyme” component to oxidize the test sample and histamine standards that can be used to prepare a histamine standard curve. For the purposes of determining if the DAO1 transgene was functional and able to oxidize histamine, the manufacturer’s protocol was modified by eliminating the use of the provided Reaction Enzyme. Briefly, 25 pg / ml histamine and phosphate buffered saline were combined with 600 pL of conditioned media (from the 293T cells transfected with the DAO1 plasmid, or the 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 conducted at 37 °C on a 150 rpm rotary shaker. The manufacturer’s detection solution was then added and stopped after 35 minutes. FIG. Il l shows the standard curve generated for histamine with an R2value of 0.998. FIGs. 14-15 demonstrate the kinetics ofhistamine degradation by increasing concentrations of recombinant porcine DA01 at 4 (FIG. 14) and 24 (FIG. 15) hours. FIGs. 14-15 demonstrate the ability of the 293T cell conditioned media transfected with the DAO1 plasmid to significantly degrade histamine after 4 (FIG. 14) 24 (FIG. 15) hours of exposure, as compared to the non-transfected negative control.

[0386] As illustrated by the provided histamine level data, transfection with a DA01 expression plasmid resulted in a substantial reduction in histamine levels as compared to the negative control (e.g., a reduction of >50% after 4 hours, and by >90% after 24 hours), confirming that transfection of 293T cells with the DAO1 plasmid resulted in the expression of functional DAO1.

[0387] Example 6: Diamine Oxidase In Vivo Study (Lacrimal Gland)

[0388] In order to test the ability of the AOC1 transgene product, diamine oxidase, to be expressed, secreted and transported to the ocular surface following the direct injection of an AAV into lacrimal gland, an in vivo study (Tables 13-15) utilizing Sus scrofa was conducted. An adeno- associated viral vector (AAV) utilizing the capsid protein from AAV9 was utilized for this study. Briefly, HEK293T cells were triple transfected with the plasmid DNA comprising the gene-of- interest (AOC1) described in Example 1, along with a helper plasmid and a AAV9 RepCap plasmid using polyethyleneimine (PEI) as the transfection reagent. AAV particles were collected and isolated 48 hours after transfection and then subjected to multiple purification steps including iodixanol gradient centrifugation to increase the percentage of full capsids in the final formulation. A PBS and 0.001% Pluronic® formulation buffer were utilized after the AAV particles were concentrated via centrifugation at a final concentration of 5xl012vector genomes per milliliter (vg / mL). A purity check was conducted using 10% SDS-PAGE and silver stain. Only VP1, VP2 and VP3 proteins in a 1 : 1 : 10 stoichiometry were detectable on the SDS-PAGE gel supporting a purity >95% (FIG. 16).Table 13: Experimental Study Design Protocol for In Vivo Porcine Study to Transduce the Lacrimal Glands of Sus scrofa domesticus and Assess Tear Protein Levels of the Transgene Product

[0389] Key Evaluations and Assessments:

[0390] In-life:• Mortality / morbidity: Twice daily• Clinical observations: Twice daily• Food consumption (quantitative): Once daily• Body weights: Prior to dosing on day 1, and on tear collection days• For all pigs (N=2): Day 0 is day of injection. Tears collected from each eye via Schirmer strips (applied for approximately 2 minutes then removed) on days 7, 14 and 30. Schirmer’s Strips immediately cut above the fluid or dye line with a pair of scissors. The bottom portion (tear saturated) of the Schirmer’s is placed in a microcentrifuge tube and kept on ice until transferred to freezer (-20C).

