CD59 to inhibit inflammasome activation

Abstract

To provide methods and kits for inhibiting inflammasome activation in cells of a subject or an inflammation-affected eye in the subject.SOLUTION: A composition, which comprises a nucleotide sequence encoding a membrane independent CD59 protein operably linked to a promoter for expression and secretion of the membrane independent CD59 protein in cells of a subject or an inflammation-affected eye in the subject or a soluble CD59 protein and inhibits inflammasome activation, is administered to the subject.SELECTED DRAWING: None

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 629,435, filed February 12, 2018; and U.S. Provisional Patent Application No. 62 / 637,460, filed March 2, 2018. GOVERNMENT SUPPORT

[0002] This invention was made with United States government support under Grant No. EY021805 awarded by the National Institutes of Health. The United States government has certain rights in this invention. [Background technology]

[0003] Complement is a key component of the innate immune system, and its role in various inflammatory diseases, including autoimmune diseases, cancer, and ischemia / reperfusion injury, among others, has been described (Non-Patent Document 1). The role of complement has also been previously described in experimental models of autoimmune uveitis (EAU) (Non-Patent Documents 2-5). Little is known about the mechanisms linking complement activation and T cell-mediated pathology in EAU. Complement activation ends with the assembly of the membrane attack complex (MAC) at the cell membrane, resulting in pore formation. At a sublytic level, pore formation promotes ion exchange and cytokine release across the plasma membrane. As a result, MAC deposition leads to cell lysis. The role of MAC in the pathophysiology of EAU remains unclear. Ca delivery to cells after MAC deposition is unclear. 2+ It is known that the entry of IL-16 activates the NLRP3 inflammasome in primary human lung epithelial cells (Non-Patent Document 6).

[0004] The NLRP3 inflammasome has been implicated in various inflammatory diseases, including age-related macular degeneration, cardiomyopathy, arthritis, chronic kidney disease, and neurodegenerative diseases (Non-Patent Document 7). Administration of lipopolysaccharide (LPS) in mice induces MAC-associated IL-1β maturation (Non-Patent Document 8). Inflammasomes are highly regulated by complement and are necessary for the resolution of tissue injury and disease; however, unregulated inflammasomes can lead to severe inflammation and damage to host tissues (Non-Patent Document 9). NLRP3 inflammasome activation is regulated by a two-step process, requiring an initial priming signal involving increased expression of the protein NLRP3, followed by a subsequent activation signal required for the formation of the inflammasome protein complex, which leads to caspase-1-mediated cleavage of pro-IL-1β to mature IL-1β (Non-Patent Document 10). Unregulated IL-1β expression can lead to the development of autoimmune and autoinflammatory diseases such as Behçet's disease, Vogt-Koyanagi-Harada disease, rheumatic diseases, autoimmune thyroid disease, insulin-dependent diabetes mellitus, gout, familial Mediterranean fever, and cryopyrin-associated periodic fever syndrome (Non-Patent Documents 11-13). Furthermore, it has been established that IL-1β is actively secreted in the retina by myeloid cells, indicating a potential pathogenic role of IL-1R signaling in EAU (Non-Patent Document 14). However, detailed mechanistic knowledge of how IL-1β is regulated in EAU remains elusive.

[0005] Uveitis is a chronic ocular inflammatory disease of the uveal and retinal layers of the eye, causing approximately 10% to 15% of all cases of blindness in the United States (Non-Patent Document 15). Uveitis is classified as infectious when an external biological agent elicits an immune response or as non-infectious when an autoimmune response is elicited. Clinical treatment of uveitis has been performed using corticosteroids, immunosuppressants, or monoclonal antibodies, but these approaches have met with limited success and are associated with significant side effects (Non-Patent Documents 16 and 17). Therefore, there is an unmet clinical need to develop effective treatments for uveitis.

Prior Technology Literature

Non-licensed literature

[0006] [Non-licensed document 1] Morgan, BP, et al. (2015) Nat. Rev. Drug Discov. 14, 857-877 [Non-licensed document 2] An, F., et al. (2009) Invest. Ophthalmol. Vis. Sci. 50, 3778-3782 [Non-licensed document 3] Copland, DA, et al. (2010) Clin. Exp. Immunol. 159, 303-314

Non-licensed Document 4

Non-licensed Document 5

Non-licensed Document 6

Non-licensed Document 7

Non-licensed literature 9

Non-licensed literature 10

[0007] Described herein are methods, compositions, and kits for treating individuals with inflammasome-related disorders using membrane-independent (soluble) CD59 protein.Membrane-independent CD59 protein can be expressed from administered nucleic acid, or can be from direct administration of protein.A nucleic acid encoding membrane-independent CD59 protein or a composition thereof is contemplated for use in treating inflammasome-related disorders.A membrane-independent CD59 protein or a composition thereof is also contemplated for use in treating inflammasome-related disorders.In some embodiments, the inflammasome-related disorder is in the eye of a subject. In some embodiments, inflammasome-associated disorders include uveitis, allergic conjunctivitis, blepharitis, chronic conjunctivitis, episcleritis, keratitis, retinitis, ocular cicatricial pemphigoid, mucous membrane pemphigoid, pterygium scleritis, Stevens-Johnson syndrome, Eales' disease, Behçet's disease, sarcoidosis, systemic lupus erythematosus, polyarteritis nodosa, Wegener's granulomatosis (granulomatosis with polyangiitis), Vogt-Koyanagi-Harada disease, sympathetic ophthalmia, and sarcoidosis. In some embodiments, the inflammasome-associated disorder includes uveitis. A protein or nucleic acid encoding soluble / membrane-independent CD59 can be administered to a subject who has tested positive for the expression or activity of at least one inflammasome activity marker. In some embodiments, the positive test is in the subject's eye. In certain embodiments, the inflammasome activity marker is selected from caspase 1, caspase 5, IL-1β, IL-β17, IL-18, apoptosis-associated speck-like protein containing a CARD (PYCARD / ASC), NACHT, LRR and PYD domains-containing protein (NALP), IFN-γ, a Th1 T cell marker or cytokine, a Th17 T cell marker or cytokine, and CD4+.In some embodiments, the inflammasome activity marker is selected from apoptosis-associated speck-like card protein (PYCARD / ASC) or NACHT, LRR and PYD domain-containing protein (NALP). In some embodiments, the NALP is NACHT, LRR and PYD domain-containing protein 3 (NLRP3).

[0008] One aspect of the present invention provides a method for inhibiting inflammasome activation in inflamed ocular cells of a subject, comprising administering to the subject a composition comprising a nucleotide sequence encoding a membrane-independent CD59 protein operably linked to a promoter for the expression and secretion of the membrane-independent CD59 protein in inflamed ocular cells, wherein the composition inhibits inflammasome activation. As used herein, the term "membrane-independent" refers to a CD59 amino acid sequence that lacks a GPI anchor or has a modified GPI anchor that lacks the function and ability to bind to cell membranes or cell membrane-associated structures (such as membrane-associated proteins). This embodiment of the method provides that membrane-independent CD59 inhibits inflammasome activation independently of MAC function.

[0009] In a method embodiment, the composition inhibits inflammasome activation independent of Membrane Attack Complex (MAC) function. In a method embodiment, the subject has at least one condition selected from uveitis, allergic conjunctivitis, blepharitis, chronic conjunctivitis, episcleritis, keratitis, retinitis, diabetic retinopathy, ocular cicatricial pemphigoid, mucous membrane pemphigoid, pterygium scleritis, Stevens-Johnson syndrome, Eales' disease, Behçet's disease, sarcoidosis, systemic lupus erythematosus, polyarteritis nodosa, Wegener's granulomatosis, Vogt-Koyanagi-Harada disease, psoriasis, immune-mediated hemolytic anemia, thrombocytopenic purpura, Alzheimer's disease, multiple sclerosis, myocardial infarction, atherosclerosis, microangiopathy, thyroiditis, inflammatory bowel disease, organ transplant rejection, membranous nephritis, sympathetic ophthalmia, and sarcoidosis.

[0010] In an embodiment of the method, the uveitis is at least one selected from anterior uveitis, intermediate uveitis, posterior uveitis, and panuveitis. The embodiment of the method further includes obtaining a sample from the subject. In an embodiment of the method, the sample is at least one selected from tears, blood, urine, eye discharge, sputum, and mucus.

[0011] Method embodiments further include measuring at least one of retinal function and inflammasome activity markers in the eye prior to administration. Method embodiments further include measuring at least one of retinal function and inflammasome activity markers in the eye after administration.

[0012] In an embodiment of the method, the inflammasome activity marker is at least one selected from caspase-1, caspase-5, IL-1β, IL-β17, IL-18, apoptosis-associated speck-like card protein (PYCARD / ASC), NACHT, LRR and PYD domain-containing protein (NALP), IFN-γ, Th1 T cell marker or cytokine, Th17 T cell marker or cytokine, and CD4+. In an embodiment of the method, the inflammasome activity marker is selected from apoptosis-associated speck-like card protein (PYCARD / ASC) or NACHT, LRR and PYD domain-containing protein (NALP). In an embodiment of the method, the NALP is NACHT, LRR and PYD domain-containing protein 3 (NLRP3).

[0013] In an embodiment of the method, measuring retinal function or inflammasome activity markers further includes performing at least one technique selected from ophthalmic examination, optical coherence tomography (OCT), conjunctival impression cytology (CIT), RTPCR, ELISA, and PCR.

[0014] Method embodiments further include administering the composition in a dosage sufficient to treat uveitis. Method embodiments further include manipulating the nucleotide sequence in the viral vector prior to administration. Method embodiments further include administering the nucleotide sequence as naked nucleic acid.

[0015] In an embodiment of the method, the viral vector is a genetically engineered genome of at least one virus selected from the group consisting of adenovirus, adeno-associated virus, herpesvirus, and lentivirus. In an embodiment of the method, the lentivirus is a retrovirus.

[0016] Embodiments of the method further include engineering the membrane-independent CD59 protein to have at least one mutation that results in a loss of function of the glycosylphosphatidylinositol (GPI) anchor domain of the translated CD59 protein. Embodiments of the method further include engineering the nucleotide sequence encoding the membrane-independent CD59 protein by deleting nucleotides encoding the region of the glycosylphosphatidylinositol (GPI) anchor domain.

[0017] In an embodiment of the method, administering further comprises injecting the composition into the eye. In an embodiment of the method, administering further comprises topically applying the composition. In one embodiment of the method, the ocular injection is selected from the group consisting of a subretinal injection, an intravitreal injection, an intraocular injection, a subconjunctival injection, and a sub-Tenon injection. In an embodiment of the method, the ocular injection further comprises administering into the outer layer of the eye.

[0018] Embodiments of the method further include administering to the eye an additional therapeutic agent, hi some embodiments of the method, the additional therapeutic agent is at least one selected from the group consisting of an anti-tumor agent, an anti-viral agent, an anti-bacterial agent, an anti-mycobacterial agent, an anti-fungal agent, an anti-proliferative agent, and an anti-apoptotic agent. In some embodiments of the method, the additional therapeutic agent is selected from the group consisting of a growth factor, an anti-inflammatory agent, a vasopressor, a collagenase inhibitor, a steroid, a matrix metalloproteinase inhibitor, ascorbate, angiotensin, calreticulin, tetracycline, fibronectin, collagen, thrombospondin, transforming growth factor (TGF), keratinocyte growth factor (KGF), fibroblast growth factor (FGF), insulin-like growth factor (IGF), IGF-binding protein (IGFBP), epidermal growth factor (EGF), platelet-derived growth factor (PDGF), neu differentiation factor (NDF), hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), heparin-binding EGF (HBEGF), thrombospondin, von Willebrand factor C, heparin, heparin sulfate, and hyaluronic acid.

[0019] One aspect of the invention herein provides a kit for inhibiting inflammasome activation in an inflamed eye of a subject, the kit comprising: a pharmaceutical composition comprising a membrane-independent CD59 protein and / or a nucleotide sequence encoding the CD59 protein, in a dosage sufficient to inhibit inflammasome activation in the inflamed eye of the subject; instructions for use; and a container.

[0020] One aspect of the invention herein provides a kit for treating uveitis in a subject, comprising: a pharmaceutical composition comprising a membrane-independent CD59 protein and / or a nucleotide sequence encoding the CD59 protein, in a dosage sufficient to treat uveitis in the subject; instructions for use; and a container.

[0021] One aspect of the present invention provides a method for inhibiting inflammasome activation in inflamed ocular cells of a subject, the method comprising administering to the subject a composition comprising a nucleotide sequence encoding a membrane-independent CD59 protein operably linked to a promoter for expression and secretion of the membrane-independent CD59 protein in inflamed ocular cells, wherein the composition inhibits inflammasome activation, and the subject exhibits a positive result for the expression or activity of at least one inflammasome activity marker in the inflamed ocular cells of the subject. In one embodiment of this method, the inflammasome activity marker is selected from caspase-1, caspase-5, IL-1β, IL-β17, IL-18, apoptosis-associated speck-like card protein (PYCARD / ASC), NACHT, LRR and PYD domain-containing protein (NALP), IFN-γ, Th1 T cell marker or cytokine, Th17 T cell marker or cytokine, and CD4+. In some embodiments of this method, the inflammasome activity marker is selected from apoptosis-associated speck-like card protein (PYCARD / ASC) or NACHT, LRR and PYD domain-containing protein (NALP). In some embodiments of this method, the NALP is NACHT, LRR and PYD domain-containing protein 3 (NLRP3). In some embodiments of this method, the method further comprises measuring the inflammasome activity marker in the eye after administering a composition comprising a nucleotide sequence encoding a membrane-independent CD59 protein operably linked to a promoter for the expression and secretion of membrane-independent CD59. In some embodiments of this method, the subject has been diagnosed with or is suspected of having uveitis, allergic conjunctivitis, blepharitis, chronic conjunctivitis, episcleritis, keratitis, retinitis, ocular cicatricial pemphigoid, mucous membrane pemphigoid, pterygium scleritis, Stevens-Johnson syndrome, Eales' disease, Behcet's disease, sarcoidosis, polyarteritis nodosa, Wegener's granulomatosis, Vogt-Koyanagi-Harada disease, sympathetic ophthalmia, or sarcoidosis. In some embodiments, the subject has been diagnosed with or is suspected of having uveitis.A positive result for the expression or activity of at least one inflammasome activity marker in the inflamed eye of a subject described herein refers to elevated expression or activity of at least one inflammasome activity marker compared to the expression or activity of at least one inflammasome activity marker in the eye of an individual not diagnosed with or afflicted with uveitis, allergic conjunctivitis, blepharitis, chronic conjunctivitis, episcleritis, keratitis, retinitis, ocular cicatricial pemphigoid, mucous membrane pemphigoid, pterygium scleritis, Stevens-Johnson syndrome, Eales disease, Behçet's disease, sarcoidosis, polyarteritis nodosa, Wegener's granulomatosis, Vogt-Koyanagi-Harada disease, sympathetic ophthalmia, or sarcoidosis. Alternatively, the expression or activity of at least one inflammasome activity marker in the inflamed eye of a subject is determined by histological scoring.

[0022] Certain aspects described herein are methods for inhibiting inflammasome activation in cells of an inflamed subject, the method comprising: (a) administering to the subject a composition comprising a nucleotide sequence encoding a membrane-independent CD59 protein operably linked to a promoter for expression and secretion of the membrane-independent CD59 protein in cells of the inflamed subject; or (b) administering to the subject a composition comprising a soluble CD59 protein, wherein the composition inhibits inflammasome activation. In some embodiments, the composition inhibits inflammasome activation independently of membrane attack complex (MAC) function. In some embodiments, the subject has at least one inflammasome-related condition selected from Alzheimer's disease, multiple sclerosis, myocardial infarction, atherosclerosis, microangiopathy, thyroiditis, inflammatory bowel disease, organ transplant rejection, membranous nephritis, sympathetic ophthalmia, and sarcoidosis. In some embodiments, the method further comprises obtaining a sample from the subject. In some embodiments, the sample is at least one selected from blood, plasma, serum, peripheral blood mononuclear cells, cerebrospinal fluid, or urine. In some embodiments, the method further comprises measuring a marker of inflammasome activity in the subject prior to administration. In some embodiments, the method further comprises measuring a marker of inflammasome activity in the subject after administration. In some embodiments, the marker of inflammasome activity is at least one selected from caspase-1, caspase-5, IL-1β, IL-β17, IL-18, apoptosis-associated speck-like card protein (PYCARD / ASC), NACHT, LRR and PYD domain-containing protein (NALP), IFN-γ, Th1 T cell marker or cytokine, Th17 T cell marker or cytokine, and CD4+. In some embodiments, the method further comprises administering the composition at a dosage sufficient to treat the inflammasome-associated condition.In some embodiments, the nucleotide sequence encoding the membrane-independent CD59 protein operably linked to a promoter for expression and secretion of the membrane-independent CD59 protein is a genetically engineered genome of at least one virus selected from the group consisting of adenovirus, adeno-associated virus, herpesvirus, and lentivirus. In some embodiments, the lentivirus is a retrovirus. In some embodiments, the administering further comprises intravenously injecting the composition. In some embodiments, the method further comprises topically applying the composition. In some embodiments, the method further comprises administering an additional therapeutic agent. In some embodiments, the additional therapeutic agent is at least one selected from the group consisting of an antitumor agent, an antiviral agent, an antibacterial agent, an antimycobacterial agent, an antifungal agent, an antiproliferative agent, and an antiapoptotic agent. In certain embodiments, the additional therapeutic agent is selected from the group consisting of growth factors, anti-inflammatory agents, vasopressors, collagenase inhibitors, steroids, matrix metalloproteinase inhibitors, ascorbate, angiotensin, calreticulin, tetracycline, fibronectin, collagen, thrombospondin, transforming growth factor (TGF), keratinocyte growth factor (KGF), fibroblast growth factor (FGF), insulin-like growth factor (IGF), IGF-binding protein (IGFBP), epidermal growth factor (EGF), platelet-derived growth factor (PDGF), neu differentiation factor (NDF), hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), heparin-binding EGF (HBEGF), thrombospondin, von Willebrand factor C, heparin, heparin sulfate, and hyaluronic acid. In another aspect, described herein is a kit for inhibiting inflammasome activation in inflamed cells of a subject, the kit including: (a) a pharmaceutical composition comprising (i) a membrane-independent CD59 protein and / or a nucleotide sequence encoding the CD59 protein; or (ii) a soluble CD59 protein; in a dosage sufficient to inhibit inflammasome activation in inflamed cells of the subject; (b) instructions for use; and (c) a container.

[0023] Another embodiment described herein is a method of treating at least one condition selected from Alzheimer's disease, multiple sclerosis, myocardial infarction, atherosclerosis, microangiopathy, thyroiditis, inflammatory bowel disease, organ transplant rejection, membranous nephritis, sympathetic ophthalmia, and sarcoidosis, the method comprising: (a) administering to a subject a composition comprising: (i) a nucleotide sequence encoding a membrane-independent CD59 protein operably linked to a promoter for expression and secretion of the membrane-independent CD59 protein in a cell; or (ii) a soluble CD59 protein; wherein the composition inhibits activation of an inflammasome. Another aspect described herein is a method for inhibiting inflammasome activation in cells of an inflamed subject, the method comprising: (a) measuring a marker of inflammasome activity in the subject; and (b) if the marker of inflammasome activity is positive, administering to the subject a composition comprising: (i) a nucleotide sequence encoding a membrane-independent CD59 protein operably linked to a promoter for expression and secretion of the membrane-independent CD59 protein in cells of the inflamed subject; or (ii) a soluble CD59 protein; wherein the composition inhibits inflammasome activation. In one embodiment, the marker of inflammasome activity is selected from caspase-1, caspase-5, IL-1β, IL-β17, IL-18, apoptosis-associated speck-like card protein (PYCARD / ASC), NACHT, LRR and PYD domain-containing protein (NALP), IFN-γ, Th1 T cell marker or cytokine, Th17 T cell marker or cytokine, and CD4+. In certain embodiments, the method further comprises measuring a marker of inflammasome activity in the subject after administration of a composition comprising a nucleotide sequence encoding a membrane-independent CD59 protein or a soluble CD59 protein operably linked to a promoter for expression and secretion of the membrane-independent CD59 protein.