[0391] Terminal (Day 28-30):• Gross pathology• Body weightsTable 14: Dose Levels and Location of the Lacrimal Gland Transduction

[0392] Method for loading syringe:1. Thaw one new stock tube of the following - AAV9-DAO. Double-check that all labels are correct, and each tube has 500pL. Thaw stock tubes on wet ice for 30 minutes. Ensure it has been thawed and then vortex for 5 seconds before aliquoting 425 pL (INJECTION STOCK) to a new labelled Eppendorf® tube. KEEP INJECTION STOCK ON WET ICE. Place the 3 original tubes (the 3 stock tubes now with 75 pL each) on dry ice for storage and shipping.2. 1 dose is 1E11 vg. Use a tuberculin syringe with a 27-29 gauge needle to accurately measure 40pL volume. The syringe will be loaded just minutes before each injection.3. Vortex INJECTION STOCK tube for 2-3 seconds before drawing 40pL into syringe. Place injection stock tube back on wet ice.4. Inject both the right (OD) and left (OS) lacrimal glands with the 40pL dose. Each AAV dose will be SLOWLY administered by direct injection (40pL) into the lacrimal gland with care taken to accurately deposit the entire vector dose into the gland. Slowly remove the needle after the injection has been deposited. Use 2 pigs injected into each left and right lacrimal gland serotypes (N=4 eyes total) and the dose is 40pL of lei 1 vg per lacrimal gland.

[0393] Method for tear collection:1. Collect tears using Schirmer strips (applied for 2min). After tear collection, immediately put the strip into to its own storage tube and immediately place these sample tubes on wet ice. After all samples for a particular day have been collected, move all sample tubes to a -80°C freezer for storage.2. Ship these frozen sample tubes (with Schirmer strips) on dry ice to KCAS for diamine oxidase ELISA.

[0394] Materials:- AAV9-DA0Wet IceDry iceGlovesOne 1.5mL microcentrifuge tube rackEppendorf DNA LoBind® Microcentrifuge 1 ,5mL tubes (Eppendorf, 022431021) TimerScissorsForcepsBenchtop vortexTuberculin syringes (27-29 gauge needle)Schirmer strips (Eagle Vision® Color Bar)Pipettes and pipette tips

[0395] Preferred Pipettes & Tips:Eppendorf Research Plus G (Sigma, EP3123000918) - 3 Eppendorf® pipettes(20pL, 200pL, lOOOpL)Eppendorf epTIPS LoRetention lOpL Tips (Sigma, ETIPLRQ10-EP) - 10 trays x 96 tipsEppendorf epTIPS LoRetention 200pL Tips (Sigma, EP022493022-960EA) - 10 trays x 96 tipsEppendorf epTIPS LoRetention lOOOpL Tips (Sigma, ETIPLRQ1000-EP) - 10 trays x 96 tips27- to 32-gauge insulin syringe

[0396] Calculation for Dilution of AAVs:1) Each stock is provided as 5 x 1012vg / mL (Cl).2) Desired dose is 1 x 1011vg, which is 2.5 x 1012vg / mL (C2)3) Formulate 300 uL of working stock (V2) for injections and allow for manipulation into syringe.4) Cl x C2 = VI x V2: (5 x 1012x C2) = 2.5 x 1012x 3005) Dilute 150 uL of 5 x 1012vg / mL stock in 150 uL of 0.9% NaCl to create working solution.Table 15: Identification (ID) Numbers for the Animals Utilized for Study and Their Body Weights at Baseline and Over the Course of the Study

[0397] Using the protocol and experimental design described above, a dose of 1 x 1011vg was injected into each lacrimal gland of Animal ID’s 23205 and 23292 on Day 1 and tears were collected using Schirmer’s Tear Strips (EagleVision® color bar) on Days 7, 14 and 21. Schirmer’s scores were recorded individually for the left (OS) and right (OD) eyes and are presented in Table 16. Tear production appeared to be normal for this species with Schirmer’s Scores all being within the expected range for this species for the duration of the study. Tear samples collected on Schirmer’ s Strips were frozen, tear proteins were extracted, and an ELISA was conducted to assess the diamine oxidase levels in the tear film at Days 7, 14 and 30. A standard curve for DAO concentration was generated ranging from 3.13 to 200 ng / mL as shown in Table 17 according to the manufacturer’s recommendations (MyBioSource™ Catalog: MBS2502445). Experimental samples generated from tear protein extraction from Schirmer’s Tear Strips at Days 7, 14 and 30 were run on the sample ELISA plate as the standard curve and blank control samples as presented in Table 18.Table 16: Schirmer’s Scores for Each Animal at Day 7, 14 and 30Table 17: Details of Data Generated for Creation of the Diamine Oxidase Enzyme LinkedImmunosorbent Assay Standard Curve Ranging from 3.13. to 200 ng / mL“RE” is relative error; “CV” is coefficient of variation.Table 18: Data Generated for the Experimental Samples Obtained From ExtractingProtein From the Schirmer’s Strips Tear Samples Collected at Days 7, 14, and 30Tear Protein Extraction Samples

[0398] Results:There were significant levels of diamine oxidase in the tears of Sus scrofa transduced with AAV- DAO at all time points collected (Table 18), demonstrating successful and robust transduction of the lacrimal gland, expression and secretion of the AOC1 transgene product, and transportation of the transgene product to the ocular surface via aqueous normally produced by lacrimal gland cells. Example 7: Diamine Oxidase In Vivo Study (Parotid Gland)

[0399] A study was conducted in order to evaluate human DAO protein levels in saliva following a one-time injection of 5 x 1012vg / mL of an AAV expressing a human DAO transgene using AAV capsid serotype 9 to transduce the porcine (Sus scrofa domesticus') parotid gland(glandula parotis). An overview of the study is provided in Table 19. In brief, two (2) domestic pigs (Sus scrofa domesticus) were divided into two experimental study groups. Each group received the same test article and concentration, AAV9 delivering a transgene encoding human DAO, in different volumes. The test article, “AAV9-DA0” in this Example 7, was prepared with the plasmid DNA comprising the gene-of-interest (AOC1) described in Example 1 herein, along with a helper plasmid and a AAV9RepCap plasmid. The test article was administered at a concentration of 5 x 1012vg / mL (“vg” is vector genomes). Single parotid gland injections via a 1 mL Terumo syringe with a 25 gauge by 5 / 8” needle into the left and right parotid glands were performed at Time point 0 hrs, and a physiological saline flush was utilized for group 1. Test groups and injection volumes are summarized in Tables 20 and 21. Saliva samples were collected via pipette on Day 7, 14, and 21. Levels of DAO protein were assessed via a human DAO ELISA kit (MyBiosource MBS2502445).Table 19: Experimental Study Design Protocol for In Vivo Porcine Study to Transduce the Parotid Gland of Sus scrofa domesticus with an AAV configured to cause expression of human DAOTable 20. Experimental DesignTable 21. Experimental Group Details

[0400] Study Formulation:

[0401] AAV9-DAO is a recombinant adeno-associated virus serotype 9 (AAV9) vector with a cytomegalovirus (CMV) immediate early enhancer, a CMV immediate early promoter driving the expression of the human diamine oxidase gene product protein. AAV9-DAO is supplied in 0.5 mL aliquots at a concentration of 5 x 1012vector genomes per milliliter (vg / mL).Table 22. AAV9-DAO, Detail of Test Article

[0402] Key Evaluations and Assessments:

[0403] In-life

[0404] Mortality / morbidity: Daily

[0405] Animal observations: Daily

[0406] Body Weights: Per Protocol

[0407] For all pigs (N=2): Day 0 is day of injection.

[0408] Saliva will be collected via pipette on days 7, 14, 21.

[0409] Method for loading syringe:

[0410] Thaw one new stock tube. Double-check that all labels are correct, and each tube has 0.5 mL. Thaw stock tubes on wet ice for 30 minutes. Ensure it has been thawed and then vortex for 5 seconds. Keep injection stock on wet ice.

[0411] Vortex injection stock tube for 2-3 seconds before drawing appropriate volume into syringe. Place injection stock tube back on wet ice.

[0412] Using a 1 mL Terumo syringe with a 25 gauge by 5 / 8” needle to measure appropriate injection volume. The syringe will be loaded just minutes before each injection.

[0413] Using portable probe ultrasound, the needle will be inserted through the skin at the base of the masseter muscle into the parotid bland. Once verification of the needle within the parotid gland is confirmed via ultrasound, the appropriate volume of AAV9-DA0 test article will be injected. Each of the test article doses will be slowly administered by direct injection into the parotid gland with care taken to accurately deposit the entire vector dose into the gland using ultrasound to confirm and guide. Slowly remove the needle after the injection has been deposited. A physiological saline flush can be used as needed.