[0024] Another aspect described herein is a method for inhibiting inflammasome activation in cells of an inflamed subject, the method comprising administering to a subject a composition comprising: (a) a nucleotide sequence encoding a membrane-independent CD59 protein operably linked to a promoter for expression and secretion of the membrane-independent CD59 protein in cells of the inflamed subject; or (b) a soluble CD59 protein; wherein the composition inhibits inflammasome activation, and the subject exhibits a positive result for the expression or activity of at least one inflammasome activity marker in the inflamed subject. In one embodiment, the inflammasome activity marker is selected from caspase-1, caspase-5, IL-1β, IL-β17, IL-18, apoptosis-associated speck-like card protein (PYCARD / ASC), NACHT, LRR and PYD domain-containing protein (NALP), IFN-γ, a Th1 T cell marker or cytokine, a Th17 T cell marker or cytokine, and CD4+. In some embodiments, the inflammasome activity marker is selected from apoptosis-associated speck-like card protein (PYCARD / ASC) or NACHT, LRR and PYD domain-containing protein (NALP). In some embodiments, the NALP is NACHT, LRR and PYD domain-containing protein 3 (NLRP3). In some embodiments, the method further comprises measuring the inflammasome activity marker in the subject after administering a composition comprising: (a) a nucleotide sequence encoding a membrane-independent CD59 protein operably linked to a promoter for the expression and secretion of the membrane-independent CD59 protein; or (b) a soluble CD59 protein. In certain embodiments, the subject has been diagnosed with or is suspected of suffering from uveitis, allergic conjunctivitis, blepharitis, chronic conjunctivitis, episcleritis, keratitis, retinitis, ocular cicatricial pemphigoid, mucous membrane pemphigoid, pterygium scleritis, Stevens-Johnson syndrome, Eales disease, Behcet's disease, sarcoidosis, systemic lupus erythematosus, polyarteritis nodosa, Wegener's granulomatosis, Vogt-Koyanagi-Harada disease, sympathetic ophthalmia, or sarcoidosis.In some embodiments, the subject is diagnosed with or suspected of suffering from uveitis.In some embodiments, the positive result of the expression or activity of at least one inflammasome activity marker in the subject with inflammation is compared with the expression or activity of at least one inflammasome activity marker in the subject who is not diagnosed with or does not suffer from Alzheimer's disease, multiple sclerosis, myocardial infarction, atherosclerosis, microangiopathy, thyroiditis, inflammatory bowel disease, organ transplant rejection, membranous nephritis, sympathetic ophthalmia and sarcoidosis.In some embodiments, the positive result of the expression or activity of at least one inflammasome activity marker in the subject with inflammation is determined by histological scoring. [Brief explanation of the drawings]

[0025] [Figure 1-1] Figures 1A-1F are a series of photomicrographs and bar graphs showing that MAC-mediated NLRP3 inflammasome activation increases IL-1β production in EAU. Values ​​are expressed as mean ± SEM. GCL: ganglion cell layer; INL: inner nuclear layer; ONL: outer nuclear layer; OS: outer segment; scale bar: 100 µm. Figure 1A is a series of photomicrographs of cryostat sections of retinas stained with 4',6-diamidino-2-phenylindole (DAPI) (left column) and C5b9 antibody (middle column), demonstrating elevated MAC formation in EAU retinas compared to normal control retinas. Retinas from C9- / - EAU mice (bottom row) were negative for C5b9 protein and therefore lacked MAC. [Figure 1-2]Figure 1B is a bar graph of IL-1β protein production measured by ELISA in EAU retinas (middle bar) and control tissues. IL-1β protein production in EAU retinas was observed to be 112% increased over control retinas as measured by ELISA and 45-fold higher by RT-PCR. Compared to normal control retinas, C9- / - EAU retinas were observed to have 37% higher IL-1β protein levels and 3.8-fold higher IL-1β mRNA. Figure 1C is a bar graph of IL-1 mRNA production measured by RT-PCR in EAU retinas (middle bar) and control tissues. IL-1β mRNA levels in EAU retinas were observed to be 4464% higher than those in normal control retinas. IL-1β mRNA levels in C9- / - EAU retinas were observed to be 285% higher than those in normal control retinas. Figure 1D is a photograph and bar graph of a Western blot showing that NLRP3 protein levels were >200% higher in EAU retinas compared to normal control retinas in mice. Slightly higher NLRP3 protein levels were observed in C9- / - EAU retinas compared to normal control retinas in mice. [Figure 1-3] Figure 1E is a photograph and bar graph of a Western blot showing that over 80% higher caspase 1 (p20) protein levels were observed in EAU mouse retinas compared to normal control retinas. A 30% higher caspase 1 (p20) protein level was observed in C9- / - EAU retinas compared to normal control mouse retinas. Figure 1F is a photograph and bar graph of a Western blot showing that ASC protein was 44% higher in EAU retinas compared to normal control mouse retinas. ASC levels were observed to be slightly elevated in C9- / - EAU mouse retinas compared to normal control retinas. Western blot and RT-PCR values ​​were normalized to β-actin. [Figure 2-1]Figures 2A-2B are a series of electroretinogram (ERG) waveforms and line graphs showing the effects of MAC formation on retinal function in EAU. The data obtained in the Examples herein demonstrate that EAU causes extensive damage to photoreceptors, which impairs retinal function compared to normal control retinas. Figure 2A shows a series of ERG responses from normal, EAU, and C9- / - EAU groups. Dark-adapted (scotopic) and light-adapted (photopic) responses were analyzed in the retinas of normal, EAU, and C9- / - EAU mice. For scotopic ERGs, flash light intensities of -20 dB (0.025 cds / m²), -10 dB (0.25 cds / m²), and 0 dB (2.5 cds / m²) were used. For photopic ERGs, flash light intensities of 0 dB (2.5 cds / m²) and 1 dB (3.15 cds / m²) were used. [Figure 2-2] Figure 2B is a series of line graphs of scotopic and light-adapted a- and b-wave ERG amplitudes plotted against intensity. For scotopic ERGs, flash light intensities of -20 dB, -10 dB, and 0 dB were used. For light-adapted ERGs, flash light intensities of 0 dB and 1 dB were used. Values ​​are expressed as mean ± SEM. *p<0.05, #p<0.05 compared to EAU. [Figure 3-1]Figures 3A-3F are a series of photomicrographs and dot plots demonstrating that C9- / - mice are not protected from EAU. In the present example, mice were subcutaneously immunized with 200 μg of IRBP (1-20) peptide in 0.2 mL of complete Freund's adjuvant (CFA) emulsion containing 2.5 mg / mL Mycobacterium tuberculosis. Simultaneously, mice were intraperitoneally injected with 1.5 μg of Bordetella pertussis toxin diluted in 100 μL of phosphate-buffered saline (PBS). Twenty-four days later, the severity of EAU in the fundus was analyzed among normal, EAU, and C9- / - EAU mice. Focal lesions, linear lesions, vasculitis, retinal hemorrhage, infiltrates, and retinal detachment were examined. Based on the severity of these findings, an EAU clinical score was assigned on a scale of 0 to 4. Figure 3A is a series of photomicrographs of fundus images from the EAU and C9- / -EAU groups showing retinal inflammatory infiltrates (white arrowheads), vasculitis (white arrows), and retinal folds (black arrows). Figure 3B is a dot plot of individual overall clinical scores. [Figure 3-2] Figures 3C-3F are dot plots of the vasculature score, cellular infiltration score, optic disc score, and structural damage score, respectively. The combination of the vasculature score (Figure 3C), cellular infiltration score (Figure 3D), optic disc score (Figure 3E), and structural damage score (Figure 3F) was calculated and plotted as an individual overall clinical score (Figure 3B). Values ​​are expressed as mean ± SEM. [Figure 4]Figures 4A-4E are a series of photomicrographs and bar graphs of retinal imaging and histology of tissues from EAU, normal control, and C9- / -EAU mice. Figure 4A is a series of photomicrographs of horizontal OCT scans showing retinal fold formation (red arrows) and vitreous cellular infiltrates (white arrows) in EAU and C9- / -EAU mice, but not in normal control mice. Figure 4B is a series of photomicrographs of retinal sections (5 μm) obtained from paraffin-embedded eyes harvested 24 days after EAU induction and stained with hematoxylin and eosin. Infiltrates of inflammatory immune cells in the vitreous (V) are indicated by black arrows; black asterisks represent retinal (R) detachment; retinal folds are indicated by white arrowheads. Figures 4C-4E are a series of bar graphs showing the degree of severity of EAU pathology, scoring cellular infiltration, vasculitis, and photoreceptor damage separately, as described in the Examples herein. Values ​​are expressed as mean ± SEM. RPE-CH, RPE and choroid; R, retina; V, vitreous; GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; OS, outer segment; OpN, optic nerve. Scale bar = 100 μm. [Figure 5-1] Figures 5A-5F are a series of photomicrographs and bar graphs showing that soluble CD59 inhibits NLRP3 inflammasome activation and IL-1β production in EAU. Figure 5A is a series of photomicrographs of retinal cryostat sections from eyes of EAU mice injected with either AAVCAGsCD59 or control AAVCAGGFP, stained with C5b9 antibody or DAPI. [Figure 5-2]Figure 5B is a bar graph of IL-1β protein levels measured by ELISA. IL-1β protein levels in the retinas of EAU mice injected with AAVCAGsCD59 were observed to be 40% lower than those in the retinas of control EAU mice injected with AAVCAGGFP. Figure 5C is a bar graph of IL-1β mRNA levels measured by RT-PCR. IL-1β mRNA levels in the retinas of EAU mice injected with AAVCAGsCD59 were observed to be 70% lower than those in the retinas of control EAU mice injected with AAVCAGGFP. Figure 5D is a photograph and bar graph of a Western blot showing that the amount of NLRP3 protein was 60% lower in the retinas of EAU mice injected with AAVCAGsCD59 compared to those in the retinas of control AAVCAGGFP. [Figure 5-3] Figure 5E is a photograph and bar graph of a Western blot showing 60% less caspase-1 (p20) protein in the retinas of EAU mice injected with AAVCAGsCD59 compared to the retinas of EAU mice injected with control AAVCAGGFP. Figure 5F is a photomicrograph and bar graph of a Western blot showing comparable ASC protein levels in the retinas of EAU mice injected with AAVCAGsCD59 and control AAVCAGGFP. Western blot and RT-PCR values ​​are normalized to β-actin. Values ​​are expressed as mean ± SEM. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; OS, outer segment; Scale bar = 100 µm. [Figure 6-1]Figure 6A-6B show a series of electroretinogram (ERG) waveforms and line graphs demonstrating that soluble CD59 improves retinal function in EAU mice. Figure 6A shows a series of ERG responses in EAU mice injected with AAVCAGsCD59 or control AAVCAGGFP. Both dark-adapted (scotopic) and light-adapted (photopic) responses were analyzed in the retinas of EAU mice injected with AAVCAGsCD59 or control AAVCAGGFP. For dark-adapted ERGs, flash light intensities of -20 dB (0.025 cds / m²), -10 dB (0.25 cds / m²), and 0 dB (2.5 cds / m²) were used. For light-adapted ERGs, flash light intensities of 0 dB (2.5 cds / m²) and 1 dB (3.15 cds / m²) were used. [Figure 6-2] Figure 6B is a series of line graphs of scotopic and light-adapted a- and b-wave ERG amplitudes plotted against intensity. For scotopic ERGs, flash light intensities of -20 dB, -10 dB, and 0 dB were used. For light-adapted ERGs, flash light intensities of 0 dB and 1 dB were used. Values ​​are expressed as mean ± SEM. *p<0.05, p<0.05 vs. #AAVCAGGFP. [Figure 7-1] Figures 7A-7F are a series of photomicrographs and dot plots showing that soluble CD59 reduces the severity of clinical scores in EAU. The severity of fundus EAU was analyzed in EAU mice injected with PBS, AAVCAGsCD59, and AAVCAGGFP. EAU clinical scores were graded on a scale of 0 to 4. Figure 7A is a series of photomicrographs of fundus images from mice injected with PBS, AAVCAGsCD59, or AAVCAGGFP, showing retinal inflammatory infiltrates (white arrowheads), vasculitis (white arrows), and papilledema (white asterisks). [Figure 7-2]Figures 7B-7F are dot plots of the individual overall clinical scores, vasculature score, cellular infiltration score, optic disc score, and structural damage score, respectively. The combination of the vasculature score, cellular infiltration score, optic disc score, and structural damage score was calculated and plotted as the individual overall clinical score. It was observed that the retinas of the EAU mice injected with AAVCAGsCD59 had statistically significantly improved individual scores of cellular infiltration, vasculature, and structural damage, as well as the overall clinical score, compared with the retinas of the control AAVCAGGFP-injected mice. [Figure 7-3] It was observed that the difference in optic disc scores (Figure 7E) remained statistically insignificant in EAU mice injected with AAVCAGsCD59 compared to control AAVCAGGFP-injected EAU mice. The difference between PBS-injected and AAVAGGFP-injected eyes remained statistically insignificant. Values ​​are expressed as mean ± SEM. [Figure 8] Figures 8A-8E are a series of photomicrographs and bar graphs showing the effects of CD59 on OCT changes and retinal histological scores in EAU mice. Figure 8A is a series of photomicrographs of horizontal OCT scans showing retinal folding (red arrows) and vitreous cellular infiltrates (white arrows) in the retinas of EAU mice injected with control AAVCAGGFP or AAVCAGsCD59 or control PBS. Figure 8B is a series of photomicrographs of retinal sections (5 μm) obtained from paraffin-embedded eyes 24 days after EAU induction and stained with hematoxylin and eosin. Inflammatory immune cell infiltration in the vitreous (V) is indicated by black arrows; retinal (R) detachment is indicated by black asterisks; retinal folds are indicated by white arrowheads. Figures 8C-8E are a series of bar graphs showing the degree of severity of EAU pathology, scoring cellular infiltration, vasculitis, and photoreceptor damage separately, as described in the Examples herein. Values ​​are expressed as mean ± SEM. RPE-CH, RPE and choroid; R, retina; V, vitreous; GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; OS, outer segment; OpN, optic nerve, scale bar = 100 μm. [Figure 9]Figure 9 is a bar graph showing elevated MAC in EAU retinas. The amount of MAC fluorescence intensity in the retinas of EAU mice was observed to be 70% higher than that in normal control retinas of mice. Values ​​are expressed as mean ± SEM. [Figure 10] Figures 10A-10D are a series of bar graphs showing the effect of MAC on Th1 and Th17 cell differentiation in EAU retinas. Freshly dissected retinas were quantified for IL-17 and IFN-γ mRNA and protein levels using ELISA and RT-PCR in control, EAU, and C9- / - EAU retinas. Figure 10A is a bar graph showing a 102% increase in IFN-γ protein levels and 200-fold higher mRNA levels in EAU mouse retinas compared to control normal mouse retinas. IFN-γ protein expression levels in C9- / - EAU retinas were observed to be 14% higher than those in control normal mouse retinas. Figure 10B is a bar graph showing that IFN-γ mRNA levels in EAU mouse retinas were 200-fold higher compared to control normal mouse retinas and 102% higher protein levels. IFN-γ mRNA levels in the C9- / -EAU retinas were observed to be 1315% higher than those in the retinas of control normal mice. Figure 10C is a bar graph showing that IL-17 protein levels in the retinas of EAU mice were observed to be 99% higher than those in the retinas of normal control mice. IL-17 protein levels in the retinas of C9- / -EAU mice were observed to be only 44% higher than those in the retinas of normal control mice. Figure 10D is a bar graph showing IL-17 mRNA levels in the retinas of EAU and C9- / -EAU mice. IL-17 mRNA levels in the retinas of normal control mice remained below the detection limit. Each experiment was repeated two to three times. Values ​​are expressed as mean ± SEM. [Figure 11-1]Figures 11A-11C are a series of scatter plots and bar graphs of the amounts of IFN-γ or IL-17 in Th1 and Th17 cells in the draining lymph nodes (DLN) of EAU mice. DLN cells were collected from EAU and C9- / -EAU mice 24 days after EAU induction. Figure 11A is a series of scatter plots of cells from DLN stained for IL-17A and IFN-γ plotted relative to CD4+ cells. [Figure 11-2] Figures 11B and 11C are bar graphs showing that IL-17-positive CD4+ cells and IFN-γ-positive CD4+ cells from draining lymph nodes were significantly increased in EAU mice compared with normal control mice. The difference in percentage of IL-17-positive CD4+ cells and IFN-γ-positive CD4+ cells from draining lymph nodes in C9- / -EAU mice compared with EAU mice was not significant. Each experiment was repeated three times. Values ​​are expressed as mean ± SEM. [Figure 12] 1 is a bar graph showing that a single intravitreal injection of AAVCAGsCD59 inhibits MAC formation in EAU retinas. The amount of MAC fluorescence intensity in the retinas of EAU mice injected with AAVCAGsCD59 was compared to the amount in the retinas of EAU mice injected with the control AAVCAGGFP, resulting in a 45% reduction in MAC formation. Values ​​are expressed as mean ± SEM. [Figure 13]Figures 13A-13D are a series of bar graphs showing that soluble CD59 inhibits the differentiation of Th1 and Th17 cells in the EAU retina. IL-17 and IFN-γ mRNA and protein levels were quantified in freshly dissected retinas using ELISA and RT-PCR in EAU mice injected with AAVCAGsCD59 or control AAVCAGGFP. Figure 13A is a bar graph showing that IFN-γ protein levels were 25% lower in the retinas of EAU mice injected with AAVCAGsCD59 compared to those in EAU mice injected with control AAVCAGGFP. Figure 13B is a bar graph showing that IFN-γ mRNA expression levels were 47% lower in the retinas of EAU mice injected with AAVCAGsCD59 compared to those in EAU mice injected with control AAVCAGGFP. Figure 13C is a bar graph showing that IL-17 protein levels in the retinas of EAU mice injected with AAVCAGsCD59 were 35% lower than those in EAU mice injected with control AAVCAGGFP. Figure 13D is a bar graph showing that IL-17 mRNA levels in the retinas of EAU mice injected with AAVCAGsCD59 were 10% lower than those in EAU mice injected with control AAVCAGGFP. The differences in IL-17 and IFN-γ mRNA levels were not statistically significant. Each experiment was repeated two to three times. Values ​​are expressed as mean ± SEM. [Figure 14]Figures 14A-14C are photomicrographs and Western blots showing the retinas of 6-week-old C57BL / 6J mice injected with 3.5 x 10 genome copies / µl of AAVCAGsCD59 or AAVCAGGFP (1 µl), subjected to EAU one week later, and maintained for 24 days. Figure 14A is a fundus image showing GFP expression in the mouse retina. Figure 14B is a retinal cryostat image of the AAAVCAGFP-injected group. The retina of a mild EAU mouse (middle row) shows strong GFP expression in the ganglion cell layer (GCL), inner plexiform layer, and inner nuclear layer, while the retina of a severe EAU mouse (bottom row) shows GFP expression in the photoreceptors and retinal pigment epithelium. Top row: control AAVCAGsCD59. Figure 14C is a Western blot showing the expression of sCD59 from mouse retinas after a single intravitreal injection of AAVCAGsCD59. Abbreviations: GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; OS, outer segment; RPE, retinal pigment epithelium. DETAILED DESCRIPTION OF THE INVENTION

[0026] Described herein are methods, compositions, and kits for treating individuals with inflammasome-related disorders using membrane-independent (soluble) CD59 protein.Membrane-independent CD59 protein can be expressed from administered nucleic acid, or can be from direct administration of protein.The nucleic acid encoding membrane-independent CD59 protein or its composition is intended for use in treating inflammasome-related disorders.Membrane-independent CD59 protein or its composition is also intended for use in treating inflammasome-related disorders.In some embodiments, the inflammasome-related disorder is in the eye of a subject. In some embodiments, inflammasome-associated disorders include uveitis, allergic conjunctivitis, blepharitis, chronic conjunctivitis, episcleritis, keratitis, retinitis, ocular cicatricial pemphigoid, mucous membrane pemphigoid, pterygium scleritis, Stevens-Johnson syndrome, Eales' disease, Behçet's disease, sarcoidosis, systemic lupus erythematosus, polyarteritis nodosa, Wegener's granulomatosis, Vogt-Koyanagi-Harada disease, sympathetic ophthalmia, and sarcoidosis. In some embodiments, the inflammasome-associated disorder includes uveitis. A protein or nucleic acid encoding soluble / membrane-independent CD59 can be administered to a subject who has tested positive for the expression or activity of at least one inflammasome activity marker. In some embodiments, the positive test is in the subject's eye. In some embodiments, the inflammasome activity marker is selected from caspase 1, caspase 5, IL-1β, IL-β17, IL-18, apoptosis-associated speck-like card protein (PYCARD / ASC), NACHT, LRR and PYD domain-containing protein (NALP), IFN-γ, Th1 T cell marker or cytokine, Th17 T cell marker or cytokine, and CD4+. In some embodiments, the inflammasome activity marker is selected from apoptosis-associated speck-like card protein (PYCARD / ASC) or NACHT, LRR and PYD domain-containing protein (NALP). In some embodiments, the NALP is NACHT, LRR and PYD domain-containing protein 3 (NLRP3).

[0027] Immunization of mice with peptides derived from retinal interphotoreceptor-binding protein (IRBP) produces a T cell-mediated autoimmune disease similar to the clinical and histological features seen in human uveitis (Caspi, RR, et al. (1988) J. Immunol 140, 1490-1495; Chan, CC, et al. (1990) J. Autoimmun. 3, 247-255). This experimental model of autoimmune uveitis (EAU) has been used as an approach for studying uveitis and developing treatments for this disease, and is used in the Examples herein.

[0028] To further understand the potential mechanisms by which MAC may be involved in the pathogenesis of EAU and elucidate the importance of NLRP3 activation and IL-1β production in the development of EAU, MAC deposition was examined in EAU retinas and determined that MAC may be required for NLRP3 inflammasome activation and subsequent IL-1β release.

[0029] The MAC contains one molecule each of C5b, C6, C7, and C8, and up to 12 molecules of C9. - / - Mice are unable to assemble a functional MAC, and therefore may be partially protected from EAU. In the examples herein, control C57BL / 6J mice and C9 - / - EAU was induced in mice, and the pathology of EAU in control mice was confirmed by C9 - / -The pathology of EAU in mice was compared with that in mice. The therapeutic potential of inhibiting C9 in EAU was investigated using a gene therapy approach using an adeno-associated virus (AAV) expressing soluble CD59 (AAVCAGsCD59), a protein that prevents C9 incorporation into preformed C5b-8 complexes. The development of EAU in AAVCAGsCD59-injected mice was monitored by fundus imaging, spectral-domain optical coherence tomography (SD-OCT), retinal histopathology, and immunohistochemistry. Retinal function was quantified by electroretinogram (ERG), and NLRP3 inflammasome activation was measured by Western blot, enzyme-linked immunosorbent assay (ELISA), and real-time polymerase chain reaction (RT-PCR). The results observed in the present examples are the first to implicate a direct role for MAC as an activator of the NLRP3 inflammasome and IL-1β production in the development of EAU. The data in the Examples herein demonstrate for the first time that AAV-mediated expression of soluble CD59 is a potential gene therapy for the treatment of uveitis.

[0030] The Examples herein investigated the direct role of MAC in NLRP3 inflammasome activation in EAU. Data from the Examples herein demonstrate that MAC is deposited in the EAU retina, leading to NLRP3 inflammasome activation and increased IL-1β production. These Examples demonstrate that C9 - / - We show that EAU mice fail to form MAC in the retina, which is accompanied by activation of the NLRP3 inflammasome, suggesting a link between MAC deposition and NLRP3 inflammasome activation in EAU. - / - Although mice cannot rescue the pathological phenotype of EAU, AAV-mediated delivery of sCD59, an inhibitor of C9 incorporation into the MAC, unexpectedly alleviates many aspects of the pathological phenotype of EAU, including activation of the NLRP3 inflammasome.