[0414] Method for saliva collection:

[0415] Collect saliva via pipette. After all samples for a particular day have been collected, move all sample tubes to a -80°C freezer for storage.

[0416] Ship frozen sample tubes on dry ice for protein analysis.

[0417] Method for quantification of DAO:

[0418] Quantify DAO using the human DAO ELISA kit (MyBiosource MBS2502445) assay kit per manufacturer’s specifications.

[0419] Results:

[0420] Following a single parotid gland administration of AAV9-DAO there were no unexpected deaths and there were no treatment related adverse events or inflammation related to the administration of the test article. Concentrations of DAO proteins measured in the saliva of each animal on Day 7, 14, and 21 are presented in Table 23 and FIG. 17. All animals demonstrated concentration levels of DAO at the earliest (Day 7) and latest (Day 21) timepoints analyzed. The levels of diamine oxidase detected are reported in nanograms per milliliter concentration in the saliva samples analyzed.

[0421] In conclusion, following a single parotid gland administration of AAV9-DAO, saliva collected from domestic pigs demonstrated expression and secretion of diamine oxidase confirmed by antibody analysis (ELISA). All study animals demonstrated detectable levels of DAO protein in saliva during the entire study period (Days 7-21). This study demonstrates the ability to transduce a salivary gland, with a protein, DAO, being expressed and secreted into the saliva providing potential therapeutic effects. The salivary presence of the DAO protein supports the ability to treat gastrointestinal diseases and disorders associated with histamine or an allergic response. Moreover, the constitutive and robust expression levels of diamine oxidase observed in this study suggest that other diseases associated with histamine that require systemic administration can be addressed due to the constant swallowing of the DAO protein via the saliva.

[0422] Table 23. DAO Protein Levels* * *

[0423] While 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, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to theembodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.SEQUENCE LISTINGIll

Claims

CLAIMSWhat is claimed is:

1. A recombinant adeno-associated virus (rAAV) vector comprising an AAV capsid and an expression cassette, the expression cassette comprising a polynucleotide encoding diamine oxidase (DA01), operatively linked to a promoter.

2. The rAAV vector of claim 1, wherein the polynucleotide encodes an amino acid sequence having at least 95% identity to an amino acid sequence selected from SEQ ID NO: 1 and 2.

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

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

5. The rAAV vector of any one of claims 1-4, wherein the promoter comprises a) a CMV promoter comprising the nucleotide sequence set forth in SEQ ID NO: 21 or a CAG promoter comprising the nucleotide sequence set forth in SEQ ID NO: 5; or b) a promoter of a gene encoding a human parotid secretory protein (“PSP”), an amylase (optionally, AMY1C), a kallikrein (optionally, KLK1), a salivary sialoperoxidase, a salivary myeloperoxidase, a P-defensin, a lingual lipase, or a lysozyme.

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

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

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

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

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

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

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

13. The rAAV vector or the composition of claim 12, wherein the ITRs are AAV2 ITRs.

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

15. The rAAV vector of any one of claims 1-10 and 12-14 or the composition of any one of claims 11-14, wherein the AAV capsid comprises a VP3 that shares at least 95%, 98%, or 100% identity with AAV2 VP3 (SEQ ID NO: 8), AAV5 VP3 (SEQ ID NO: 10), AAV8 VP3 (SEQ ID NO: 12), or AAV9 VP3 (SEQ ID NO: 14).

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

17. A composition comprising 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 comprising a nucleotide sequence sharing at least 95% identity to a nucleotide sequence selected from SEQ ID NOs: 29-31, and wherein the polynucleotide is linked to a promoter.

18. 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 sequence sharing at least 95% identity to SEQ ID NO: 23.

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

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

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

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

23. The pharmaceutical composition of claim 22, wherein the composition comprises about 1 x 109to about 1 x 1013genome copies per milliliter of the rAAV vector.

24. The pharmaceutical composition of claim 22, wherein the composition comprises about 1 x 1013to about 1 x 1014genome copies per milliliter of the rAAV vector.

25. The pharmaceutical composition of any one of claims 22-24, wherein the composition is formulated for administration into a human salivary gland, optionally wherein the human salivary gland is a parotid gland.

26. The pharmaceutical composition of any one of claims 22-25, wherein the composition is formulated for administration by direct injection, ballistic delivery, as a sustained release dosage form, as a subcutaneous implant, as an oral spray, as a temperature-shifting gel spray, as a direct topical application, or as a multiple-tine injection.

27. The pharmaceutical composition of any one of claims 22-26, wherein the composition is formulated for use, or adaptable for use, in the treatment of a disease, disorder, or condition associated with excess histamine or histamine-induced inflammation.

28. A method of treating a condition in a subject in need thereof, the method comprising administering an effective amount of the pharmaceutical composition of any one of claims 22-27 to a salivary gland or an oral cavity of the subject.

29. The method of claim 28, wherein the pharmaceutical composition is delivered to a salivary gland of the subject.

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

31. The method of claims 29 or 30, wherein cells within the salivary gland or the parotid gland are transduced by the rAAV vector.

32. The method of claim 31, wherein the transduced cells within the salivary gland or the parotid gland express a) an effective amount of DAO 1 in the subject’s saliva; b) an amount of DAO 1 sufficient to increase a plasma level of DAO 1 in the subject by at least 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1,000-fold compared to a plasma level of DAO1 in the subject measured prior to administration of the pharmaceutical composition.

33. The method of claim 32, wherein the transduced cells within the salivary gland or the parotid gland express an amount of DAO 1 sufficient to increase a plasma level of DAO 1 in the subject by at least 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1,000-fold compared to a plasma level of DAO 1 in the subject measured within one day, week, or month, of the administration of the pharmaceutical composition.

34. The method of any one of claims 28-33, wherein the pharmaceutical composition is delivered to the salivary gland or the parotid gland by direct injection.

35. The method of any one of claims 28-34, wherein about 1 x 109to about 1 x IO10, about 1 x IO10to about 1 x 1011, about 1 x 1011to about 1 x 1012, about 1 x 1012to about 1 x 1013, or about 1 x 1013to about 1 x 1015genome copies of the rAAV vector are administered.

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

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

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

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

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

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

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

43. The method of any one of claims 28-38, wherein the condition comprises histamine intolerance syndrome (“HITS”).

44. The method of any one of claims 28-37, wherein the condition comprises eosinophilic esophagitis.

45. The method of any one of claims 28-37, wherein the condition comprises a migraine.

46. The method of any one of claims 28-45, wherein the administration results in expression of DAO 1 in the cells of a salivary gland and / or a parotid gland of the subject.

47. The method of any one of claims 28-46, wherein the administration results in secretion of DA01 into saliva of the subject.

48. The method of claim 47, wherein secretion of DA01 into the saliva is stimulated by a cholinergic agonist.

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

50. The method of any one of claim 49, wherein the improvement is measured about 1 months, 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.

51. The method of claims 49 or 50, 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, at least 5 years, at least 6 years, at least 7 years, at least 8 years, at least 9 years, or at least 10 years, after the administration.

52. The method of any one of claims 49-51, further comprising administering one or more additional therapeutic agents to the subject.

53. The method of any one of claims 28-52, wherein the subject is human.

54. The pharmaceutical composition of any one of claims 22-27 for use in a method of treating a condition in a subject in need thereof comprising administering an effective amount of the pharmaceutical composition to a salivary gland, a parotid gland, or an oral cavity or a surface thereof, of the subject.

55. The pharmaceutical composition of any one of claims 22-27 for use in the manufacture of a medicament for treating a condition in a subject in need thereof.

56. A kit comprising a pharmaceutical composition of rAAV vector of any one of claims 1- 10 and 12-16 or the composition of any one of claims 11 or 17-21, and a pharmaceutically acceptable carrier, and instructions for use in treating a condition in a subject, comprising administering the pharmaceutical composition to a salivary gland, a parotid gland, or an oral cavity or a surface thereof, of the subject.

57. A kit comprising a pharmaceutical composition of rAAV vector of any one of claims 1- 10 and 12-16 or the composition of any one of claims 11 or 17-21, 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, comprising administering the pharmaceutical composition to a salivary gland, a parotid gland, or an oral cavity, of the subject.