[0031] The complement system is a major component of innate immunity and is composed of a diverse group of plasma and membrane-bound proteins. These membrane-bound proteins play a central role in defense against pathogens and in regulating immune and inflammatory processes. Complement activation against pathogens is necessary for host defense, but excessive complement activation can damage host tissues (Morgan, BP, et al. (2015) Nat. Rev. Drug Discov. 14, 857-877; Carroll, MV, et al. (2011) Adv. Drug Deliv. Rev. 63, 965-975). Therefore, complement regulatory proteins must maintain a balance between complement activation and complement inhibition. An overactive and unregulated complement system results in the formation of MAC, which has been shown to be involved in various ocular diseases, including EAU (An, F., et al. (2009) Invest. Ophthalmol. Vis. Sci. 50, 3778-3782; Copland, DA, et al. (2010) Clin. Exp. Immunol. 159, 303-314; Read, RW, et al. (2006) Exp. Eye Res. 82, 389-394; Gehrs, KM, et al. (2010) Arch. Ophthalmol. 128, 349-358; Yanai, R., et al. (2012) Adv. Exp. Med. Biol. 946, 161-183). However, the direct role of MAC deposition in the pathogenesis of EAU remains unknown. In the examples herein, it was observed that MAC was deposited in EAU, which resulted in increased IL-1β production. However, C9, which lacks the ability to form MAC, - / - EAU mice were observed to have reduced IL-1β levels as a result.

[0032] Although MAC deposition at the plasma membrane ultimately leads to lytic cell death, sublytic MAC on the plasma membrane is involved in cell cycle and proliferation control, apoptosis, cytokine production, and initiation of downstream signaling cascades (Morgan, B.P., et al. (2015) Nat. Rev. Drug Discov. 14, 857-877). The NLRP3 inflammasome complex is a cytoplasmic protein complex consisting of the key regulatory subunit NLRP3, the adaptor subunit ASC, and the effector subunit caspase-1, which converts pro-IL-1β to active IL-1β (Latz, E., et al. (2013) Nat. Rev. Immunol.13, 397-411; Broz, P., et al. (2016) Nat. Rev. Immunol. 16, 407-420). Recent studies in LPS-primed models have demonstrated that sublytic MAC-induced pore formation is associated with intracellular Ca2+ upregulation. 2+It has been shown that this leads to the accumulation of NLRP3, which subsequently activates the NLRP3 inflammasome (Triantafilou, K., et al. (2013) J. Cell Sci. 126, 2903-2913; Laudisi, F., et al. (2013) J. Immunol. 191, 1006-1010). The NLRP3 inflammasome has previously been linked to several ocular diseases (Devi, TS, et al. (2012) Exp. Diab. Res. 2012, 438238; Tseng, WA, et al. (2013) Invest Ophthalmol Vis Sci 54, 110-120). The results in the Examples herein demonstrate that attenuation of NLRP3 inflammasome activation is a novel therapeutic approach for the treatment of uveitis. The Examples herein demonstrate for the first time the direct role of MAC and NLRP3 inflammasome activation in an EAU mouse model. The data obtained in the Examples herein demonstrate that MAC directly activated and increased the protein expression of NLRP3, caspase-1, and ASC subunits. These activated subunits form the NLRP3 inflammasome complex, which produces active IL-1β. C9 - / - EAU mice did not activate the NLRP3 inflammasome and IL-1β remained near basal levels, confirming that NLRP3 inflammasome activation is a MAC-dependent pathway.

[0033] Autoreactive effector CD4+ T cells have been implicated in the pathogenesis of EAU. Both Th1 and Th17 lineages are particularly involved in the development of EAU and have been reported in patients with uveitis (Amadi-Obi, A., et al. (2007) Nat. Med. 13, 711-718; Caspi, RR, et al. (1996) J. Immunol. 157, 2668-2675). Furthermore, IL-1β signaling promotes the differentiation of CD4+ T cells into Th17 cells (Chung, Y., et al. (2009) Immunity 30, 576-587). Recent studies have shown that blocking the IL-1 signaling pathway may be effective in treating EAU in mice (Wan, CK, et al. (2016) J. Immunol. 196, 543-546). Anakinra, an IL-1R antagonist, rilonacept, a soluble decoy IL-1R, and canakinumab, an IL-1β neutralizing antibody, have been approved for the treatment of uveitis (Knickelbein, JE, et al. (2017) Handb. Exp. Pharmacol. 242, 231-268). However, their use is limited due to various side effects and a short duration of action. In the examples herein, increased IL-1β production was observed in the retinas of mice with EAU; however, the production of IL-1β was not significantly increased in C9 mice with EAU. - / - Lower IL-1β levels were observed in the retinas of mice with EAU. Furthermore, enhanced differentiation of Th1 and Th17 cells was observed in the retinas of mice with EAU; however, C9 mice with EAU showed no significant difference. - / - In the mouse retina, a decrease in Th1 and Th17 cells was observed, as indicated by the protein and mRNA levels of IL17 and IFN-γ, respectively. Furthermore, an increase in the levels of Th1 and Th17 positive CD4 cells was observed in the DLN from EAU mice. However, C9 - / -We observed that levels of Th1- and Th17-positive CD4 cells remained unchanged in DLN from EAU mice. Without being limited to a particular theory or mechanism of action, we predict that MAC-induced IL-1β production and differentiation of Th1 and Th17 cells in the EAU retina are local effects.

[0034] Many complement regulatory proteins are secreted or found on the cell surface and prevent complement-mediated damage to host tissues. These proteins include factor H, decay accelerating factor (CD55), membrane cofactor protein (CD46), and protectin (CD59). Decreased or absent activity of these complement regulatory proteins can lead to immunopathologies, including EAU and experimental autoimmune anterior uveitis (Morgan, BP, et al. (2015) Nat. Rev. Drug Discov. 14, 857-877; An, F., et al. (2009) Invest. Ophthalmol. Vis. Sci. 50, 3778-3782; Carroll, MV, et al. (2011) Adv. Drug Deliv. Rev. 63, 965-975; Jha, P., et al. (2006) J. Immunol. 176, 7221-7231). Autoimmune uveitis is a chronic, multifactorial disease associated with systemic disease. Various studies have shown that inhibiting complement activation can help ameliorate EAU pathology in mice (An, F., et al. (2009) Invest. Ophthalmol. Vis. Sci. 50, 3778-3782; Copland, DA, et al. (2010) Clin. Exp. Immunol. 159, 303-314; Read, RW, et al. (2006) Exp. Eye Res. 82, 389-394). Embodiments of the methods described herein demonstrate a long-acting gene therapy approach using AAV to deliver sCD59 to EAU mice. This is the first time that AAVCAGsCD59 has been demonstrated to inhibit MAC deposition in EAU retinas. The examples herein demonstrate that AAVCAGsCD59 inhibited NLRP3 inflammasome activation and attenuated IL-1β production.

[0035] Furthermore, the Examples herein demonstrate that a single intravitreal injection of AAVCAGsCD59 inhibited phenotypic pathology in EAU mice. These mice showed significant improvement in clinical signs associated with EAU, including reduced inflammation, less immune cell infiltrate, and reduced vasculitis. The data obtained in the Examples herein demonstrate that injection of AAVCAGsCD59 resulted in improvement of the loss of retinal function associated with EAU, as measured by both dark-adapted and light-adapted ERGs. The Examples herein demonstrate that injection of C9 - / - Mice were observed to have significantly inhibited NLRP3 inflammasome activation and reduced IL-1β upregulation. There was a trend toward improved histological scores and improved retinal function. - / - It was observed in EAU mice that, except for a few data points in the ERG, the differences did not reach a significant level of statistical significance.

[0036] Unexpectedly, our study suggests that AAVCAGsCD59 may attenuate inflammation in a manner other than blocking the incorporation of C9 into the C5b-8 complex. GPI-anchored CD59 has previously been reported to bind to CD2 and transmit activation signals within T cells (Deckert, M. et al. (1995) Eur J Immunol. 25, 1815-22). CD59 cross-linking induces the T cell receptor zeta (ζ) / ZAP-70 signaling cascade and interleukin-2 (IL-2) synthesis. IL-2 has been shown to effectively regulate the immune system in diseases such as type 1 diabetes and vasculitis (Hartemann, A. et al. (2013) The Lancet. Diabetes & Endocrinology. 1, 295-305), supporting the hypothesis that sCD59 may attenuate inflammation in a MAC-independent manner. The EAU model used herein to analyze uveitis has inconsistencies related to the development of mild to moderate EAU. These minor changes can have a significant impact on the results.

[0037] Uveitis is a chronic inflammatory disease, and many patients face recurrent episodes, some of which are refractory to available treatments. Overall, conventional treatments are limited to managing severe and advanced uveitis in patients with systemic autoimmune diseases (e.g., Behçet's disease, Vogt-Koyanagi disease) due to significant side effects and short-term efficacy. Treatments for managing uveitis have not made significant advances over the past few decades (Knickelbein, JE, et al. (2017) Handb. Exp. Pharmacol. 242, 231-268). Given these concerns, developing long-acting therapies, such as continuous inhibition of MAC-dependent activation of the inflammasome, in patients with uveitis is practical. Therefore, a single intravitreal injection of AAVsCD59 holds great potential and advantages over the short-term efficacy of current treatments. AAV-dependent gene therapy has been shown to be successful in treating ocular diseases in animal models (Adhi, M., et al. (2013) PLoS One 8, e79661; Ildefonso, CJ, et al. (2015) Hum. Gene Ther. 26, 59-68). Furthermore, AAV-mediated gene therapy has successfully progressed into human clinical trials for age-related macular degeneration (Mingozzi, F., et al. (2011) Nat. Rev.

[0038] The examples herein demonstrate that MAC is a key regulator of NLRP3 inflammasome activation and IL-1β production in EAU. MAC plays an important role in Th1 and Th17 cell differentiation by increasing IL-1β production. AAV-mediated expression of sCD59 was observed to effectively inhibit MAC deposition and subsequent NLRP3 inflammasome activation. A single intravitreal injection of AAVCAGsCD59 was observed to effectively inhibit the development of EAU in mice.

[0039] CD59 protein CD59 is a membrane-bound glycoprotein found in association with the membranes of cells such as human erythrocytes, lymphocytes, and vascular endothelial cells. The CD59 protein inhibits the assembly of a functional MAC, thus protecting cells from complement-mediated activation and / or lysis.

[0040] Without being limited to a particular theory or mechanism of action, it is believed that the plasma membrane of cells is normally protected from the effects of complement by cell surface proteins, such as CD59, which specifically inhibit the activation of the C5b-9 pore by binding of the C9 complement protein to membrane C5b-8 (Holguin et al. 1989 J. Clin. Invest. 84: 7-17; Sims et al. 1989 J. Biol. Chem. 264: 19228-19235; Davies et al. 1989 J. Exp. Med. 170: 637-654; Rollins et al. 1990 J. Immunol. 144: 3478-3483; and Hamilton et al. 1990 Blood 76: 2572-2577). CD59 competes with the C9 complement protein for binding to the C8 complement protein in the C5b-8 complex, thereby reducing or preventing the formation of the C5b-9 membrane attack complex. Thus, CD59 acts to reduce both cell activation and cell lysis by the terminal complement MAC.

[0041] The mature human CD59 protein consists of 77 amino acids and has a molecular weight of 18-21 kD. The precursor human CD59 protein contains a 25-amino acid amino-terminal signal peptide and a 26-amino acid carboxy-terminal peptide that confers membrane anchor attachment. Exemplary amino acid sequences of precursor human CD59, mature human CD59, and CD59 sequences from other mammals (e.g., baboon, African green monkey, owl monkey, marmoset, HVS-15, pig, rabbit, rat, and mouse) are set forth in U.S. Patent No. 7,166,568, issued January 23, 2007, to Sims et al. (incorporated by reference).

[0042] The protein structure of CD59 contains a single cysteine-rich domain, a hydrophobic core with three loops and a small fourth helical loop (Yu et al. 1997 Journal of Experimental Medicine 185(4): 745-753).

[0043] The structure and sequence of the gene encoding CD59 have been characterized (Fodor et al., U.S. Patent No. 5,624,837, issued April 29, 1997, incorporated by reference). This gene is located on the short arm of human chromosome 11, specifically chromosomes 11p13 and 11p14 (Online Mendelian Inheritance in Man, accession number 107271), and is composed of four exons spanning 20 kb (Petranka et al. 1992 Proc. Nat. Acad. Sci. 89: 7876-7879). The untranslated first exon is preceded by a G- and C-rich promoter region lacking consensus TATA or CAAT motifs. The second exon encodes the hydrophobic leader sequence of the protein, and the third exon encodes the N-terminal portion of the mature protein. The fourth exon encodes the remainder of the mature protein, including the hydrophobic sequence for glycosylphosphatidylinositol (GPI) anchor attachment to the cell membrane.

[0044] CD59 is a glycosylphosphatidylinositol-anchored glycoprotein expressed in human peripheral blood leukocytes, erythrocytes, and many cell lines. This protein is expressed in both hematopoietic and non-hematopoietic cells, such as endothelial cells, peripheral nerve fibers, neurons, microglia, oligodendrocytes (or oligodendrocytes), astrocytes, ependymal cells, epithelial cells, salivary gland acinar cells, bronchial epithelium, renal tubules, and squamous epithelium. See Nose, M. et al. 1990 Immunology 70(2): 145-149; Vedeler, C. et al. 1994 Immunology 82(4): 542-547; and Hidestima, T. et al. 1990 Immunology 69(3): 396:401, each of which is incorporated herein by reference in its entirety. A cDNA encoding CD59 was reported in Sawada, R. et al. 1989 Nucleic Acids Res 17(16): 6728. cDNA encoding CD59 has also been cloned from human T-cell leukemia (YT) and human erythroleukemia (K562) cell lines, and CD59 has been transiently expressed in COS cells (Walsh, LA et al. 1990 Eur J. Immol 21(3): 847-850). Human CD59 contains 26 amino acids located at the C-terminus, which specify a signal sequence for attachment of a glycosylphosphatidylinositol anchor (GPI anchor) at the 77th amino acid, asparagine. The cDNA sequence of CD59 is shown in U.S. Patent No. 5,624,837 to Fodor et al., issued April 29, 1997, which is incorporated herein by reference in its entirety.

[0045] Analysis of the physical binding of MAC components to CD59 indicates that distinct binding sites for CD59 are contained within the α chains of human C8 and human C9. The binding site for human CD59 interaction with human C9 has been identified as amino acid residues 42-58 of the mature human CD59 sequence, which binds to the region of human C9 corresponding to human amino acid residues 334-418 of the human C9 protein, more specifically, human C9 amino acid residues 359-384, immediately C-terminal to the predicted membrane insertion domain of C9 (Sims et al., PCT / US96 / 17940, filed November 8, 1996, incorporated herein by reference in its entirety).

[0046] Based on the solution structure of mature human CD59 from published NMR data and knowledge of the active portion of the CD59 molecule, the active surface-exposed amino acid residue side chains available for binding to C8 / C9 were identified as histidine at position 44, asparagine at position 48, aspartic acid at position 49, threonine at positions 51 and 52, arginine at position 55, and glutamic acid at position 58. The NMR structures of CD59 are available from the deposit by Kieffer et al., Human complement regulatory protein CD59 (extracellular domain, residue 170; NMR, 10 structures), MMDB Id:891, PDB Id:1ERH; the deposit by Kieffer et al., Human complement regulatory protein CD59 (extracellular domain, residue 170; NMR, constraint), MMDB Id:890, PDB Id:1ERG; the deposit by Fletcher et al., CD59 in complex with Glcnac-Beta-1,4-(Fuc-Alpha-1,6)-Glcnac-Beta-1 (NMR, 10 structures), MMDB Id:498, PDB Id:1CDS; and the deposit by Fletcher et al., CD59 in complex with Glcnac-Beta-1,4-Glcnac-Beta-1 (NMR, 10 structures), MMDB Id:497, PDB Id:1CDR is described in the deposition of Id:1CDR by Fletcher et al. Id:1CDR and Id:1CDR. Amino acid sequences of CD59 presenting these side chains in the same relative positions function similarly to human CD59 (Sims et al.), and such variants are within the scope of the methods, kits, and pharmaceutical compositions herein.

[0047] The relationship between complement activation and abnormal levels of autoantibodies has been analyzed in relation to ocular diseases such as macular degeneration and other conditions. U.S. Patent Application Publication No. 2005 / 0287601, published December 29, 2005, by Hageman et al. (incorporated by reference), proposes diagnosing macular degeneration by measuring the presence of autoantibodies specific to retinal proteins (RPE and choroidal proteins) in samples from AMD patients.

[0048] Theories have linked complement to the causative role of Alzheimer's disease and age-related macular degeneration, with prevention by inhibiting the activation of the complement system and the formation of MAC. U.S. Patent Application Publication No. 2007 / 0196367 to Dinu, published August 23, 2007 (incorporated by reference), proposes the prevention of debris formation by inhibiting complement as a treatment for Alzheimer's disease and AMD. U.S. Patent Application Publication No. 2007 / 0203190 to Patil et al., published August 30, 2007 (incorporated by reference), describes hydroxylamine compounds (e.g., TEMPOL-H, TEMPO-H, and OXANO-H) or ester derivatives as putative inhibitors of complement activation.

[0049] U.S. Patent Application Publication No. 2005 / 0265995, published December 1, 2005 by Tomlinson et al. (incorporated by reference), inhibits complement-directed proteinuria in rats using a drug created by linking the complement inhibitors Crry and CD59, respectively, at their amino termini to single-chain antibodies (scFv) that bind to rat glomerular epithelial cells and proximal tubule epithelial cells. This reference states that soluble CD59 is ineffective as an inhibitor. Bora et al. (2007 J. Immunol 178: 1783-1790) used recombinant methods to fuse the binding arm Fc of immunoglobulin G (IgG1) to the CD59 protein (rsCD59a-Fc) to create a membrane-targeting composition, which was then injected into mice intravenously, intraocularly (intravitreal), and intraperitoneally. The number of CNV-positive spots was reduced in subjects treated with fusion proteins via the intraperitoneal route compared with the intravitreal and intravenous routes. Fc functionality can result in immediate binding when the fusion protein contacts cells generally and nonspecifically after administration. Administration of purified proteins is further limited by metabolism and half-life due to the presence of proteases and peptidases. Tomlinson et al. (Tomlinson et al. 2009 IOVS 50(7): 3056-3064) reduced the size of CNV spots in a mouse model by intravenously injecting animals with CNV spots with a plasmid encoding a fusion protein complement inhibitor created by linking the N-terminal binding domain of factor H to a fragment of complement receptor 2 (CR2) that targets cell membrane molecules.

[0050] The CD59 compositions provided herein lack the essential amino acid sequence of a functional GPI anchor. Functionally equivalent proteins include amino acid sequences of modified GPI anchor domains that are functionally defective and lack the ability to target membranes. sCD59 is an example of recombinant membrane-independent CD59 (rmiCD59). Additional methods for obtaining membrane-independent CD59 include non-recombinant methods, such as providing an inhibitor of membrane association, for example, synthesizing CD59 in vivo or in vitro so that it lacks a GPI anchor. Methods for obtaining membrane-independent CD59 are shown in the Examples herein. Additional recombinant techniques for modifying the nucleic acid and amino acid sequences of molecules are well known in the fields of genetics and molecular biology.

[0051] In various embodiments, the CD59 protein described herein is a soluble CD59 protein. The compositions provide CD59 proteins, including full-length CD59 nucleic acids modified to remove the signal sequence for GPI anchor attachment at the nucleotide encoding the asparagine amino acid at position 77. Alternatively, the CD59 nucleic acid sequence is modified by point mutation, substitution, or deletion to obtain a nucleic acid sequence encoding an amino acid sequence with a modified amino acid sequence at the GPI anchor position, such that the protein cannot attach to a cell membrane.

[0052] As used herein, the term "membrane-independent" refers to a CD59 amino acid sequence that lacks a GPI anchor or has a modified GPI anchor that lacks the function and ability to bind to cell membranes or cell membrane-associated structures such as membrane-associated proteins. The GPI anchor can be modified by recombinant DNA techniques to remove the GPI anchor domain or to introduce one or more mutations to disrupt GPI function. These one or more mutations can include the insertion of one or more amino acids, the deletion of one or more amino acids, or the substitution of one or more amino acids.

[0053] GPI anchoring involves a multistep pathway in the endoplasmic reticulum, involving the interaction of multiple gene products. Many proteins, including CD59, are expressed on the cell surface and require GPI for effective function. The mechanism by which the structure of a protein signal encodes the attachment of a GPI anchor is reviewed in Orlean et al. 2007 JLR 48: 993-1011. GPI attachment requires an amino acid sequence containing a hydrophobic N-terminal secretion signal that targets the protein to the ER and a C-terminal GPI signal anchor sequence. In addition to the native CD59 secretion signal, which is located at the amino terminus of the protein and is cleaved in vivo, other secretion signals are suitable for the CD59 protein and are within the scope of the methods described herein. Common eukaryotic secretion signals suitable for use in mammalian cells herein are described, for example, in U.S. Patent No. 6,733,997, issued May 11, 2004 to Ding et al.; Tan et al. 2002 Protein Engineering 15(4): 337-345; and Tan et al. 1999 Biochim. Biophys. Acta 1452: 103-120, each of which is incorporated herein by reference in its entirety.