58. A kit comprising a pharmaceutical composition of rAAV vector of any one of claims 1- 10 and 12-16 or the composition of any one of claims 11 or 17-21, and a pharmaceutically acceptable carrier, and instructions for use in treating an autoimmune condition in a subject, comprising administering the pharmaceutical composition to a salivary gland, a parotid gland, or an oral cavity or a surface thereof, of the subject.

59. A kit comprising a pharmaceutical composition of rAAV vector of any one of claims 1- 10 and 12-16 or the composition of any one of claims 11 or 17-21, and a pharmaceutically acceptable carrier, and instructions for use in treating an allergy condition in a subject, comprising administering the pharmaceutical composition to a salivary gland, a parotid gland, or an oral cavity or a surface thereof, of the subject.

60. A pharmaceutical composition, comprising a) a polypeptide comprising a diamine oxidase (“DAO1”) enzyme, or a functional variant thereof, optionally wherein the polypeptide has an amino acid sequence having at least 90% identity to an amino acid sequence selected from SEQ ID NO: 1 and 2; andb) a pharmaceutically-acceptable carrier suitable for administration to a salivary gland, a parotid gland, or an oral cavity or a surface thereof, of a human subject.

61. A pharmaceutical composition, comprising a) a vector comprising a polynucleotide that encodes a polypeptide comprising a diamine oxidase (“DA01”) enzyme, or a functional variant thereof, optionally wherein the polypeptide has an amino acid sequence having at least 90% identity to an amino acid sequence selected from SEQ ID NO: 1 and 2; and b) a pharmaceutically-acceptable carrier suitable for administration to a salivary gland, a parotid gland, or an oral cavity or a surface thereof, of a human subject.

62. The pharmaceutical composition of claims 60 or 61, wherein the pharmaceutically- acceptable carrier comprises water; sterile water; pyrogen-free water; phosphate-buffered saline; HEPES-buffered saline; an isotonic sodium chloride solution; a balanced salt solution; a wetting agent; a surfactant; a tonicity agent; a pH modifier; a viscosity -modifying agent; a buffering agent; a disaccharide, optionally, sucrose or trehalose; a cellulose and / or a derivate thereof; an amino acid, optionally histidine; or any combination thereof.

63. The pharmaceutical composition of any one of claims 60-62, 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.

64. The pharmaceutical composition of any one of claims 60-63, wherein the polypeptide comprises at least 20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, or 500 amino acids.

65. The pharmaceutical composition of any one of claims 60-64, wherein the formulation is a liquid formulated for direct injection into a salivary gland or a parotid gland of the human subject, or a solid or gel formulated for sublingual administration.

66. The pharmaceutical composition of any one of claims 61-65, comprising the vector, wherein the vector is present in the composition in an amount effective to express from 100pg / mL to 50 pg / mL of the polypeptide in saliva of a subject subsequent to administration of the composition to the subject.

67. The pharmaceutical composition of any one of claims 61-66, comprising the vector, wherein the polynucleotide is operably linked to a promotor.

68. The pharmaceutical composition of any one of claims 61-67, comprising the vector, wherein the vector is engineered to constitutively express the polypeptide having an amino acid sequence having at least 90% identity to an amino acid sequence selected from SEQ ID NO: 1 and 2.

69. The pharmaceutical composition of any one of claims 61-68, comprising the vector, wherein the vector comprises a virus, optionally an adenoviral vector or a lentiviral vector; a plasmid; an episome; or an artificial chromosome; and optionally comprises one or more lipids, polycations, DNA-carrier proteins, histones, pseudocapsids, chimeric proteins, or endocytosis receptor proteins.

70. The pharmaceutical composition of any one of claims 60, or 62-65, comprising the polypeptide, wherein the polypeptide is present in the pharmaceutical composition at a concentration of 100 pg / mL to 50 pg / mL.

71. The pharmaceutical composition of any one of claims 60, 62-65, or 70, comprising the polypeptide, wherein the polypeptide is present in the pharmaceutical composition in an amount of 500 ng to 5 pg.

72. The pharmaceutical composition of any one of claims 60, 62-65, 70, or 71, comprising the polypeptide, wherein the polypeptide is present in the pharmaceutical composition in a unit dose amount.