[0054] The amino acid to which the GPI is attached is called the omega (ω) residue; the amino acid N-terminal to the omega residue is called omega minus (ω-), and the amino acid C-terminal to the omega residue is called omega plus (ω+). The GPI anchor sequence contains a stretch of approximately 10 polar amino acids (i.e., ω-10 to ω-1), such as arginine, lysine, aspartic acid, glutamic acid, asparagine, or glutamic acid, forming a flexible linker region. The ω residue has been identified as either glycine, alanine, serine, asparagine, aspartic acid, or cysteine. Mutations, such as substitutions and deletions of the nucleic acids encoding the amino acids at the omega positions, can be used to reduce or eliminate GPI anchor attachment or the effective function of the GPI anchor. For example, such variations include substituting the hydrophobic leucine-encoding nucleic acids (e.g., CTG nucleic acid) and alanine-encoding nucleic acids (e.g., GCA nucleic acid) with the less hydrophobic (i.e., more hydrophilic) amino acids glutamine-encoding nucleic acids (e.g., CAG nucleic acid) and glutamic acid-encoding nucleic acids (e.g., GAA nucleic acid), or glycine-encoding nucleic acids (e.g., GGN nucleic acid). Alternatively, variations include substituting the ω residue with another amino acid, e.g., substituting tyrosine for glycine.

[0055] Other promoter sequences useful for regulating the transcription of the CD59 gene sequence are also within the scope of the expression vector of the present invention.These promoters include, for example, constitutive promoters, cell cycle-specific promoters, ubiquitous promoters, tissue-specific promoters, metabolically regulated promoters, inducible promoters, and promoters found in specific subjects, including humans and animals.Examples of promoters and promoter systems are shown in, for example, U.S. Patent No. 6,677,311 issued to Evans et al. on January 13, 2004; U.S. Patent No. 7,109,029 issued to Clark et al. on September 19, 2006; and U.S. Patent No. 5,998,205 issued to Hallenbeck et al. on December 7, 1999, each of which is incorporated herein by reference in its entirety.

[0056] In the remaining amino acid sequences of the CD59 protein portions not involved in GPI anchoring, the scope of the CD59 protein herein is intended to include conservative sequence modifications. As used herein, the term "conservative sequence modification" refers to an amino acid modification that does not significantly affect or change the properties of the CD59 protein or membrane-independent CD59, including its amino acid sequence, i.e., an amino acid modification in which the amino acid sequence of CD59 presents its side chains in the same relative positions functions similarly to human CD59. Such conservative modifications include amino acid substitutions, additions, and deletions. Modification of the amino acid sequence of CD59 can be achieved using any technique known in the art, such as site-directed mutagenesis or PCR-based mutagenesis. Such techniques are described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Plainview, NY, 1989, Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY, 1989, Molecular Biomethods Handbook, 2nd ed., JM Walker et al., Humana Press, 2008, and Handbook of Molec. and Cellul. Methods in Biol. and Med., 3rd ed., LJ Ceske et al., CRC Press, 2011.

[0057] Conservative amino acid substitutions are those in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), amino acids with nonpolar side chains (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0058] In some embodiments, a CD59 amino acid sequence is substantially identical to the amino acid sequence of a wild-type sequence. The term "substantially identical" is used herein to refer to a first amino acid sequence that contains a sufficient or minimal number of amino acid residues identical to aligned amino acid residues of a second amino acid sequence such that the first and second amino acid sequences share a common structural domain and / or a common functional activity. For example, an amino acid sequence that contains a common structural domain with at least about 60% identity, or at least 75%, 80%, 85%, 90%, 95%, 96%, 98%, or 99% identity. In some embodiments, a CD59 amino acid sequence is "identical" to the amino acid sequence of a wild-type sequence. An identical sequence is one that exhibits 100% identity with the wild-type sequence of CD59. In some embodiments, an identical sequence may have one or more amino acids located at the N- or C-terminus. For example, one or more amino acids may comprise an epitope tag, a purification tag, the remainder of a cleavage site introduced to allow removal of a purification tag, or one or more modifications to improve the stability or bioavailability of the protein.

[0059] As used herein, the term "about" refers to a number, measurement, or amount that is close to the stated amount by no more than 10%.

[0060] CD59 polypeptides are useful for treating inflammasome-mediated disorders. The amino acid sequence of a human CD59 polypeptide useful in the methods described herein is represented by the following sequence: MGIQGGSVLFGLLLVLAVFCHSGHSLQCYNCPNPTADCKTAVNCSSDFDACLITKAGLQVYNKCWKFEHCNFNDVTTRLRENELTYYCCKKDLCNFNEQLENGGTSLSEKTVLLLVTPFLAAAWSLHP (SEQ ID NO: 1). The signal sequence of CD59 is cleaved before secretion from cells and is not necessary for therapeutic utility. However, nucleic acids encoding CD59 with a signal sequence enhance secretion of the CD59 polypeptide, and therefore, a signal sequence may be desirable to include in the CD59 polypeptide encoded by the nucleic acid. A signal sequence-truncated human CD59 polypeptide useful in the methods described herein is represented by the following sequence: LQCYNCPNPTADCKTAVNCSSDFDACLITKAGLQVYNKCWKFEHCNFNDVTTRLRENELTYYCCKKDLCNFNEQLENGGTSLSEKTVLLLVTPFLAAAWSLHP (SEQ ID NO: 2). However, the exact signal sequence end can differ from SEQ ID NO: 2 by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 amino acids from the first leucine of SEQ ID NO: 2, either toward the N-terminus or C-terminus of the complete CD59 polypeptide shown in SEQ ID NO: 1. An additional deletion of the N-terminal 26 amino acids removes the GPI anchor domain of human CD59. A signal sequence-cleaved soluble human CD59 polypeptide useful in the methods described herein is represented by the following sequence: LQCYNCPNPTADCKTAVNCSSDFDACLITKAGLQVYNKCWKFEHCNFNDVTTRLRENELTYYCCKKDLCNFNEQLEN (SEQ ID NO: 3).This soluble version can also vary by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 amino acids at its exact N-terminus, which may result in further deletions from or restoration of its N-terminus. In one embodiment, the soluble version of CD59 comprises the amino acid sequence set forth in SEQ ID NO:2 with one or more mutations to residues required for GPI attachment. In one embodiment, the residues are either or both of the asparagines at amino acids 70 or 77 of SEQ ID NO:2. In certain embodiments, a CD59 polypeptide useful in the methods described herein comprises a deletion of 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 amino acids from the N-terminus, C-terminus, or both termini of any of SEQ ID NOs: 1, 2, or 3. The CD59 polypeptides used in the methods described herein may comprise or consist of an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the sequence set forth in any of SEQ ID NOs: 1, 2, or 3. Furthermore, the CD59 polypeptides used herein may comprise additional non-CD59-derived sequences, such as a purification tag, a remnant of a cleaved purification tag, an additional complement inhibitory polypeptide, an anti-inflammatory polypeptide, or a polypeptide that enhances the stability or bioavailability of the CD59 polypeptide. Nucleic acids encoding polypeptides comprising or consisting of the CD59 polypeptides described herein are also useful for treating inflammasome-mediated disorders. Accordingly, one or more nucleic acids encoding a polypeptide comprising or consisting of the CD59 polypeptides described herein are also contemplated. The nucleic acids may be suitably contained in a recombinant vector, such as a viral or plasmid vector, suitable for administration to a subject.These vectors can additionally be included in pharmaceutical compositions for administration to individuals with inflammasome-mediated disorders.

[0061] A polypeptide comprising or consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 1, 2, or 3 can be used in the methods for treating an inflammasome disorder in a subject described herein. In one embodiment, a polypeptide comprising or consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 1, 2, or 3 is used in a method for treating an inflammasome disorder in the eye of a subject. In one embodiment, the inflammasome disorder is uveitis. In one embodiment, the subject shows a positive result for the expression or activity of at least one inflammasome activity marker. In one embodiment, the inflammasome activity marker is selected from caspase 1, caspase 5, IL-1β, IL-β17, IL-18, apoptosis-associated speck-like card protein (PYCARD / ASC), NACHT, LRR and PYD domain-containing protein (NALP), IFN-γ, Th1 T cell markers or cytokines, Th17 T cell markers or cytokines, and CD4+. In some embodiments, the inflammasome activity marker is selected from apoptosis-associated speck-like card protein (PYCARD / ASC) or NACHT, LRR, and PYD domain-containing protein (NALP). In some embodiments, the NALP is NACHT, LRR, and PYD domain-containing protein 3 (NLRP3). In some embodiments, the methods described herein include administering a human CD59 polypeptide to an individual who has tested positive for the expression or activity of at least one inflammasome activity marker. The CD59 polypeptide may comprise an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the sequence set forth in any one of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

[0062] A nucleic acid or nucleic acids encoding a polypeptide comprising or consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 1, 2, or 3 can be used in a method for treating an inflammasome disorder in a subject. In one embodiment, a nucleic acid or nucleic acids encoding a polypeptide comprising or consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 1, 2, or 3 is used in a method for treating an inflammasome disorder in the eye of a subject. In one embodiment, the inflammasome disorder is uveitis. In one embodiment, the subject shows a positive result for the expression or activity of at least one inflammasome activity marker. In one embodiment, the inflammasome activity marker is selected from caspase 1, caspase 5, IL-1β, IL-β17, IL-18, apoptosis-associated speck-like card protein (PYCARD / ASC), NACHT, LRR and PYD domain-containing protein (NALP), IFN-γ, Th1 T cell markers or cytokines, Th17 T cell markers or cytokines, and CD4+. In some embodiments, the inflammasome activity marker is selected from apoptosis-associated speck-like card protein (PYCARD / ASC) or NACHT, LRR and PYD domain-containing protein (NALP). In some embodiments, the NALP is NACHT, LRR and PYD domain-containing protein 3 (NLRP3).

[0063] In certain embodiments, the methods described herein comprise administering a human CD59 polypeptide to an individual who tests positive for expression or activity of at least one marker of inflammasome activity. The CD59 polypeptide can comprise or consist of an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the sequence set forth in any one of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. In certain embodiments, the marker of inflammasome activity is selected from caspase-1, caspase-5, IL-1β, IL-β17, IL-18, apoptosis-associated speck-like card protein (PYCARD / ASC), NACHT, LRR- and PYD-domain-containing protein (NALP), IFN-γ, a Th1 T cell marker or cytokine, a Th17 T cell marker or cytokine, and CD4+. In some embodiments, the inflammasome activity marker is selected from apoptosis-associated speck-like card protein (PYCARD / ASC) or NACHT, LRR and PYD domain-containing protein (NALP). In some embodiments, the NALP is NACHT, LRR and PYD domain-containing protein 3 (NLRP3). In some embodiments, the positive result is obtained from the individual's eye.

[0064] In some embodiments, the methods described herein include administering a human CD59 polypeptide to the eye of an individual that has tested positive for expression or activity of at least one marker of inflammasome activity. The CD59 polypeptide can comprise or consist of an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the sequence set forth in any one of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. In some embodiments, the marker of inflammasome activity is selected from caspase-1, caspase-5, IL-1β, IL-β17, IL-18, apoptosis-associated speck-like card protein (PYCARD / ASC), NACHT, LRR- and PYD-domain-containing protein (NALP), IFN-γ, a Th1 T cell marker or cytokine, a Th17 T cell marker or cytokine, and CD4+. In some embodiments, the inflammasome activity marker is selected from apoptosis-associated speck-like card protein (PYCARD / ASC) or NACHT, LRR and PYD domain-containing protein (NALP). In some embodiments, the NALP is NACHT, LRR and PYD domain-containing protein 3 (NLRP3). In some embodiments, the positive result is obtained from the individual's eye.

[0065] In certain embodiments, the methods described herein comprise administering a human CD59 polypeptide to an individual suffering from uveitis. The CD59 polypeptide can comprise or consist of an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the sequence set forth in any one of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. In certain embodiments, the polypeptide is administered to the affected eye of the individual.

[0066] In some embodiments, the methods described herein involve administering a nucleic acid encoding a human CD59 polypeptide to an individual who has tested positive for the expression or activity of at least one inflammasome activity marker. The nucleic acid can encode a CD59 polypeptide comprising or consisting of an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. In some embodiments, the inflammasome activity marker is selected from caspase 1, caspase 5, IL-1β, IL-β17, IL-18, apoptosis-associated speck-like card protein (PYCARD / ASC), NACHT, LRR- and PYD-domain-containing protein (NALP), IFN-γ, Th1 T cell marker or cytokine, Th17 T cell marker or cytokine, and CD4+. In some embodiments, the inflammasome activity marker is selected from apoptosis-associated speck-like card protein (PYCARD / ASC), or NACHT, LRR- and PYD-domain-containing protein (NALP). In some embodiments, the NALP is NACHT, LRR and PYD domain-containing protein 3 (NLRP3). In some embodiments, the positive result is obtained from the eye of the individual.

[0067] In some embodiments, the methods described herein involve administering a nucleic acid encoding a human CD59 polypeptide to the eye of an individual who has tested positive for expression or activity of at least one marker of inflammasome activity. The nucleic acid can encode a CD59 polypeptide comprising or consisting of an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. In some embodiments, the marker of inflammasome activity is selected from caspase-1, caspase-5, IL-1β, IL-β17, IL-18, apoptosis-associated speck-like card protein (PYCARD / ASC), NACHT, LRR- and PYD-domain-containing protein (NALP), IFN-γ, a Th1 T cell marker or cytokine, a Th17 T cell marker or cytokine, and CD4+. In some embodiments, the inflammasome activity marker is selected from apoptosis-associated speck-like card protein (PYCARD / ASC) or NACHT, LRR and PYD domain-containing protein (NALP). In some embodiments, the NALP is NACHT, LRR and PYD domain-containing protein 3 (NLRP3). In some embodiments, the positive result is obtained from the individual's eye.

[0068] In certain embodiments, the methods described herein comprise administering to an individual suffering from uveitis a nucleic acid encoding a human CD59 polypeptide. The nucleic acid can encode a CD59 polypeptide comprising or consisting of an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. In certain embodiments, the nucleic acid is administered to the affected eye of the individual.

[0069] Calculation of sequence identity between sequences is performed as follows: To determine the percent identity of two amino acid sequences, the sequences are aligned for optimal comparison (e.g., gaps may be introduced into one or both of the first and second amino acid sequences for optimal alignment). The amino acid residues at corresponding amino acid positions or nucleotide positions are then compared. If a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the proteins are identical at that position. The percent identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps that need to be introduced for optimal alignment of the two sequences and the length of each gap.

[0070] The comparison of sequences and determination of percent identity between two sequences is accomplished using a mathematical algorithm. The percent identity between two amino acid sequences is determined using an alignment software program using default parameters. Suitable programs include, for example, CLUSTAL W by Thompson et al., Nuc. Acids Research 22:4673, 1994 (www.ebi.ac.uk / clustalw); BL2SEQ by Tatusova and Madden, FEMS Microbiol. Lett. 174:247, 1999 (www.ncbi.nlm.nih.gov / blast / bl2seq / bl2.html); SAGA by Notredame and Higgins, Nuc. Acids Research 24:1515, 1996 (igs-server.cnrs-mrs.fr / ~cnotred); and DIALIGN by Morgenstern et al., Bioinformatics 14:290, 1998 (bibiserv.techfak.uni-bielefeld.de / dialign).

[0071] vector The term "recombinant" refers to a protein produced by the manipulation of a genetically modified organism (e.g., a microorganism).

[0072] According to the present invention, sources of CD59 include, for example, polynucleotide sequences encoding the CD59 protein engineered into recombinant DNA molecules to direct the expression of the CD59 protein in a suitable host cell. To express a biologically active CD59 protein, the nucleotide sequence encoding the CD59 protein, or a functional equivalent, is inserted into a suitable expression vector (i.e., a vector containing the necessary nucleic acid encoding elements to regulate the transcription and translation of the inserted coding sequence, operably linked to a nucleotide sequence encoding the amino acid sequence of the CD59 protein).

[0073] Use methods well known to those skilled in the art to construct an expression vector comprising a sequence encoding CD59 protein, operably linked to suitable transcriptional and translational regulatory elements.These methods include in vitro recombinant DNA technology, synthetic technology, and in vivo recombination or genetic recombination.Such techniques are described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Plainview, NY, 1989, Molecular Biomethods Handbook, 2nd ed., JM Walker et al., Humana Press, 2008, and Handbook of Molec. and Cellul. Methods in Biol. and Med., 3rd ed., LJ Ceske et al., CRC Press, 2011.

[0074] A variety of commercially available expression vector / host systems are useful for containing and expressing sequences encoding CD59 proteins. These include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems contacted with viral expression vectors (e.g., baculovirus); plant cell systems transfected with viral expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with bacterial expression vectors (e.g., Ti, pBR322, or pET25b plasmids); or animal cell systems.

[0075] Viral vectors include, but are not limited to, adenoviral vectors, lentiviral vectors, adeno-associated virus (AAV) vectors, and helper-dependent adenoviral vectors. As shown herein, viral vectors deliver the nucleic acid sequence encoding the CD59 protein, which prevents the harmful effects of MAC in the pathogenesis of AMD. Adenoviral packaging vectors are commercially available from the American Type Tissue Culture Collection (Manassas, Virginia). Methods for constructing and using adenoviral vectors are described in Klein et al. 2007 Ophthalmology 114: 253-262, and van Leeuwen et al. 2003 Eur. J. Epidemiol. 18: 845-854.

[0076] Adenoviral vectors have been used in eukaryotic gene expression (Levrero et al. 1991 Gene, 101: 195-202) and vaccine development (Graham et al. 1991 Methods in Molecular Biology: Gene Transfer and Expression Protocols 7, (Murray, Ed.), Humana Press, Clifton, NJ, 109-128). Additionally, recombinant adenoviral vectors are used in gene therapy (U.S. Patent No. 7,235,391, issued June 26, 2007 to Wu et al., incorporated herein by reference in its entirety).

[0077] Recombinant adenovirus vectors are produced, for example, by homologous recombination between shuttle vectors and provirus vectors (U.S. Patent No. 7,235,391, issued June 26, 2007 to Wu et al.; incorporated by reference). The adenovirus vectors herein are replication-deficient, for example, conditionally defective, lacking the adenovirus E1 region, and a polynucleotide encoding CD59 is introduced into the position where the E1 coding sequence has been removed. Alternatively, a polynucleotide encoding the CD59 gene is inserted into the E3 region.

[0078] Helper cell lines may be derived from human cells such as 293 human embryonic kidney cells, muscle cells, hematopoietic cells, or other human embryonic mesenchymal or epithelial cells. Alternatively, helper cells may be derived from cells of other mammalian species that are permissive for human adenovirus, such as Vero cells or other monkey embryonic mesenchymal or epithelial cells. The production and propagation of these replication-defective adenoviral vectors using helper cell lines is described in Graham et al. 1977 J. Gen. Virol. 36: 59-72.

[0079] Lentiviral vector packaging vectors are commercially available from Invitrogen Corporation (Carlsbad, CA). HIV-based packaging systems for lentiviral vector production are prepared using the constructs described in Naldini et al. 1996 Science 272: 263-267; Zufferey et al. 1997 Nature Biotechnol. 15: 871-875; and Dull et al. 1998 J. Virol. 72: 8463-8471.

[0080] Many vector constructs are available for packaging using systems based on third-generation lentiviral SIN vector backbones (Dull et al. 1998 J. Virol. 72: 8463-8471). For example, the vector construct pRRLsinCMVGFPpre contains a 5' LTR in which the HIV promoter sequence has been replaced with that of Rous sarcoma virus (RSV), a self-inactivating 3' LTR containing a deletion in the U3 promoter region, an HIV packaging signal, an RRE sequence linked to a marker gene cassette consisting of Aequorea victoria green fluorescent protein (GFP) driven by the CMV promoter, and a woodchuck hepatitis virus PRE element that appears to enhance nuclear export. The GFP marker gene allows quantification of transfection or transduction efficiency by direct observation with a UV fluorescence microscope or flow cytometry (Kafri et al. 1997 Nature Genet. 17: 314-317; and Sakoda et al. 1999 J. Mol. Cell. Cardiol. 31: 2037-2047).

[0081] Manipulation of retroviral nucleic acid to construct retroviral vectors and packaging cells containing a gene encoding the CD59 protein can be accomplished using techniques known in the art (see, for example, Ausubel et al., 1992, Volume 1, Section III (units 9.10.1-9.14.3); Sambrook et al., 1989. Molecular Cloning: A Laboratory Manual. Second Edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Miller et al., Biotechniques. 7:981-990, 1989; Molecular Biomethods Handbook, 2nd ed., JM Walker et al., Humana Press, 2008; Handbook of Molec. and Cellul. Methods in Biol. and Med., 3rd ed., LJ Ceske et al., CRC Press, 2011; Eglitis et al., Biotechniques. 6:608-614, 1988; U.S. Patent Nos. 4,650,764, 4,861,719, 4,980,289, 5,122,767, and 5,124,263; and WO 85 / 05629, WO 89 / 07150, WO 90 / 02797, WO 90 / 02806, WO 90 / 13641, WO 92 / 05266, WO 92 / 07943, WO 92 / 14829, and WO 93 / 14188, each of which is incorporated by reference in its entirety.

[0082] Retroviral vectors are constructed and packaged into non-infectious transducing viral particles (virions) using amphotropic packaging systems. Examples of such packaging systems are described, for example, in Markowitz et al. 1988 J. Virol. 62:1120-1124; Cosset et al. 1990 J. Virol. 64: 1070-1078; U.S. Pat. Nos. 4,650,764, 4,861,719, 4,980,289, 5,122,767, and 5,124,263, and WO 85 / 05629, WO 89 / 07150, WO 90 / 02797, WO 90 / 02806, WO 90 / 13641, WO 92 / 05266, WO 92 / 07943, WO 92 / 14829, and WO 93 / 14188, each of which is incorporated herein by reference in its entirety.

[0083] The generation of "producer cells" is achieved by introducing a retroviral vector into packaging cells. Examples of such retroviral vectors are described, for example, in Korman et al. 1987 Proc. Natl. Acad. Sci. USA. 84: 2150-2154; Morgenstern et al. 1990 Nucleic Acids Res. 18: 3587-3596; U.S. Patent Nos. 4,405,712, 4,980,289, and 5,112,767; and WO 85 / 05629, WO 90 / 02797, and WO 92 / 07943, each of which is incorporated herein by reference in its entirety.

[0084] Herpesvirus packaging vectors are commercially available from Invitrogen Corporation (Carlsbad, CA). Examples of herpesviruses include α-herpesviruses such as Varicella-Zoster virus or pseudorabies virus; herpes simplex viruses such as HSV-1 or HSV-2; or herpesviruses such as Epstein-Barr virus. A method for preparing empty herpesvirus particles that can be packaged with a desired nucleotide fragment (e.g., a CD59 nucleotide or polynucleotide sequence) in the absence of a helper virus, useful for most herpesviruses, is described in U.S. Patent No. 5,998,208, issued December 7, 1999, to Fraefel et al. (incorporated by reference in its entirety).

[0085] Herpesvirus DNA vectors can be constructed using techniques well known to those skilled in the art. For example, the DNA fragment encoding the entire genome of herpesvirus is divided into many vectors that can carry large DNA fragments, such as cosmids (Evans, et al., Gene 79, 9-20, 1989), yeast artificial chromosomes (YACS) (Sambrook, J. et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Edition, Cold Spring Harbor Press, Cold Spring Harbor, NY, 1989), or E. coli F factor plasmids (O'Conner et al. 1989 Science 244:1307-1313).

[0086] For example, sets of cosmids containing overlapping clones representing the entire genomes of various herpesviruses, including Epstein-Barr virus, varicella-zoster virus, pseudorabies virus, and HSV-1, have been isolated (see M. van Zijl et al. 1988 J. Virol. 62: 2191; Cohen et al. 1993 Proc. Nat'l Acad. Sci. USA 90: 7376; Tomkinson et al. 1993 J. Virol. 67: 7298; and Cunningham et al. 1993 Virology 197: 116).

[0087] AAV is a dependent parvovirus that relies on coinfection with another virus (adenovirus or a virus from the Herpesviridae family) to productively infect cultured cells (Muzyczka 1992 Curr Top Microbiol Immunol, 158: 97-129). For example, recombinant AAV (rAAV) virus is produced by cotransfecting a plasmid containing a gene of interest (e.g., the CD59 gene) flanked by two AAV terminal repeats (McLaughlin et al. 1988 J. Virol., 62(6): 1963-1973; Samulski et al. 1989 J. Virol., 63: 3822-3828) with an expression plasmid containing a wild-type AAV coding sequence without the terminal repeats. Cells are also contacted with or transfected with adenovirus or plasmids carrying adenoviral genes required for AAV helper function. Recombinant AAV virus stocks produced in such a manner contain adenovirus, which must be physically separated from the recombinant AAV particles (eg, by cesium chloride density centrifugation).

[0088] Adeno-associated virus (AAV) packaging vectors are commercially available from GeneDetect (Auckland, New Zealand). AAV has been shown to have a high integration frequency and can infect non-dividing cells, making it useful for delivering genes to mammalian cells in tissue culture (Muzyczka 1992 Curr Top Microbiol Immunol 158: 97-129). AAV has a wide host range of infection (Tratschin et al. 1984 Mol. Cell. Biol. 4: 2072-2081; Laughlin et al. 1986 J. Virol., 60(2): 515-524; Lebkowski et al. 1988 Mol. Cell. Biol. 8(10): 3988-3996; McLaughlin et al. 1988 J. Virol. 62(6): 1963-1973).

[0089] Methods for constructing and using AAV vectors are described, for example, in U.S. Patent No. 5,139,941 (Wu et al.), issued June 26, 2007, and U.S. Patent No. 4,797,368 (Carter et al.), issued January 10, 1989, each of which is incorporated herein by reference. The use of AAV in gene delivery is further described in LaFace et al. 1988 Virology 162(2): 483-486; Zhou et al. 1993 Exp. Hematol, 21: 928-933; Flotte et al. 1992 Am. J. Respir. Cell Mol. Biol. 7(3): 349-356; and Walsh et al. 1994 J. Clin. Invest 94: 1440-1448.

[0090] Recombinant AAV vectors can be used for in vitro and in vivo transduction of marker genes (Kaplitt et al. 1994 Nat Genet., 8(2):148-154; Lebkowski et al. 1988 Mol. Cell. Biol. 8(10): 3988-3996; Samulski et al. 1991 EMBO J. 10: 3941-3950; Shelling and Smith 1994 Gene Therapy, 1: 165-169; Yoder et al. 1994 Blood, 82 (Supp.): 1: 347A; Zhou et al. 1993 Exp. Hematol 21: 928-933; Tratschin et al. 1985 Mol. Cell. Biol. 5: 3258-3260; McLaughlin et al. 1988 J. Virol. 62(6): 1963-1973), and for the transduction of genes involved in human disease (Flotte et al. 1992 Am. J. Respir. Cell Mol. Biol. 7(3): 349-356; Ohi et al. 1990 Gene, 89(2): 279-282; Walsh et al. 1994 J. Clin. Invest. 94: 1440-1448; and Wei et al. 1994 Gene Therapy, 1: 261-268).

[0091] In some embodiments, the vector is a non-viral vector, e.g., a synthetic gene delivery vehicle or vector that specifically delivers CD59 gene-encoding material to target cells or tissues, unrelated to viral particles. Examples of non-viral vectors include liposomes, peptides, nanoparticles, emulsions, or encapsulated two or more phase systems or other suitable preparations. Thus, in some embodiments, the method, kit, or composition includes a non-viral vector containing a nucleic acid that is loaded and contacted with a tissue or cell. For example, naked DNA encoding a membrane-independent CD59 protein with a modified GPI anchor that does not target the membrane, or a gene encoding a membrane-independent CD59 protein without a GPI anchor, is encapsulated in a liposome, and the liposome is contacted with the tissue or cell to effectively deliver the nucleic acid to the tissue or cell for the treatment of complement-related diseases.

[0092] antibody The term "antibody" as referred to herein includes whole antibodies and any antigen-binding fragment (i.e., "antigen-binding portion") or single chains thereof. Naturally occurring "antibodies" are glycoproteins comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds.

[0093] As used herein, an antibody that "specifically binds to human MAC" is one that is 5×10 -9 M or less, 2×10 -9 M or less, or 1 x 10 -10 K below M D The term "monoclonal antibody" is intended to refer to an antibody that binds to human MAC at a specific site. For example, the antibody may be monoclonal or polyclonal. As used herein, the term "monoclonal antibody" or "monoclonal antibody composition" refers to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition exhibits a single binding specificity and affinity for a MAC or a particular epitope of a MAC. The antibody may be IgM, IgE, or IgG (such as IgG1 or IgG4).

[0094] Also useful for MAC assays are antibodies that are recombinant antibodies. As used herein, the term "recombinant human antibody" includes all antibodies prepared, expressed, produced, or isolated by recombinant means, such as antibodies isolated from animals (e.g., mice). Mammalian host cells for expressing recombinant antibodies used in the methods herein include Chinese hamster ovary cells (CHO cells), such as dhfr-CHO cells, NSO myeloma cells, COS cells, and SP2 cells, used with a DHFR selectable marker, as described in Urlaub and Chasin, 1980 Proc. Natl. Acad. Sci. USA 77: 4216-4220, for example, as described in RJ Kaufman and PA Sharp, 1982 Mol. Biol. 159:601-621. In particular, another expression system for use in NSO myeloma cells is the GS gene expression system shown in WO 87 / 04462, WO 89 / 01036, and EP 338,841. To produce antibodies, an expression vector encoding an intact protein of interest or a portion of the protein of interest is introduced into mammalian host cells, and the host cells are cultured for a time sufficient to allow expression of the antibody in the host cells or secretion of the antibody into the culture medium in which the host cells are grown. Antibodies can be recovered from the culture medium using standard protein purification methods.

[0095] Standard assays for evaluating the binding ability of antibodies to targets of various species are known in the art, including, for example, ELISA, Western blot and RIA. The binding kinetics (e.g., binding affinity) of antibodies can also be evaluated by standard assays known in the art, such as Biacore analysis.

[0096] A general methodology for antibody production, including criteria to consider when selecting animals for antiserum production, is described in Harlow et al. (1988 Antibodies, Cold Spring Harbor Laboratory, pp. 93-117). For example, an animal of appropriate size, such as a goat, dog, sheep, mouse, or camel, is immunized by administering an immunogen, such as an intact protein or a portion thereof containing an epitope derived from human MAC, that is effective in generating an immune response. An exemplary protocol is as follows: 100 μg to 100 mg of antigen, depending on the size of the animal, is injected subcutaneously into the back of the animal. Three weeks later, 100 μg to 100 mg of immunogen containing an adjuvant (e.g., complete Freund's adjuvant), depending on the size of the animal, is injected intraperitoneally. Further intraperitoneal injections of adjuvant (e.g., complete Freund's adjuvant) are given every two weeks until the antibody in the animal's blood reaches an appropriate titer. Exemplary titers include titers of at least about 1:5000 or titers of 1:100,000 or greater (i.e., the dilution at which detectable activity exists). Antibodies are purified, for example, by affinity purification on a column containing human MAC.

[0097] The technique of in vitro immunization of human lymphocytes is used to produce monoclonal antibodies. Techniques for in vitro immunization of human lymphocytes are well known to those skilled in the art. For example, see Inai et al. May 1993 Histochemistry, 99(5): 335-362; Mulder et al. 1993 Hum. Immunol. 36(3): 186-192; Harada, et al. 1993 J. Oral Pathol. Med., 22(4): 145-152; Stauber, et al. 1993 J. Immunol. Methods 161(2): 157-168; and Venkateswaran, et al. 1992 Hybridoma, 11(6): 729-739. These techniques can be used to produce antigen-reactive monoclonal antibodies, such as antigen-specific IgG and IgM monoclonal antibodies. Any antibody or fragment thereof that has affinity for and is specific for human MAC is within the scope of the assays for MAC deposition provided herein.

[0098] In the examples herein, contact with CD59 is achieved by injecting cells or tissues with a vector encoding the CD59 gene.

[0099] In this example, cell lysis is measured by the uptake of propidium iodide (PI). PI is commercially available, for example, from Fluka BioChemica (Buchs, Switzerland). PI is an intercalating agent that fluoresces when bound to DNA. PI is membrane-impermeable and generally excluded from viable cells, therefore, PI is generally used to identify and / or determine the amount of non-viable cells in a mixed population.

[0100] In the examples and in certain embodiments herein, the detectable protein is a fluorescent protein, such as green fluorescent protein, aequorin, cyan fluorescent protein, DsRed fluorescent protein, enhanced green fluorescent protein, and yellow fluorescent protein. Green fluorescent protein (GFP) and aequorin are bioluminescent compounds isolated from the jellyfish Aequorea victoria. When calcium ions bind to aequorin, the complex breaks down into apoaequorin and a luminescent compound, which emits blue light. Synthetic aequorin is commercially available from Sealite, Sciences (Bogart, GA) under the trademark AQUALITE®. GFP emits light in the lower green portion of the visible spectrum, and synthetic GFP is commercially available from Clontech (Mountain View, CA).

[0101] Mutations to the amino acid sequence of GFP have been made to generate derivatives of GFP that emit different colors of fluorescence, such as cyan fluorescent protein, DsRed fluorescent protein, enhanced green fluorescent protein, and yellow fluorescent protein. Synthetic cyan fluorescent protein, synthetic DsRed fluorescent protein, synthetic enhanced green fluorescent protein, and synthetic yellow fluorescent protein are all commercially available from Clontech (Mountain View, CA).

[0102] In another embodiment, the detectable substance is a fluorescent substance that is not a fluorescent protein, such as indocyanine green, doxorubicin, riboflavin, chlorophyll, and porphyrin.

[0103] Indocyanine green (ICG) is a tricarbocyanine dye that emits light at about 800 nm, about 820 nm, about 840 nm, or about 860 nm upon excitation. ICG is commercially available from HW Sands Corp. (Jupiter, Florida). Doxorubicin is fluorescent, emitting light at wavelengths of, for example, about 550 nm, 600 nm, or 650 nm. Doxorubicin is commercially available from Sigma-Aldrich (St. Louis, Missouri). Riboflavin is commercially available from Sigma-Aldrich (St. Louis, Missouri) and is fluorescent, emitting light at wavelengths of, for example, about 450 nm, about 550 nm, about 650 nm, or about 750 nm. Chlorophyll A is a green photosynthetic pigment that emits light at wavelengths of, for example, about 600 nm, about 700 nm, or about 800 nm. Chlorophyll A is commercially available from suppliers such as Sigma Chemical (St. Louis, MO) and Turner Designs (Sunnyvale, CA). Porphyrins are heterocyclic macrocycles made up of four pyrrole subunits linked on both sides through four methine bridges (=CH-). Due to the large conjugated structure of porphyrins, the compounds are colored, i.e., they fluoresce at wavelengths of, for example, about 600 nm, about 650 nm, or about 700 nm. Porphyrins are commercially available from Sigma-Aldrich (St. Louis, MO).

[0104] In other alternative embodiments, the detectable substance is an enzymatic agent, which may be a protein such as β-galactosidase or alkaline phosphatase, and may be expressed in a nucleotide vector.

[0105] β-Galactosidase is a hydrolase that catalyzes the hydrolysis of β-galactosides into monosaccharides. A luminescent β-galactosidase detection kit is commercially available from Clontech (Mountain View, CA). Alkaline phosphatase is a hydrolase that removes phosphate groups from various types of molecules, including nucleotides, proteins, and alkaloids. A luminescent alkaline phosphatase detection kit is commercially available from Sigma-Aldrich (St. Louis, MO).

[0106] Pharmaceutical Composition One aspect of the present invention provides methods of using a nucleic acid encoding a CD59 protein or a soluble CD59 protein to inhibit inflammasome activation in inflamed ocular cells of a subject. In various embodiments, the CD59 protein comprises a membrane-independent (soluble) CD59 protein. In certain embodiments, the CD59 composition is formulated for intravenous administration. In certain embodiments, the composition is formulated as an ophthalmic formulation for administration to the eye, and may be formulated to enhance delivery to the fundus to provide localized sustained release at the retina, or to provide effective treatment of blood vessels and / or tissues involved in ocular diseases, including macular degeneration. In related embodiments, the pharmaceutical composition is formulated with sufficient purity for administration to a human subject, e.g., to the circulation or eye of a human subject. In certain embodiments, these compositions optionally further comprise one or more additional therapeutic agents. In certain embodiments, the additional therapeutic agent is selected from the group consisting of growth factors, anti-inflammatory agents, vasopressors including but not limited to nitric oxide and calcium channel blockers, collagenase inhibitors, topical steroids, matrix metalloproteinase inhibitors, ascorbate, angiotensin II, angiotensin III, calreticulin, tetracycline, fibronectin, collagen, thrombospondin, transforming growth factor (TGF), keratinocyte growth factor (KGF), fibroblast growth factor (FGF), insulin-like growth factor (IGF), IGF-binding protein (IGFBP), epidermal growth factor (EGF), platelet-derived growth factor (PDGF), neuronal differentiation factor (NDF), hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), heparin-binding EGF (HBEGF), thrombospondin, von Willebrand factor C, heparin and heparin sulfate, and hyaluronic acid.

[0107] In other embodiments, the additional agent is a compound, composition, biological agent, or the like that enhances, stabilizes, synergizes with, or even replaces the ability of the CD59 protein to protect cells from MAC deposition. Also included are therapeutic agents that can be advantageously or conveniently provided simultaneously with the CD59 protein, such as, for example, agents used to treat the same, concurrent, or related symptoms, conditions, or diseases. In some embodiments, the drug may include, but is not limited to, an antitumor agent, an antiviral agent, an antibacterial agent, an antimycobacterial agent, an antifungal agent, an antiproliferative agent, or an antiapoptotic agent. Drugs that can be included in the compositions of the present invention are well known in the art. See, e.g., Goodman & Gilman's *The Pharmacological Basis of Therapeutics*, 9th Ed., Hardman, et al., eds., McGraw-Hill, 1996 (the contents of which are incorporated herein by reference).

[0108] As used herein, the term "pharmaceutically acceptable carrier" includes any and all solvents, diluents, or other liquid vehicles, dispersing or suspending aids, surfactants, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants, and the like, that are appropriate for the particular dosage form desired. Remington's Pharmaceutical Sciences Ed. by Gennaro, Mack Publishing, Easton, PA, 1995 provides various carriers used in formulating pharmaceutical compositions, as well as known techniques for their preparation. Some examples of materials which may serve as pharmaceutically acceptable carriers include, but are not limited to, sugars such as glucose and sucrose; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, soybean oil, and the like; glycols such as propylene glycol; esters such as ethyl oleate, ethyl laurate, and the like; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer, and other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate; further, coloring agents, releasing agents, coating agents, preservatives, and antioxidants can also be present in the composition, according to the judgment of the formulator.

[0109] Therapeutically Effective Dose Treatment of an inflamed eye using the methods provided herein involves contacting tissues or cells with a pharmaceutical composition, e.g., administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition having as an active agent a nucleic acid encoding a CD59 protein or a source of expression of a CD59 protein, in an amount and for a time necessary to achieve the desired result. Methods include, for example, treating uveitis by contacting ocular tissues or cells with a CD59 protein or a vector encoding a CD59 protein.

[0110] The compositions according to the methods of the present invention can be administered using any amount and any route of administration effective for treating uveitis or other complement-related diseases and conditions. Accordingly, as used herein, the phrase "an amount effective to treat uveitis" refers to an amount of a composition sufficient to beneficially prevent or ameliorate the symptoms of uveitis.

[0111] The exact dosage will be selected by the individual physician, taking into account the patient receiving treatment. Dosage and administration are adjusted to provide sufficient levels of the active substance or maintain the desired effect. Additional factors that may be considered include the severity of the condition, such as intermediate or advanced uveitis; the patient's age, weight, and sex; diet, time and frequency of administration; route of administration; drug combinations; reaction sensitivities; and tolerance / response to treatment. Long-acting pharmaceutical compositions may be administered hourly, twice hourly, every 3-4 hours, daily, twice daily, every 3-4 days, weekly, or once every two weeks, depending on the half-life and clearance rate of the particular composition.

[0112] The active substances of the present invention are preferably formulated in dosage unit form for ease of administration and uniformity of dosage. As used herein, the expression "dosage unit form" refers to a physically discrete unit of active substance appropriate for the patient being treated. However, it will be understood that the total daily usage amount of the compositions of the present invention will be determined by the attending physician within the scope of sound medical judgment. For any active substance, the therapeutically effective dose can be initially estimated either in cell culture assays or animal models (usually mice, as provided herein, but also rats, rabbits, dogs, or pigs). The animal cell models provided herein are also used to achieve the desired concentration and total dosage range and route of administration. Such information can be used to determine useful doses and routes for administration to humans.

[0113] A therapeutically effective dose refers to the amount of an active substance that improves symptoms or conditions or prevents the progression of uveitis. The therapeutic efficacy and toxicity of an active substance can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, such as ED50 (the dose that is therapeutically effective in 50% of the population) and LD50 (the dose that is lethal to 50% of the population). The ratio of the toxic dose to the therapeutically effective dose is the therapeutic index, which can be expressed as the ratio LD50 / ED50. Pharmaceutical compositions that exhibit a large therapeutic index are preferred. Data obtained from cell culture assays and animal experiments are used to formulate a range of dosages for human use.

[0114] The daily dosage of the product may vary over a wide range, such as from 0.001 to 100 mg per adult per day. For ocular administration, the composition is preferably provided in the form of a solution containing 0.001, 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100.0, 250.0, or 500.0 μg of the active ingredient for symptomatic adjustment of the dosage to the patient being treated.

[0115] The dose of soluble CD59 protein administered intravenously can range from 0.01 mg / kg / dose to about 10 mg / kg / dose, 0.1 mg / kg / dose to about 5 mg / kg / dose, or 1.0 mg / kg / dose to about 5 mg / kg / dose. Dosing protocols include daily, twice weekly, once weekly, once every two weeks, once every three weeks, or once every four weeks.

[0116] A unit dose typically contains about 0.001 μg to about 500 μg of active ingredient, preferably about 0.1 μg to about 100 μg, and more preferably about 1.0 μg to about 10 μg. An effective amount of the drug is usually provided at a dosage level of about 0.0001 mg / kg body weight / day to about 25 mg / kg body weight / day. For example, the range is about 0.001 to 10 mg / kg body weight / day, or about 0.001 mg / kg body weight / day to 1 mg / kg body weight / day. The composition may be administered, for example, in a regimen of one to four or more times per day. The unit dose may also be divided, e.g., administered in two or more separate doses.

[0117] The administration of a viral vector or nucleic acid vector as a source of expression of CD59 protein is a dose containing at least about 50, 100, 500, 1000, or at least about 5000 particles per treated cell. Cell numbers can be calculated from the retinal area requiring treatment by methods known to those skilled in the art of treating uveitis or ocular diseases.

[0118] Administration of the Pharmaceutical Composition When formulated with a suitable pharmaceutically acceptable carrier in a desired dosage, the pharmaceutical compositions provided herein are administered to humans and other mammals topically, such as ophthalmically (as a solution, ointment, or eye drop), intranasally, bucally, orally, rectally, parenterally, intracisternally, intravaginally, or intraperitoneally.

[0119] Intraocular injections include intraocular injections into the aqueous or vitreous humor, or into the outer layers of the eye, such as by subconjunctival or sub-Tenon injection.

[0120] Liquid dosage forms for ophthalmic, oral, intravenous, or other systemic administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs. In addition to the active substance, liquid dosage forms can contain inert diluents commonly used in the art, such as water or other solvents, solubilizers and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (especially cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil, and sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol, and fatty acid esters of sorbitan, and mixtures thereof. In addition to inert diluents, ophthalmic compositions, oral compositions, or other systemically delivered compositions can also contain adjuvants such as wetting agents, emulsifiers, and suspending agents.