73. A method of treating a disease, disorder, or condition in a subject in need thereof, the method comprising administering an effective amount of the pharmaceutical composition of anyone of claims 64-76 to a salivary gland, a parotid gland, or an oral cavity or surface thereof, of the subject.

74. The method of claim 73, wherein the disease, disorder, or condition is associated with increased histamine production and / or increased histamine signaling.

75. The method of claims 73 or 74, wherein the condition is an inflammatory condition.

76. The method of any one of claims 73-75, wherein the condition is an autoimmune condition.

77. The method of any one of claims 73-76, wherein the condition is an allergic condition.

78. The method of any one of claims 73-77, wherein the condition is an allergic reaction to a therapeutic agent.

79. The method of any one of claims 73-78, wherein the condition is an allergic reaction to a microbial agent.

80. The method of any one of claims 73-79, wherein the condition comprises vernal keratoconj uncti viti s .

81. The method of any one of claims 73-80, wherein the condition comprises atopic keratoconj uncti viti s .

82. The method of any one of claims 73-81, wherein the condition comprises seasonal or perennial allergic conjunctivitis.

83. The method of any one of claims 73-82, wherein the administration results in expression of a functional diamine oxidase in one or more cells of a main salivary gland of the subject.

84. The method of any one of claims 73-83, wherein the administration results in secretion of a functional diamine oxidase into saliva of the subject.

85. The method of claim 84, wherein secretion of the functional diamine oxidase into the saliva is stimulated by a cholinergic agonist.

86. The method of any one of claims 73-85, wherein the administration is to an oral cavity or a surface thereof, or into a salivary gland or a parotid gland of the subject.

87. The method of any one of claims 73-86, wherein the subject is a human subject.

88. A kit comprising the pharmaceutical composition of any one of claims 60-72 and instructions for use in treating a condition in a human subject, comprising administering the pharmaceutical composition to a salivary gland, a parotid gland, or an oral cavity or a surface thereof, of the human subject.

89. The pharmaceutical composition of any one of claims 60-72 for use in the manufacture of a medicament for treating a condition in a human subject in need thereof.

90. A method of reducing a likelihood of miscarriage in a pregnant human subject, comprising administering an effective amount of the pharmaceutical composition of any one of claims 22-27 to a salivary gland or an oral cavity of the pregnant human subject.

91. A method of increasing a likelihood of achieving a full-term pregnancy in a pregnant human subject, comprising administering an effective amount of the pharmaceutical composition of any one of claims 22-27 to a salivary gland or an oral cavity of the pregnant human subject.

92. The method of claims 90 or 91, wherein the pharmaceutical composition is delivered to a salivary gland of the pregnant human subject.

93. The method of claims 90 or 91, wherein the pharmaceutical composition is delivered to a parotid gland of the pregnant human subject.

94. The method of claims 90 or 91, wherein cells within the salivary gland or the parotid gland are transduced by the rAAV vector.

95. The method of claim 94, wherein the transduced cells within the salivary gland or the parotid gland express a) an effective amount of DAO 1 in the pregnant human subject’s saliva; b) an amount of DAO 1 sufficient to increase a plasma level of DAO 1 in the pregnant human subject by at least 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1,000-fold compared to a plasma level of DAO 1 in the subject measured prior to administration of the pharmaceutical composition.

96. The method of claim 95, wherein the transduced cells within the salivary gland or the parotid gland express an amount of DAO 1 sufficient to increase a plasma level of DAO 1 in the pregnant human subject by at least 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1,000-fold compared to a plasma level of DAO 1 in the pregnant human subject measured within one day, week, or month, of the administration of the pharmaceutical composition.

97. The method of any one of claims 90-96, wherein the pharmaceutical composition is delivered to the salivary gland or the parotid gland of the pregnant human subject by direct injection.

98. The method of any one of claims 90-97, wherein about 1 x 109to about 1 x 1010, about 1 x 1010to about 1 x 1011, about 1 x 1011to about 1 x 1012, about 1 x 1012to about 1 x 1013, or about 1 x 1013to about 1 x 1015genome copies of the rAAV vector are administered to the pregnant human subject.