[0121] Dosage forms for topical or transdermal administration of the pharmaceutical compositions of the present invention include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants, or patches. The active substance is mixed under sterile conditions with a pharmaceutically acceptable carrier and, if necessary, any necessary preservatives or buffers. For example, administration to the eye or skin is achieved by drops, mists, emulsions, or creams. Administration can be therapeutic or prophylactic. The present invention includes ophthalmic, surgical, and audiological devices or products (e.g., gauze bandages or strips) containing the disclosed compositions, as well as methods for making or using such devices or products. These devices can be coated, impregnated, bonded, or otherwise treated with the compositions described herein.

[0122] Transdermal patches have the additional advantage of providing controlled delivery of active ingredients to the body.Such dosage forms can be prepared by dissolving or dispersing the compound in a suitable medium.Absorption enhancers can also be used to increase the flux of the compound through the skin.The rate can be controlled by providing a rate-controlling membrane or dispersing the compound in a polymer matrix or gel.

[0123] Injectable preparations, for example, sterile injectable aqueous or oily suspensions, can be formulated according to known techniques using appropriate dispersing or wetting agents and suspending agents. Sterile injectable preparations can be sterile injectable solutions, suspensions, or emulsions in non-toxic parenterally acceptable diluents or solvents, such as solutions in 1,3-butanediol. Acceptable vehicles and solvents that can be used include water, Ringer's solution, USP, and isotonic sodium chloride solution. Additionally, sterile, fixed oils are conventionally used as solvents or suspending media. For this purpose, any bland, fixed oil, including synthetic mono- or diglycerides, can be used. Additionally, fatty acids, such as oleic acid, are used in the preparation of injectable preparations. Injectable preparations can be sterilized, for example, by filtration through a bacteria-retaining filter, or by incorporating a sterilizing agent in the form of a sterile solid composition that can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use. To prolong the effect of an active substance, it is often desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. Delayed absorption of parenterally administered active substances can be achieved by dissolving or suspending the drug in an oil vehicle. Injectable depot forms are made by forming microencapsule matrices of the drug in biodegradable polymers such as polylactide-polyglycolide. The release rate of the active substance can be controlled depending on the ratio of active substance to polymer and the nature of the particular polymer used. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Injectable depot formulations can also be prepared by entrapping the drug in liposomes or microemulsions that are compatible with body tissues.

[0124] Compositions for rectal or vaginal administration are preferably suppositories which can be prepared by mixing the active agents of the invention with a suitable non-irritating excipient or carrier, such as cocoa butter, polyethylene glycol or a suppository wax, which are solid at ordinary temperatures but liquid at body temperature and therefore melt in the rectum or vaginal cavity to release the active agent.

[0125] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active agent is mixed with at least one inert pharmaceutically acceptable excipient or carrier, such as sodium citrate or dicalcium phosphate, and / or a) fillers or extenders, such as starches, sucrose, glucose, mannitol, and silicic acid; b) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia; c) humectants, such as glycerol; d) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; e) solution retarding agents, such as paraffin; f) absorption accelerators, such as quaternary ammonium compounds; g) humectants, such as cetyl alcohol and glycerol monostearate; h) adsorbents, such as kaolin and bentonite clay; i) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof.

[0126] Similar types of solid compositions can also be used as fillers for soft and hard-filled gelatin capsules, using excipients such as lactose and high molecular weight polyethylene glycols. Solid dosage forms such as tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells, such as enteric coatings, release-controlling coatings, and other coatings well known in the pharmaceutical formulation art. In such solid dosage forms, the active ingredient can be mixed with at least one inert diluent, such as sucrose or starch. Such dosage forms can also contain, as is common practice, additional substances other than inert diluents, such as tableting lubricants and other tableting aids, such as magnesium stearate and microcrystalline cellulose. In the case of capsules, tablets, and pills, the formulations can also contain buffering agents. They can optionally contain opacifying agents, and can be of a composition that releases the active ingredient only or preferentially in a certain part of the intestinal tract, optionally in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes.

[0127] A portion of this work, attached hereto as Appendix A, has been prepared for publication as a manuscript entitled "Complement-Mediated Activation of the NLRP3 Inflammasome and Its Inhibition by AAV-Mediated Delivery of CD59 in a Model of Uveitis" by co-authors and co-inventors Binit Kumar, Siobhan M. Cashman, and Rajendra Kumar-Singh, which is incorporated herein by reference in its entirety.

[0128] Selection of patients for disease monitoring and treatment The CD59 nucleic acid and protein compositions described herein are useful for treating inflammatory and inflammasome-related diseases. The described compositions can be administered to individuals selected for treatment based on a positive result in a test assaying inflammasome activity. Such tests are not limited to direct measurements of inflammasome activation, but also include indirect measurements. Indirect measurements can be, for example, measurements of cytokines, chemokines, cell surface markers in inflamed or diseased tissue, cell activation of specific immune types, or the presence or absence of specific immune types. For example, cytokines such as IL-1α, IL-1β, and IL-18 are elevated in response to inflammasome activation. Thus, selected individuals may exhibit elevated levels of these cytokines in plasma, serum, blood, tissue sections, mRNA, or cell extracts of diseased tissue, or elevated levels of expression or secretion by immune cells isolated from blood or diseased tissue. Selection criteria for treatment with a CD59 nucleic acid or protein composition can be an increase in the level or activity of any one or more of caspase 1, caspase 5, IL-1β, IL-β17, IL-18, apoptosis-associated speck-like card protein (PYCARD / ASC), NACHT, LRR and PYD domain-containing protein (NALP), IFN-γ, Th1 T cell markers or cytokines, Th17 T cell markers or cytokines, and CD4+.

[0129] Elevated levels of cytokines IL-1α, IL-1β, IL-β17, IL-18, or IFN-γ can be assessed, for example, by ELISA, AlphaLISA®, immunohistochemistry, flow cytometry analysis of relevant cell populations, or quantitative RT-PCR. Activation of caspases and inflammasomes can be determined by immunohistochemical staining of biopsy specimens using antibodies specific for activated caspase and inflammasome components, such as caspase-1, caspase-5, apoptosis-associated speck-like card protein (PYCARD / ASC), NACHT, and LRR and PYD domain-containing protein (NALP). Th1 and Th17 cells can be distinguished using flow cytometry for the cell surface markers CXCR3 or CCR5, or the transcription factor TBX21 in the case of Th1, or intracellular staining for the transcription factor RORg / gt in the case of Th17.

[0130] The described CD59 nucleic acid and protein compositions are administered to individuals selected for treatment based on a diagnosis of an inflammasome-associated disorder, such as Alzheimer's disease, multiple sclerosis, myocardial infarction, atherosclerotic vascular disease, microangiopathy, thyroiditis, inflammatory bowel disease, organ transplant rejection, membranous nephritis, sympathetic ophthalmia, and sarcoidosis. These include inflammasome-associated ophthalmic disorders such as uveitis, allergic conjunctivitis, blepharitis, chronic conjunctivitis, episcleritis, keratitis, retinitis, ocular cicatricial pemphigoid, mucous membrane pemphigoid, pterygium scleritis, Stevens-Johnson syndrome, Eales' disease, Behçet's disease, sarcoidosis, systemic lupus erythematosus, polyarteritis nodosa, Wegener's granulomatosis, Vogt-Koyanagi-Harada disease, sympathetic ophthalmia, and sarcoidosis.

[0131] In certain embodiments, CD59 nucleic acid or protein compositions can be administered after monitoring the individual for response to at least one administration of such composition or to another treatment to reduce inflammation or resolve an inflammasome-associated disease. This cycle of administration and monitoring can be repeated 1, 2, 3, 4, 5, 6, 7, 8, 9, or more times, or until an appropriate clinical response is achieved or inflammasome activity declines. In certain embodiments, CD59 nucleic acid and protein compositions are 1) administered to an individual selected as having an inflammasome-associated disease or having a positive result for inflammasome activity; 2) the individual can then be monitored and given at least one or more subsequent administrations of the composition based on a worsening, unchanged, or suboptimal response as determined by testing for inflammasome activity or diagnostic criteria for an inflammasome-associated disease. In certain embodiments, CD59 nucleic acid and protein compositions are 1) administered to an individual, and 2) the individual can then be monitored and given at least one or more subsequent doses of the composition based on a worsening, unchanged, or suboptimal or greater response as determined by testing for inflammasome activity or diagnostic criteria for an inflammasome-associated disease.

[0132] The following number of embodiments encompassing CD59 polypeptide / nucleic acid methods and compositions are contemplated herein:

[0133] 1. A method for inhibiting inflammasome activation in inflamed ocular cells of a subject, comprising administering to the subject a composition comprising a nucleotide sequence encoding a membrane-independent CD59 protein operably linked to a promoter for expression and secretion of the membrane-independent CD59 protein in inflamed ocular cells, wherein the composition inhibits inflammasome activation.

[0134] 2. The method of embodiment 1, wherein the composition inhibits inflammasome activation independently of membrane attack complex (MAC) function.

[0135] 3. The method of embodiment 1, wherein the subject has at least one condition selected from uveitis, allergic conjunctivitis, blepharitis, chronic conjunctivitis, episcleritis, keratitis, retinitis, ocular cicatricial pemphigoid, mucous membrane pemphigoid, pterygium scleritis, Stevens-Johnson syndrome, Eales disease, Behcet's disease, sarcoidosis, systemic lupus erythematosus, polyarteritis nodosa, Wegener's granulomatosis, Vogt-Koyanagi-Harada disease, sympathetic ophthalmia, and sarcoidosis.

[0136] 4. The method of embodiment 3, wherein the uveitis is at least one selected from anterior uveitis, intermediate uveitis, posterior uveitis, and panuveitis.

[0137] 5. The method of embodiment 1, further comprising obtaining a sample from the subject.

[0138] 6. The method of embodiment 5, wherein the sample is at least one selected from tears, blood, urine, ocular secretions, sputum, and mucus.

[0139] 7. The method of any one of embodiments 1-6, further comprising measuring at least one of retinal function and inflammasome activity markers in the eye prior to administration.

[0140] 8. The method of any one of embodiments 1 to 7, further comprising measuring at least one of retinal function and inflammasome activity markers in the eye after administration.

[0141] 9. The method of embodiment 7 or 8, wherein the inflammasome activity marker is at least one selected from caspase 1, caspase 5, IL-1β, IL-β17, IL-18, apoptosis-associated speck-like card protein (PYCARD / ASC), NACHT, LRR and PYD domain-containing protein (NALP), IFN-γ, Th1 T cell markers or cytokines, Th17 T cell markers or cytokines, and CD4+.

[0142] 10. The method of embodiment 7 or 8, wherein measuring retinal function or inflammasome activity markers further comprises performing at least one technique selected from eye examination, optical coherence tomography (OCT), conjunctival impression cytology, RTPCR, ELISA, and PCR.

[0143] 11. The method of embodiment 3, further comprising administering the composition in a dosage sufficient to treat uveitis.

[0144] 12. The method of embodiment 1, further comprising manipulating a nucleotide sequence in the viral vector prior to administration.

[0145] 13. The method of embodiment 1 or 12, further comprising administering the nucleotide sequence as naked nucleic acid.

[0146] 14. The method of embodiment 13, wherein the viral vector is a genetically engineered genome of at least one virus selected from the group consisting of adenovirus, adeno-associated virus, herpesvirus, and lentivirus.

[0147] 15. The method of embodiment 14, wherein the lentivirus is a retrovirus.

[0148] 16. The method of embodiment 1, further comprising engineering a membrane-independent CD59 protein to have at least one mutation that results in loss of function of the glycosylphosphatidylinositol (GPI) anchor domain of the translated CD59 protein.

[0149] 17. The method of embodiment 1, further comprising manipulating the nucleotide sequence encoding the membrane-independent CD59 protein by deleting nucleotides encoding the region of the glycosylphosphatidylinositol (GPI) anchor domain.

[0150] 18. The method of embodiment 1, wherein administering further comprises injecting the composition into the eye.

[0151] 19. The method of embodiment 1, wherein administering further comprises topically applying the composition.

[0152] 20. The method of embodiment 18, wherein the ocular injection is selected from the group consisting of subretinal injection, intravitreal injection, intraocular injection, subconjunctival injection, and subtenon injection.

[0153] 21. The method of embodiment 18, wherein the ocular injection further comprises administering to the outer layer of the eye.

[0154] 22. The method of embodiment 1, further comprising administering an additional therapeutic agent to the eye.

[0155] 23. The method of embodiment 22, wherein the additional therapeutic agent is at least one selected from the group consisting of an antitumor agent, an antiviral agent, an antibacterial agent, an antimycobacterial agent, an antifungal agent, an antiproliferative agent, and an antiapoptotic agent.

[0156] 24. The method of embodiment 22, wherein the additional therapeutic agent is selected from the group consisting of a growth factor, an anti-inflammatory agent, a vasopressor, a collagenase inhibitor, a steroid, a matrix metalloproteinase inhibitor, ascorbate, angiotensin, calreticulin, tetracycline, fibronectin, collagen, thrombospondin, transforming growth factor (TGF), keratinocyte growth factor (KGF), fibroblast growth factor (FGF), insulin-like growth factor (IGF), IGF-binding protein (IGFBP), epidermal growth factor (EGF), platelet-derived growth factor (PDGF), neu differentiation factor (NDF), hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), heparin-binding EGF (HBEGF), thrombospondin, von Willebrand factor C, heparin, heparin sulfate, and hyaluronic acid.

[0157] 25. A kit for inhibiting inflammasome activation in an inflamed eye of a subject, comprising: a. a pharmaceutical composition comprising a membrane-independent CD59 protein and / or a nucleotide sequence encoding a CD59 protein, in a dosage sufficient to inhibit inflammasomes in the inflamed eye of a subject; b. Instructions for use; and c. Container Includes a kit.

[0158] 26. A kit for treating uveitis in a subject, comprising: a. a pharmaceutical composition comprising a membrane-independent CD59 protein and / or a nucleotide sequence encoding a CD59 protein, in a dosage sufficient to treat uveitis in a subject; b. Instructions for use, and c. Container Includes a kit.

[0159] 27. A method of treating at least one condition selected from uveitis, allergic conjunctivitis, blepharitis, chronic conjunctivitis, episcleritis, keratitis, retinitis, ocular cicatricial pemphigoid, mucous membrane pemphigoid, pterygium sclerosis, Stevens-Johnson syndrome, Eales' disease, Behcet's disease, sarcoidosis, polyarteritis nodosa, Wegener's granulomatosis, Vogt-Koyanagi-Harada disease, sympathetic ophthalmia, and sarcoidosis, comprising: a) a nucleotide sequence encoding a membrane-independent CD59 protein operably linked to a promoter for expression and secretion of the membrane-independent CD59 protein in cells of the inflamed eye; or b) Membrane-independent CD59 protein The method includes administering to a subject a composition comprising:

[0160] 28. A method for inhibiting inflammasome activation in inflamed eye cells of a subject, comprising: a) measuring inflammasome activity markers in the eye; and b) if the marker of inflammasome activity is positive, administering to the subject a composition comprising a nucleotide sequence encoding a membrane-independent CD59 protein operably linked to a promoter for expression and secretion of the membrane-independent CD59 protein in cells of the inflamed eye. wherein the composition inhibits inflammasome activation.

[0161] 29. The method of embodiment 28, wherein the inflammasome activity marker is selected from caspase 1, caspase 5, IL-1β, IL-β17, IL-18, apoptosis-associated speck-like card protein (PYCARD / ASC), NACHT, LRR and PYD domain-containing protein (NALP), IFN-γ, Th1 T cell markers or cytokines, Th17 T cell markers or cytokines, and CD4+.

[0162] 30. The method of embodiment 28 or 29, further comprising measuring an inflammasome activity marker in the eye after administration of a composition comprising a nucleotide sequence encoding a membrane-independent CD59 protein operably linked to a promoter for expression and secretion of the membrane-independent CD59 protein.

[0163] 31. A method for inhibiting inflammasome activation in cells of an inflamed subject, comprising: a. administering to a subject a composition comprising a nucleotide sequence encoding a membrane-independent CD59 protein operably linked to a promoter for expression and secretion of the membrane-independent CD59 protein in cells of the inflamed subject; or b. Administering a composition comprising a soluble CD59 protein to a subject wherein the composition inhibits inflammasome activation.

[0164] 32. The method of embodiment 31, wherein the composition inhibits inflammasome activation independently of membrane attack complex (MAC) function.

[0165] 33. The method of embodiment 31, wherein the subject has at least one inflammasome-associated condition selected from Alzheimer's disease, multiple sclerosis, myocardial infarction, atherosclerosis, microangiopathy, thyroiditis, inflammatory bowel disease, organ transplant rejection, membranous nephritis, sympathetic ophthalmia, and sarcoidosis.

[0166] 34. The method of embodiment 31, further comprising obtaining a sample from the subject.

[0167] 35. The method of embodiment 34, wherein the sample is at least one selected from blood, plasma, serum, peripheral blood mononuclear cells, cerebrospinal fluid, and urine.

[0168] 36. The method of any of embodiments 31-35, further comprising measuring a marker of inflammasome activity in the subject prior to administration.

[0169] 37. The method of any of embodiments 31-35, further comprising measuring a marker of inflammasome activity in the subject after administration.

[0170] 38. The method of embodiment 36 or 37, wherein the inflammasome activity marker is selected from caspase 1, caspase 5, IL-1β, IL-β17, IL-18, apoptosis-associated speck-like card protein (PYCARD / ASC), NACHT, LRR and PYD domain-containing protein (NALP), IFN-γ, Th1 T cell markers or cytokines, Th17 T cell markers or cytokines, and CD4+.

[0171] 39. The method of embodiment 38, further comprising administering the composition in a dosage sufficient to treat the inflammasome-associated condition.

[0172] 40. The method of embodiment 39, wherein the nucleotide sequence encoding the membrane-independent CD59 protein operably linked to a promoter for expression and secretion of the membrane-independent CD59 protein is carried in a vector comprising a genetically engineered genome of at least one virus selected from the group consisting of adenovirus, adeno-associated virus, herpesvirus, and lentivirus.

[0173] 41. The method of embodiment 40, wherein the lentivirus is a retrovirus.

[0174] 42. The method of embodiment 31, wherein administering further comprises injecting the composition intravenously.

[0175] 43. The method of embodiment 31, wherein administering further comprises topically applying the composition.

[0176] 44. The method of embodiment 31, further comprising administering an additional therapeutic agent.

[0177] 45. The method of embodiment 44, wherein the additional therapeutic agent is at least one selected from the group consisting of an antitumor agent, an antiviral agent, an antibacterial agent, an antimycobacterial agent, an antifungal agent, an antiproliferative agent, and an antiapoptotic agent.

[0178] 46. ​​The method of embodiment 44, wherein the additional therapeutic agent is selected from the group consisting of a growth factor, an anti-inflammatory agent, a vasopressor, a collagenase inhibitor, a steroid, a matrix metalloproteinase inhibitor, ascorbate, angiotensin, calreticulin, tetracycline, fibronectin, collagen, thrombospondin, transforming growth factor (TGF), keratinocyte growth factor (KGF), fibroblast growth factor (FGF), insulin-like growth factor (IGF), IGF-binding protein (IGFBP), epidermal growth factor (EGF), platelet-derived growth factor (PDGF), neu differentiation factor (NDF), hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), heparin-binding EGF (HBEGF), thrombospondin, von Willebrand factor C, heparin, heparin sulfate, and hyaluronic acid.

[0179] 47. A kit for inhibiting inflammasome activation in inflamed cells of a subject, comprising: c. (i) a membrane-independent CD59 protein and / or a nucleotide sequence encoding a CD59 protein; or (ii) soluble CD59 protein; in a dosage sufficient to inhibit inflammasomes in inflamed cells of a subject; d. Instructions for use; and e. Container Includes a kit.

[0180] 48. A method of treating at least one condition selected from Alzheimer's disease, multiple sclerosis, myocardial infarction, atherosclerosis, microangiopathy, thyroiditis, inflammatory bowel disease, organ transplant rejection, membranous nephritis, sympathetic ophthalmia, and sarcoidosis, comprising: The method includes administering to a subject a composition comprising: a nucleotide sequence encoding a membrane-independent CD59 protein operably linked to a promoter for expression and secretion of the membrane-independent CD59 protein from a cell; or a soluble CD59 protein; wherein the composition inhibits inflammasome activation.

[0181] The invention now fully described is further illustrated by the following examples and claims, which are illustrative and not meant to be further limiting. [Example]

[0182] Example 1: Mice C57BL / 6J and C9 on the C57BL / 6J background - / - Mice were purchased from the Jackson Laboratory (Bar Harbor, ME) and maintained in the animal facility at Tufts University School of Medicine in Boston. All animal study protocols adhered to the recommendations of the Association for Research in Vision and Ophthalmology resolution on the use of animals in vision research and the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals.

[0183] Example 2: AAV constructs and intravitreal injections AAV serotype 2 vectors (AAVCAGsCD59) expressing truncated forms of human CD59 or protectin lacking the glycosylphosphatidylinositol (GPI) anchoring signal were constructed according to the protocol described by Cashman, SM, et al. (2011) PLoS One 6, e19078. Soluble CD59 (sCD59) was expressed from the chicken β-actin promoter. As a negative control, a similar vector expressing green fluorescent protein (AAVCAGGFP) was used. Six-week-old C57BL / 6J mice were transfected with 3.5 × 10 9AAVCAGsCD59 or AAVCAGGFP at genome copies / μl, or PBS (1 μl) were injected, and one week later, the mice were subjected to EAU as described in the Examples herein.

[0184] Example 3: Induction of EAU by active immunization Six-week-old C57BL / 6J and C9 mice on the same genetic background - / - Mice were immunized with 200 μg of human interphotoreceptor retinoid-binding protein (IRBP) peptide 1-20 (amino acid sequence: GPTHLFQPSLVLDMAKVLLD, SEQ ID NO: 1; Biomatik Corporation, Cambridge, Ontario, Canada) emulsified in 200 μl of 1:1 (v / v) complete Freund's adjuvant (CFA) containing Mycobacterium tuberculosis strain H37RA (2.5 mg / mL). Simultaneously, mice were administered 1.5 μg of Bordetella pertussis toxin diluted in 100 μl of PBS via intraperitoneal injection (Agarwal, RK, et al. (2012) Methods Mol. Biol. 900, 443-469).

[0185] Example 4: Immunohistochemistry Cryostat retinal sections (10 μm) were rehydrated in PBS for 15 minutes, blocked with 6% normal goat serum in PBS for 1 hour, and incubated overnight in a moist chamber with rabbit anti-human C5b-9 primary antibody (Complement Technology, Inc., Tyler, TX; dilution: 1:800, diluted in PBS containing 2% normal goat serum). The sections were then washed and incubated in a Cy3-conjugated anti-rabbit secondary antibody (Molecular Probes, Eugene, OR) to localize C5b-9 in the retinal sections. Slides were mounted with a DAPI-containing antifade mounting medium (Vectashield-DAPI; Vector Laboratories, Burlingame, CA) to counterstain nuclei, and images were acquired using a Leica confocal microscope. The intensity of C5b-9 staining across the sections was quantified using ImageJ software (National Institutes of Health; Bethesda, MD).

[0186] Example 5: Western blot analysis Mouse retinas were harvested and homogenized in ice-cold RIPA buffer containing 50 mM Tris-HCl (pH 7.4), 250 mM NaCl, 1% Nonidet P-40, and a protease inhibitor cocktail. Each protein sample (35 μg) was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (Any kD™ Mini-PROTEAN® Precast Gel, Biorad, CA) for NLRP3, caspase-1, and ASC, and then transferred to a nitrocellulose membrane. After blocking with blocking buffer (LI-COR, Lincoln, NE, USA) mixed with 0.1% Tween-20, immunoblots were incubated overnight at 4°C with mouse anti-NLRP3 monoclonal antibody, mouse anti-caspase-1 monoclonal antibody, and rabbit anti-ASC polyclonal antibody (Adipogen Corporation, San Diego, CA; dilution 1:500) as primary antibodies. After incubation with the appropriate secondary antibodies, immunoreactive bands were visualized using a LI-COR-Odyssey infrared scanner (LI-COR). Blots were reprobed with β-actin as a loading control.

[0187] Example 6: Enzyme-linked immunosorbent assay Cytokines were quantified by sandwich ELISA for murine IL-1β, IFN-γ, and IL-17 (PeproTech, Rocky Hills, NJ) using 20 μg of retinal protein supernatant according to the manufacturer's instructions. Supernatants were added in duplicate, and monoclonal antibodies conjugated to horseradish peroxidase were used to reveal the cytokines being measured. Cytokine concentrations were expressed in pg / mL.

[0188] Example 7: Gene Expression Total RNA was isolated from mouse retinas using the RNeasy Mini Kit (QIAGEN, Valencia, CA) according to the manufacturer's protocol. RNA was quantified by absorbance at 260 nm in a Nanodrop™, and 1 μg of RNA was used for cDNA synthesis using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA).

[0189] Real-time polymerase chain reaction (RT-PCR) was performed using predesigned TaqMan primers for β-actin (Mm02619580_g1), IL-1β (Mm00434228_m1), IL-17 (Mm00439619_m1), and IFN-γ (Mm01168134_m1). Denaturation was performed at 95°C for 10 minutes, followed by 40 cycles of 95°C for 15 seconds, and annealing and extension at 60°C for 60 seconds. The final PCR products were electrophoresed on a 2% agarose gel to confirm PCR specificity. Ct values ​​obtained from RT-PCR were normalized to the Ct value from β-actin of the same sample using the ddCt method to obtain fold-change data for gene expression. IL-17 gene expression was quantified semiquantitatively.

[0190] Example 8: Flow cytometry Draining lymph node cells were isolated from mice and single-cell suspensions were obtained by passing the cells through a 40 μm nylon mesh. Prior to intracellular cytokine staining, the draining lymph node cells were stimulated with 50 ng / mL PMA and 500 ng / mL ionomycin (Sigma-Aldrich, St. Louis, MO) for 4 hours in the presence of GolgiStop (BD Biosciences, San Jose, CA). Cells were stained for the surface marker CD4 and then fixed with Cytofix / Cytoperm Buffer (BD Biosciences, San Jose, CA) for cytokine staining. Cells were stained with appropriately diluted fluorophore-conjugated antibodies against intracellular targets (IFNγ, IL-17; eBiosciences, Inc., San Diego, CA) and their respective isotype controls in Perm / Wash Buffer (BD Biosciences, San Jose, CA). Flow cytometry was performed on a FACS Calibur (BD Biosciences), and data were analyzed using FlowJo software (Tree Star, Ashland, OR). Gates were set based on appropriate isotype controls. Where indicated, percentages of positive cells represent the percentage of the gated population relative to normal controls.

[0191] Example 9: Electroretinogram Scotopic and photopic electroretinogram (ERG) analysis was used to measure the loss of rod and cone function. Three weeks after EAU induction, ERG tests were recorded using a UTAS system equipped with a BigShot Ganzfeld (LKC Technologies; Gaithersburg, MD). After overnight dark adaptation, mice were anesthetized under dark conditions with an intraperitoneal injection of ketamine (100 mg / kg) / xylazine (10 mg / kg). Pupils were dilated with 1% tropicamide and 2.5% phenylephrine hydrochloride. An ERG-active gold contact lens electrode was gently placed on the center of the cornea with a drop of lubricant (GenTeal, Alcon, Inc., Fort Worth, TX) to maintain corneal hydration and good electrical conductivity. Reference and ground electrodes were inserted subcutaneously at the back of the neck and near the base of the tail, respectively. Scotopic ERGs were recorded at 0 dB (5 cds / m). 2 ), -10dB(25cds / m 2 ), -20dB(25cds / m 2 The photopic ERG was elicited with a 10-msec flash of white light at 0 dB and 1 dB (3.15 cds / m). After a 2-min white-light bleaching period, a simultaneous photopic ERG was obtained. The photopic response was measured at 0 dB and 1 dB (3.15 cds / m). 2 ) and elicited with a white light flash at an intensity of 100 Hz. Ten responses were averaged for each flash intensity. The amplitude of the a-wave was measured from baseline to the negative peak of the a-wave, and the b-wave was measured from the negative peak of the a-wave to the peak of the b-wave.

[0192] Example 10: Spectral Domain Optical Coherence Tomography (OCT) and Fundus Imaging Mice were anesthetized with a cocktail of ketamine and xylazine, and pupils were dilated with drops of 1% tropicamide and 2.5% phenylephrine. The cornea was kept moist by topical application of ocular lubricant (GenTeal, Alcon, Inc., Fort Worth, TX). Optical coherence tomography (OCT) images were acquired using a Bioptigen Spectral Domain Ophthalmic Imaging System (Bioptigen Envisu R2300, Morrisville, NC). Averaged single B-scans and volume scans were acquired with the optic nerve head centered, as described in Chen, J., et al. (2013) PLoS One 8, e63904.

[0193] Twenty-four days after EAU, fundus imaging was performed. Images were acquired using a Micron III Retinal Imaging Microscope and StreamPix software (Phoenix Research Labs, Pleasanton, CA). Specifically, eyes were examined for physiological and pathological symptoms, including infiltrates, perivascular inflammatory cell infiltration (cuffing), white line lesions, retinal dystrophy, subretinal hemorrhage, and retinal detachment. Individual scores were assessed on a scale of 0-4 by two independent observers in a blinded manner for each of the following parameters: retinal infiltrates, optic disc changes, vascularity, and structural damage. The clinical score was calculated by averaging the scores for each of these four criteria (Xu, H., et al. (2008) Exp. Eye Res. 87, 319-326).

[0194] Example 11: Histopathological examination Eyes for histological examination from all groups were harvested 24 days after immunization and fixed in 10% buffered formalin. After 2 days of fixation, specimens were dehydrated through graded alcohol steps and embedded in paraffin blocks. Six vertical sections (5 μm) were cut at six different planes, including the optic nerve region, and stained with hematoxylin and eosin. The detailed severity of EAU was assessed on a scale of 0–4 for photoreceptor damage, infiltration, and vasculitis, as previously described by Caspi, RR, et al. (1988) J. Immunol 140, 1490–1495. Based on these criteria, the photoreceptor damage score was calculated as a composite score of photoreceptor loss, retinal folds, and retinal detachment. Similarly, the infiltration score consisted of a combination of granulomas, hemorrhages, DF nodules, and infiltrates. The vasculitis score represented the extent of perivasculitis and perivascular CD4 cell infiltration, thrombus formation, and retinal vasculature involvement.

[0195] Example 12: Statistical Analysis Results are shown as mean ± SEM. The Mann-Whitney test was used for clinical scores, histological scores, and FACS analysis. Statistical differences between two groups were analyzed using an unpaired t-test. For comparisons between three or more groups, a one-way analysis of variance was performed. A p value of 0.05 or less was considered statistically significant.

[0196] Example 13: MAC deposition in EAU The final step in complement activation results in the deposition of MAC on the cell surface. Recruitment of multiple C9 molecules to the preformed C5b-8 complex is the final required step in the assembly of MAC (C5b-9). Therefore, C9-deficient mice are unable to form functional MAC complexes.

[0197] To determine whether MAC forms on the surface of retinal cells in EAU, we examined frozen retinal sections 24 days after induction of EAU in C57BL / 6J mice by staining with an antibody against C5b-9. We observed that MAC deposition in the retinas of EAU mice was 70% greater than that in the retinas of normal control mice.- / - No significant MAC deposition was observed on the surface of the EAU retina (Figure 1A and Figure 9). The data obtained indicated that complement is activated in the EAU, and the reaction is completed to form MAC on the retinal tissue. The data showed that C9 - / - They also show that EAU mice fail to form MAC on the retina.

[0198] Example 14: Inflammasome activation in EAU Deposition of MAC on the cell surface leads to the formation of pores, which results in an increase in intracellular calcium. In the Examples herein, we investigated whether MAC deposition triggers activation of the NLRP3 inflammasome and subsequent secretion of IL-1β.

[0199] The data obtained found that EAU in C57BL / 6J mice resulted in 112% more IL-1β protein and a corresponding 45-fold more IL-1β mRNA than normal mouse controls. - / - In EAU mice, 37% more IL-1β protein and a corresponding 3.8-fold more IL-1β mRNA were observed (Figure 1B and C). Western blot analysis showed 200% more NLRP3 protein in EAU mice, suggesting a significant increase in C9 - / - EAU mice had 8% more NLRP3 protein ( Fig. 1 D).

[0200] A key component of the inflammasome complex is caspase-1, which generally occurs in the form of an inactive precursor (zymogen) and is activated by the NLRP3 complex and proteolytically converted to the active p10 and p20 subunits, creating the final multimeric inflammasome complex. Western blot analysis showed 80% higher caspase-1 activation (p20) in the retinas of EAU mice than in normal mice; however, C9 - / -In the retinas of EAU mice, 25% higher activation of caspase-1 (p20) was observed compared to normal mice (Figure 1E). Expression of ASC protein, an inflammasome adaptor protein, in the retinas of EAU mice was measured by Western blot. Western blot analysis showed that ASC protein was 44% higher in EAU mice than normal mice, and C9 - / - We showed that EAU mice had 18% more ASC protein than normal mice (Fig. 1F).

[0201] Accordingly, IL-1β, NLRP3, caspase 1, and ASC were each observed to be increased in EAU, whereas C9 - / - EAU mice were partially protected from such increases, indicating that MAC deposition is a key player in inflammasome activation in EAU.

[0202] Example 15: Role of MAC on retinal function in EAU EAU mice and C9 - / - Retinal function in EAU mice was measured using electroretinograms (ERGs). Compared to control C57BL / 6J mice, dark-adapted a-wave amplitudes were observed to be reduced by 33%, 50%, and 41% at flash intensities of -20 dB, -10 dB, and 0 dB, respectively. C9 - / - Retinal function in EAU mice was observed to be such that the a-wave amplitude was 9%, 40%, and 27% less than that of normal control C57BL / 6J mice at flash intensities of -20 dB, -10 dB, and 0 dB, respectively (Figures 2A and 2B).

[0203] The dark-adapted b-wave amplitude in EAU mice was observed to be reduced by 47%, 52%, and 49% at flash intensities of -20 dB, -10 dB, and 0 dB, respectively, compared with that in control C57BL / 6J mice. - / -EAU mice were observed to have dark-adapted b-wave amplitudes that were 32%, 33%, and 17% lower at flash intensities of -20 dB, -10 dB, and 0 dB, respectively, compared to control C57BL / 6J mice. Differences were observed in both the a-wave and b-wave of the dark-adapted ERG, but were statistically significant (p<0.05) at 0 dB (Figures 2A and 2B).

[0204] EAU mice exhibited a 44% and 37% reduction in light-adapted b-wave amplitude at 0 dB and 1 dB flash intensities, respectively, compared with control C57BL / 6J mice. - / - EAU mice exhibited a 5% and 15% reduction in b-wave amplitude at 0 dB and 1 dB flash intensities, respectively, compared with control C57BL / 6J mice. Differences in b-wave amplitude at light adaptation were observed, but the difference at 0 dB was statistically significant (p<0.05) (Figures 2A and 2B).

[0205] Example 16: C9 - / - No significant preservation of retinal structures in EAU EAU and C9 - / - The retinal structure and pathological severity of disease in EAU mice were quantified using fundus imaging, optical coherence tomography (OCT), and histological examination 24 days after EAU induction. Based on the clinical scoring criteria developed by Xu, H. et al. (2008) Exp. Eye Res. 87, 319-326, fundus imaging showed that EAU mice had severe inflammation, specifically an overall clinical score 16-fold higher than controls, including 38-fold higher vascular inflammation, 8-fold greater immune cell infiltration, 13-fold greater optic disc damage, and 34-fold greater structural damage compared to control C57BL / 6J mice. Unexpectedly, the uveitis-inducing C9 mice were significantly more severe than control C57BL / 6J mice. - / - Mice were observed to have pathological outcomes that were statistically equivalent to EAU mice (Figure 3).

[0206] Twenty-four days after immunization, EAU mice exhibited severe inflammatory cell infiltration in the vitreous and choroid, retinal vasculitis, retinal edema, and moderate to severe retinal folds and infiltrates, as observed by OCT imaging and histopathological examination, compared with C57BL / 6J control mice (Figure 4A and Figure 4B). - / - Detailed histological analysis was performed on paraffin-embedded sections from the retinas of EAU mice and scored based on the criteria described in Caspi, RR, et al. (1988) J. Immunol 140, 1490-1495. EAU mice were observed to have approximately 27 times more infiltrates (Figure 4C), increased vasculitis (Figure 4D), and 78 times greater photoreceptor damage (Figure 4D) than C57BL / 6J control mice. - / - Histological scores in EAU mice showed 28% less infiltration, 35% less photoreceptor damage, and 31% less vasculitis compared with EAU mice, although no statistically significant differences were observed ( Fig. 4B and C ).

[0207] The photoreceptor damage score was calculated as a composite score of photoreceptor loss, retinal folds, and retinal detachment based on the criteria described by Caspi, RR, et al. (1988) J. Immunol 140, 1490-1495. Similarly, the infiltration score consisted of a combination of granulomas, hemorrhages, DF nodules, and infiltrates. The vasculitis score represented the extent of perivasculitis and perivascular CD4 cell infiltration, thrombus formation, and retinal vasculature involvement.

[0208] Example 17: Soluble CD59-mediated inhibition of MAC deposition in EAU The data obtained in the Examples herein show that a genetic defect in C9 - / - These mice were unable to form MAC and activate inflammasomes, suggesting they were protected from some of the hallmarks of uveitis. - / - It was observed that the retina of EAU mice was not protected from the histological pathology associated with EAU.

[0209] CD59 is a GPI-anchored protein located on the membrane of most nucleated cells. The primary function of CD59 is to prevent the recruitment of C9 to preformed C5b-8 complexes. A recombinant adeno-associated viral vector (AAVCAGsCD59) expressing a truncated CD59 lacking the GPI anchor signal, allowing it to be secreted and diffuse throughout the retina, is described in Cashman, SM, et al. (2011) PLoS One 6, e19078.

[0210] Mice were intravitreally injected with AAVCAGsCD59, and one week later (to allow for optimal transgene levels), they underwent EAU as described in the Examples herein. For a negative control, mice were intravitreally injected with AAVCAGGFP (a virus expressing green fluorescent protein (GFP)). Twenty-four days after EAU induction, frozen retinal sections from both sets of mice were examined by staining with C5b-9 antibody. In mice intravitreally injected with AAVCAGGFP and subsequently subjected to EAU, transgene expression was observed in the ganglion cell layer, inner plexiform layer, and inner nuclear layer (Figure 14B). In mice with phenotypically more severe EAU, transgene expression was also observed in the RPE and photoreceptors (Figure 14B, bottom). Approximately 45% less MAC deposition was observed in EAU mice injected with AAVCAGsCD59 compared to EAU mice injected with AAVCAGGFP (Figures 5A and 12). Accordingly, intravitreally injected AAVCAGsCD59 was observed to inhibit the formation of MACs in the retinas of EAU mice.

[0211] Example 18: Soluble CD59-mediated inhibition of inflammasomes in EAU In the examples herein, C9 - / - EAU mice were observed to have significantly reduced inflammasome activation compared with EAU control C57BL / 6J mice. NLRP3 / caspase-1-mediated IL-1β secretion in EAU mice pre-injected with either AAVCAGsCD59 or AAVCAGGFP was measured as described in the Examples herein.

[0212] As measured by ELISA and RT-PCR, AAVCAGsCD59-injected EAU mice had 40% less IL-1β protein and 70% less IL-1β mRNA compared with AAAVAGsCD59-injected EAU mice (Figures 5B and 5C). Similarly, NLRP3 protein and caspase-1 p20 levels were reduced by 60% in AAVCAGsCD59-injected EAU mice compared with control AAVCAGGFP-injected EAU mice (Figures 5D and 5E). No substantial difference in the amount of ASCs was observed in AAVCAGsCD59-injected EAU mice compared with control AAVCAGGFP-injected EAU mice (Figure 5F). Thus, the data obtained indicate that sCD59 inhibits NLRP3-mediated IL-1β production by inhibiting MAC deposition in uveitis.

[0213] Example 19: Role of MAC-mediated NLRP3 inflammasome activation in T cell differentiation in EAU CD4+ infiltrating T cells enter the retina either in an undifferentiated state or differentiated into Th1 (IFN-γ-producing) and Th17 (IL-17-producing) cells. To investigate the potential role of MAC in T cell differentiation, we used EAU and C9 mice. - / - The levels of IFN-γ and IL-17 in the retinas of EAU mice were measured, respectively.

[0214] Using ELISA, a 102% increased amount of IFN-γ protein was observed in EAU retinas compared to control C57BL / 6J retinas. - / - 14% less IFN-γ protein was observed in EAU retinas compared to control C57BL / 6J retinas. RT-PCR data also showed that EAU retinas and C9 - / - We demonstrated that IFN-γ mRNA in EAU retinas was increased by more than 200-fold and more than 14-fold, respectively (Figures 10A and 10B). Similarly, IL-17 protein expression was observed to be 99% higher in EAU retinas compared to control C57BL / 6J retinas, whereas C9 - / -In EAU retinas, IL-17 protein expression was observed to be 44% higher than in control C57BL / 6J retinas. - / - No statistical difference was observed between IL-17 mRNA expression levels in EAU retinas. IL-17 mRNA levels remained below the detection limit in C57BL / 6J retinas (Figures 10C and 10D). These data indicate that MAC plays a role in T cell differentiation in EAU.

[0215] Control C57BL / 6J mice, EAU mice, and C9 - / - In EAU mice, the levels of Th1 CD4+ cells and Th17 CD4+ cells were measured in the draining lymph nodes (DLN) 24 days after EAU. Compared with control C57BL / 6J mice, higher numbers of IL-17- and IFN-γ-positive CD4+ cells were observed in the DLN of EAU mice (Figures 11A-11C). - / - No statistically significant differences were observed in the levels of IFN-γ and IL-17-positive CD4+ cells in the DLN between EAU mice (Figures 11A-C). These data suggest that MAC plays an important role in T cell differentiation in the EAU retina and that MAC may not play an important role in the DLN.

[0216] Example 20: Effect of soluble CD59 on T cell differentiation in EAU To examine the potential impact on T cell differentiation in sCD59-expressing eyes affected by EAU, we measured the protein and mRNA levels of IFN-γ and IL-17 in AAVCAGsCD59-injected and AAVCAGGFP-injected EAU mice.

[0217] The protein levels of IFN-γ and IL-17 were significantly lower in AAVCAGsCD59-injected EAU retinas than in control AAVCAGGFP-injected EAU retinas by more than 25% and more than 35%, respectively (Figures 13A and 13C). Furthermore, the mRNA levels of IFN-γ and IL-17 in freshly isolated AAVCAGsCD59-injected EAU retinas were observed to be 47% and 10%, respectively, lower than in control AAVCAGGFP-injected EAU retinas. The mRNA differences were not observed to be statistically significant (Figures 13B and 13D).

[0218] Example 21: Soluble CD59-mediated protection of retinal function in EAU To determine whether sCD59 can protect retinal function in EAU, we performed ERGs in AAVCAGsCD59- or AAVCAGGFP-injected mice with uveitis. AAVCAGsCD59-injected EAU mice were observed to have 45%, 49%, and 51% greater dark-adapted a-wave amplitudes at flash intensities of -20 dB, -10 dB, and 0 dB, respectively, compared to control AAVCAGGFP-injected EAU mice. Similarly, dark-adapted b-wave amplitudes in AAVCAGsCD59-injected mice were observed to be 55%, 48%, and 40% greater at flash intensities of -20 dB, -10 dB, and 0 dB, respectively, compared to control AAVCAGGFP-injected EAU eyes (Figure 6). Furthermore, photopic b-wave amplitude at 0 dB and 1 dB flash intensities in AAVCAGsCD59-injected EAU eyes was protected by 12% and 39% compared with control AAVCAGGFP-injected EAU eyes (Figure 6). An important and potentially therapeutic role of recombinant sCD59 in protecting retinal function in the inner and outer retina in uveitis was observed.

[0219] Example 22: Soluble CD59-mediated protection of retinal structure and pathology in EAU To determine whether sCD59 could protect retinal structure and reduce the pathology associated with uveitis, fundus imaging was performed on AAVCAGsCD59- or AAVCAGGFP-injected EAU mice. Based on the scoring criteria described in Xu, H., et al. (2008) Exp. Eye Res. 87, 319-326, the overall clinical score of AAVCAGsCD59-injected EAU mice was determined to be 22% lower compared to control AAVCAGGFP-injected EAU mice. Overall clinical scores included 24% less appearance of inflammatory infiltrates, 27% less structural damage, 22% reduced vasculitis and vascular cuffing, and 13% less damage to the optic nerve head in AAVCAGsCD59-injected EAU mice compared to AAVCAGGFP-injected EAU mice (Figures 7A-F). No significant differences were observed between PBS vehicle controls and AAVCAGGFP-injected EAU retinas. The vascular, infiltration, and structural damage scores were statistically significantly different (p<0.05), but the optic disc score did not reach statistical significance (p=0.1). Thus, the data obtained indicate that sCD59 expression reduced retinal inflammation and immune cell infiltration in uveitis.

[0220] The progression and severity of EAU lesions were observed to be more severe in AAVCAGGFP-injected EAU mice compared with AAVCAGsCD59-injected EAU mice, as documented and observed with optical coherence tomography (OCT) scans (Figure 8A). Detailed histological analysis of paraffin sections taken from the retinas of AAVCAGsCD59-injected EAU mice or control AAVCAGGFP-injected EAU mice was performed. Similarly, based on the criteria described by Caspi, RR, et al. (1988) J. Immunol 140, 1490-1495, AAVCAGsCD59-injected EAU mice exhibited approximately 76% less infiltrates, 69% less photoreceptor damage, and 78% less vasculitis compared with control AAVCAGGFP-injected EAU mice (Figure 8B-E). The differences in photoreceptor damage and infiltrates were statistically significant (p = 0.03 and p = 0.01, respectively), but the vasculitis score was not statistically significant (p = 0.2).

[0221] Example 23: Treatment and subsequent monitoring of individuals with atherosclerosis using compositions of soluble CD59 protein An individual is diagnosed with atherosclerosis based on an ultrasound examination showing an increase in intima-media thickness (IMT), after which the individual is administered a composition containing soluble CD59 protein via intravenous infusion. The patient receives three consecutive doses, administered weekly. After administration, the individual is re-evaluated by ultrasound examination, and if the IMT remains unchanged, the individual receives four additional doses of CD59 protein, administered intravenously, once weekly, and is then re-evaluated. At follow-up, the IMT thickness has decreased to an acceptable level, and no further CD59 treatment is administered.

[0222] Example 24: Treatment and subsequent monitoring of individuals with sarcoidosis using compositions of soluble CD59 protein An individual presents to their physician with symptoms consistent with sarcoidosis, lung x-rays show growths consistent with such a diagnosis, a biopsy sample is taken, and elevated levels of activated caspase-1 are identified by comparison with healthy tissue controls. The individual is then administered a composition containing soluble CD59 protein via intravenous infusion. Following administration, the individual is re-evaluated with x-rays, which show partial remission of sarcoidosis. The patient is again administered the soluble CD59 protein composition, and x-ray re-evaluation shows a complete response, and no further CD59 treatment is administered.

[0223] As used herein, the terms "individual," "patient," or "subject" refer to an individual who has been diagnosed with, is suspected of having, or is at risk of developing at least one disease for which the described compositions and methods are useful for treating. In some embodiments, the individual is a mammal. In some embodiments, the mammal is a mouse, rat, rabbit, dog, cat, horse, cow, sheep, pig, goat, llama, alpaca, or yak. ​​In some embodiments, the individual is a human.

[0224] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will occur to those skilled in the art without departing from the invention. It will be understood that various alternatives to the embodiments of the invention described herein may be used in practicing the invention.

[0225] All publications, patent applications, issued patents, and other documents mentioned in this specification are incorporated herein by reference as if each individual publication, patent application, issued patent, or other document were expressly indicated to be incorporated by reference in its entirety. Definitions contained in text incorporated by reference are excluded only to the extent that they conflict with definitions in this disclosure. Finally, preferred embodiments of the present invention are described in sections.

[0226] [Embodiment 1] A method for inhibiting inflammasome activation in cells of an inflamed eye of a subject, comprising administering to the subject a composition comprising a nucleotide sequence encoding a membrane-independent CD59 protein operably linked to a promoter for expression and secretion of the membrane-independent CD59 protein in cells of the inflamed eye, wherein the composition inhibits inflammasome activation, and the subject exhibits a positive result for the expression or activity of at least one inflammasome activity marker in the inflamed eye of the subject.

[0227] [Embodiment 2] 2. The method of embodiment 1, wherein the inflammasome activity marker is selected from caspase 1, caspase 5, IL-1β, IL-β17, IL-18, apoptosis-associated speck-like card protein (PYCARD / ASC), NACHT, LRR- and PYD domain-containing protein (NALP), IFN-γ, Th1 T cell markers or cytokines, Th17 T cell markers or cytokines, and CD4+.

[0228] [Embodiment 3] 2. The method of embodiment 1, wherein the inflammasome activity marker is selected from apoptosis-associated speck-like card protein (PYCARD / ASC), or NACHT, LRR and PYD domain-containing protein (NALP).

[0229] [Embodiment 4] The method of embodiment 3, wherein the NALP is NACHT, LRR and PYD domain-containing protein 3 (NLRP3).

[0230] [Embodiment 5] 5. The method of any of embodiments 1-4, further comprising measuring a marker of inflammasome activity in the eye after administration of a composition comprising a nucleotide sequence encoding a membrane-independent CD59 protein operably linked to a promoter for expression and secretion of said membrane-independent CD59 protein.

[0231] [Embodiment 6] 6. The method of any of embodiments 1-5, wherein the subject has been diagnosed with or is suspected of suffering from uveitis, allergic conjunctivitis, blepharitis, chronic conjunctivitis, episcleritis, keratitis, retinitis, ocular cicatricial pemphigoid, mucous membrane pemphigoid, pterygium scleritis, Stevens-Johnson syndrome, Eales disease, Behcet's disease, sarcoidosis, systemic lupus erythematosus, polyarteritis nodosa, Wegener's granulomatosis, Vogt-Koyanagi-Harada disease, sympathetic ophthalmia, or sarcoidosis.

[0232] [Embodiment 7] The method of embodiment 6, wherein the subject has been diagnosed with or is suspected of having uveitis.

[0233] [Embodiment 8] 8. The method of any of embodiments 1-7, wherein the positive result for expression or activity of at least one inflammasome activity marker in the inflamed eye of the subject is increased expression or activity of at least one inflammasome activity marker compared to expression or activity of at least one inflammasome activity marker in the eye of an individual who has not been diagnosed with or is not afflicted with uveitis, allergic conjunctivitis, blepharitis, chronic conjunctivitis, episcleritis, keratitis, retinitis, ocular cicatricial pemphigoid, mucous membrane pemphigoid, pterygium scleritis, Stevens-Johnson syndrome, Eales disease, Behcet's disease, sarcoidosis, systemic lupus erythematosus, polyarteritis nodosa, Wegener's granulomatosis, Vogt-Koyanagi-Harada disease, sympathetic ophthalmia, or sarcoidosis.

[0234] [Embodiment 9] The method of embodiment 8, wherein a positive result for the expression or activity of at least one inflammasome activity marker in the inflamed eye of the subject is an increase in the expression or activity of at least one inflammasome activity marker compared to the expression or activity of at least one inflammasome activity marker in the eye of an individual who has not been diagnosed with or is not suffering from uveitis.

[0235] [Embodiment 10] A method according to any one of embodiments 1 to 9, wherein a positive result for the expression or activity of at least one inflammasome activity marker in the inflamed eye of the subject is determined by a histological score.

[0236] [Embodiment 11] 1. A method of inhibiting inflammasome activation in cells of an inflamed subject, comprising: a. Measuring a marker of inflammasome activity in a subject; and b. If the inflammasome activation marker is positive, (i) a nucleotide sequence encoding a membrane-independent CD59 protein operably linked to a promoter for expression and secretion of the membrane-independent CD59 protein in cells of an inflamed subject; or (ii) Soluble CD59 protein administering to the subject a composition comprising wherein the composition inhibits inflammasome activation.

[0237] [Embodiment 12] 12. The method of embodiment 11, wherein the inflammasome activity marker is selected from caspase 1, caspase 5, IL-1β, IL-β17, IL-18, apoptosis-associated speck-like card protein (PYCARD / ASC), NACHT, LRR- and PYD domain-containing protein (NALP), IFN-γ, Th1 T cell markers or cytokines, Th17 T cell markers or cytokines, and CD4+.

[0238] [Embodiment 13] 12. The method of embodiment 11, wherein the inflammasome activity marker is selected from apoptosis-associated speck-like card protein (PYCARD / ASC), or NACHT, LRR and PYD domain-containing protein (NALP).

[0239] [Embodiment 14] The method of embodiment 13, wherein the NALP is NACHT, LRR and PYD domain-containing protein 3 (NLRP3).

[0240] [Embodiment 15] A method according to any one of embodiments 11 to 14, wherein the inflammasome activity marker is measured in the eye of the subject.

[0241] [Embodiment 16] 16. The method of any of embodiments 11-15, further comprising measuring a marker of inflammasome activity in the subject after administration of a composition comprising a nucleotide sequence encoding a membrane-independent CD59 protein or a soluble CD59 protein operably linked to a promoter for expression and secretion of said membrane-independent CD59 protein.

[0242] [Embodiment 17] 17. The method of any of embodiments 11-16, wherein the subject has been diagnosed with or is suspected of suffering from uveitis, allergic conjunctivitis, blepharitis, chronic conjunctivitis, episcleritis, keratitis, retinitis, ocular cicatricial pemphigoid, mucous membrane pemphigoid, pterygium scleritis, Stevens-Johnson syndrome, Eales disease, Behcet's disease, sarcoidosis, systemic lupus erythematosus, polyarteritis nodosa, Wegener's granulomatosis, Vogt-Koyanagi-Harada disease, sympathetic ophthalmia, or sarcoidosis.

[0243] [Embodiment 18] 18. The method of embodiment 17, wherein the subject has been diagnosed with or is suspected of having uveitis.

[0244] [Embodiment 19] 19. The method of any of embodiments 11 to 18, wherein the positive inflammasome activity marker is an increase in the expression or activity of at least one inflammasome activity marker compared to the expression or activity of at least one inflammasome activity marker in a subject who has not been diagnosed with or does not suffer from Alzheimer's disease, multiple sclerosis, myocardial infarction, atherosclerosis, microangiopathy, thyroiditis, inflammatory bowel disease, organ transplant rejection, membranous nephritis, sympathetic ophthalmia, uveitis, or sarcoidosis.

[0245] [Embodiment 20] The method of embodiment 19, wherein the positive inflammasome activity marker is an increase in the expression or activity of at least one inflammasome activity marker compared to the expression or activity of at least one inflammasome activity marker in a subject not diagnosed with uveitis.

[0246] [Embodiment 21] A method according to any one of embodiments 11 to 20, wherein the positive inflammasome activity marker in the inflamed subject is determined by a histological score.

[0247] [Embodiment 22] a. a nucleotide sequence encoding a membrane-independent CD59 protein operably linked to a promoter for expression and secretion of the membrane-independent CD59 protein in cells of an inflamed subject; or B soluble CD59 protein 1. A method of inhibiting inflammasome activation in cells of an inflamed subject by administering to the subject a composition comprising: the composition inhibits inflammasome activation; The method, wherein the subject has a positive result for expression or activity of at least one inflammasome activity marker in an inflamed subject.

[0248] [Embodiment 23] 23. The method of embodiment 22, wherein the inflammasome activity marker is selected from caspase 1, caspase 5, IL-1β, IL-β17, IL-18, apoptosis-associated speck-like card protein (PYCARD / ASC), NACHT, LRR- and PYD domain-containing protein (NALP), IFN-γ, Th1 T cell markers or cytokines, Th17 T cell markers or cytokines, and CD4+.

[0249] [Embodiment 24] 23. The method of embodiment 22, wherein the inflammasome activity marker is selected from apoptosis-associated speck-like card protein (PYCARD / ASC), or NACHT, LRR and PYD domain-containing protein (NALP).

[0250] [Embodiment 25] 25. The method of embodiment 24, wherein the NALP is NACHT, LRR and PYD domain-containing protein 3 (NLRP3).

[0251] [Embodiment 26] A method according to any one of embodiments 22 to 25, wherein the positive result for the expression or activity of at least one inflammasome activity marker in the inflamed subject is in the eye of the inflamed subject.

[0252] [Embodiment 27] 27. The method of any of embodiments 22-26, wherein the subject has been diagnosed with or is suspected of suffering from uveitis, allergic conjunctivitis, blepharitis, chronic conjunctivitis, episcleritis, keratitis, retinitis, ocular cicatricial pemphigoid, mucous membrane pemphigoid, pterygium scleritis, Stevens-Johnson syndrome, Eales disease, Behcet's disease, sarcoidosis, systemic lupus erythematosus, polyarteritis nodosa, Wegener's granulomatosis, Vogt-Koyanagi-Harada disease, sympathetic ophthalmia, or sarcoidosis.

[0253] [Embodiment 28] 28. The method of embodiment 27, wherein the subject has been diagnosed with or is suspected of having uveitis.

[0254] [Embodiment 29] 29. The method of any one of embodiments 22 to 28, wherein the positive result for the expression or activity of at least one inflammasome activity marker in the inflamed subject is an increase in the expression or activity of at least one inflammasome activity marker compared to the expression or activity of at least one inflammasome activity marker in a subject who has not been diagnosed with or does not suffer from Alzheimer's disease, multiple sclerosis, myocardial infarction, atherosclerosis, microangiopathy, thyroiditis, inflammatory bowel disease, organ transplant rejection, membranous nephritis, sympathetic ophthalmia, uveitis, or sarcoidosis.

[0255] [Embodiment 30] The method of embodiment 29, wherein a positive result for the expression or activity of at least one inflammasome activity marker in the inflamed subject is an increase in the expression or activity of at least one inflammasome activity marker compared to the expression or activity of the inflammasome activity marker in a subject not diagnosed with uveitis.

[0256] [Embodiment 31] A method according to any one of embodiments 22 to 30, wherein a positive result for the expression or activity of at least one inflammasome activity marker in the inflamed subject is determined by a histological score.

[0257] [Embodiment 32] a. a nucleotide sequence encoding a membrane-independent CD59 protein operably linked to a promoter for expression and secretion of the membrane-independent CD59 protein in cells of an inflamed subject; or B soluble CD59 protein A method for inhibiting inflammasome activation in cells of a subject suffering from uveitis by administering to the subject a composition comprising:

[0258] [Embodiment 33] The method of embodiment 32, wherein the subject has a positive result for the expression or activity of at least one inflammasome activity marker in the inflamed subject's eye.

[0259] [Embodiment 34] 34. The method of embodiment 33, wherein the inflammasome activity marker is selected from caspase 1, caspase 5, IL-1β, IL-β17, IL-18, apoptosis-associated speck-like card protein (PYCARD / ASC), NACHT, LRR- and PYD domain-containing protein (NALP), IFN-γ, Th1 T cell markers or cytokines, Th17 T cell markers or cytokines, and CD4+.

[0260] [Embodiment 35] 34. The method of embodiment 33, wherein the inflammasome activity marker is selected from apoptosis-associated speck-like card protein (PYCARD / ASC), or NACHT, LRR and PYD domain-containing protein (NALP).

[0261] [Embodiment 36] The method of embodiment 35, wherein the NALP is NACHT, LRR and PYD domain-containing protein 3 (NLRP3).

[0262] [Embodiment 37] 37. The method of any of embodiments 33-36, wherein the subject has been diagnosed with or is suspected of suffering from uveitis, allergic conjunctivitis, blepharitis, chronic conjunctivitis, episcleritis, keratitis, retinitis, ocular cicatricial pemphigoid, mucous membrane pemphigoid, pterygium scleritis, Stevens-Johnson syndrome, Eales disease, Behcet's disease, sarcoidosis, systemic lupus erythematosus, polyarteritis nodosa, Wegener's granulomatosis, Vogt-Koyanagi-Harada disease, sympathetic ophthalmia, or sarcoidosis.

[0263] [Embodiment 38] 38. The method of embodiment 37, wherein the subject has been diagnosed with or is suspected of having uveitis.

[0264] [Embodiment 39] The method of any of embodiments 33 to 38, wherein the positive result for the expression or activity of at least one inflammasome activity marker in the inflamed subject is an increase in the expression or activity of at least one inflammasome activity marker compared to the expression or activity of at least one inflammasome activity marker in a subject who has not been diagnosed with or does not suffer from Alzheimer's disease, multiple sclerosis, myocardial infarction, atherosclerosis, microangiopathy, thyroiditis, inflammatory bowel disease, organ transplant rejection, membranous nephritis, sympathetic ophthalmia, uveitis, or sarcoidosis.

[0265] [Embodiment 40] The method described in embodiment 39, wherein a positive result for the expression or activity of at least one inflammasome activity marker in the inflamed subject is an increase in the expression or activity of at least one inflammasome activity marker compared to the expression or activity of the inflammasome activity marker in a subject not diagnosed with uveitis.

[0266] [Embodiment 41] A method according to any one of embodiments 33 to 40, wherein a positive result for the expression or activity of at least one inflammasome activity marker in the inflamed subject is determined by a histological score.

[0267] [Embodiment 42] A method for inhibiting inflammasome activation in cells of an inflamed subject, comprising administering to the subject a composition comprising a nucleotide sequence encoding a membrane-independent CD59 protein operably linked to a promoter for expression and secretion of the membrane-independent CD59 protein in cells of the inflamed subject, wherein the composition inhibits inflammasome activation, and the subject exhibits a positive result for the expression or activity of at least one inflammasome activity marker.

[0268] [Embodiment 43] 43. The method of embodiment 42, wherein the inflammasome activity marker is selected from caspase 1, caspase 5, IL-1β, IL-β17, IL-18, apoptosis-associated speck-like card protein (PYCARD / ASC), NACHT, LRR and PYD domain-containing protein (NALP), IFN-γ, Th1 T cell markers or cytokines, Th17 T cell markers or cytokines, and CD4+.

[0269] [Embodiment 44] 43. The method of embodiment 42, wherein the inflammasome activity marker is selected from apoptosis-associated speck-like card protein (PYCARD / ASC), or NACHT, LRR and PYD domain-containing protein (NALP).

[0270] [Embodiment 45] The method of embodiment 44, wherein the NALP is NACHT, LRR and PYD domain-containing protein 3 (NLRP3).

[0271] [Embodiment 46] A method described in any one of embodiments 42 to 45, wherein the subject has a positive result for the expression or activity of at least one inflammasome activity marker in the subject's eye.

[0272] [Embodiment 47] 47. The method of any of embodiments 42-46, further comprising measuring an inflammasome activity marker in the inflamed subject after administration of a composition comprising a nucleotide sequence encoding a membrane-independent CD59 protein operably linked to a promoter for expression and secretion of said membrane-independent CD59 protein.

[0273] [Embodiment 48] 48. The method of any of embodiments 42-47, wherein the subject has been diagnosed with or is suspected of suffering from uveitis, allergic conjunctivitis, blepharitis, chronic conjunctivitis, episcleritis, keratitis, retinitis, ocular cicatricial pemphigoid, mucous membrane pemphigoid, pterygium scleritis, Stevens-Johnson syndrome, Eales disease, Behcet's disease, sarcoidosis, systemic lupus erythematosus, polyarteritis nodosa, Wegener's granulomatosis, Vogt-Koyanagi-Harada disease, sympathetic ophthalmia, or sarcoidosis.

[0274] [Embodiment 49] The method of embodiment 48, wherein the subject has been diagnosed with or is suspected of having uveitis.

[0275] [Embodiment 50] 50. The method of any of embodiments 42-49, wherein the positive result for expression or activity of at least one inflammasome activity marker in the inflamed subject is increased expression or activity of at least one inflammasome activity marker compared to expression or activity of at least one inflammasome activity marker in a subject not diagnosed with or afflicted with uveitis, allergic conjunctivitis, blepharitis, chronic conjunctivitis, episcleritis, keratitis, retinitis, ocular cicatricial pemphigoid, mucous membrane pemphigoid, pterygium scleritis, Stevens-Johnson syndrome, Eales disease, Behcet's disease, sarcoidosis, systemic lupus erythematosus, polyarteritis nodosa, Wegener's granulomatosis, Vogt-Koyanagi-Harada disease, sympathetic ophthalmia, or sarcoidosis.

[0276] [Embodiment 51] The method of embodiment 50, wherein a positive result for the expression or activity of at least one inflammasome activity marker in inflamed cells of the subject is an increase in the expression or activity of at least one inflammasome activity marker compared to the expression or activity of at least one inflammasome activity marker in a subject not diagnosed with or not suffering from uveitis.

[0277] [Embodiment 52] A method according to any one of embodiments 42 to 51, wherein a positive result for the expression or activity of at least one inflammasome activity marker in the inflamed subject is determined by a histological score.

Claims

1. A gene therapy composition for treating uveitis in a subject suffering from autoimmune uveitis, comprising a viral expression vector encoding a membrane-independent CD59 protein operably linked to a promoter for expression and secretion of the membrane-independent CD59 protein in cells of the inflamed eye, wherein the composition inhibits inflammasome activation, and the subject has shown a positive result for expression or activity of at least one inflammasome activity marker in the inflamed eye.

2. The composition described in claim 1, wherein an inflammasome activity marker is measured after administration of the composition.

3. A gene therapy composition for treating uveitis in an inflamed subject suffering from autoimmune uveitis, comprising: a viral expression vector encoding a membrane-independent CD59 protein operably linked to a promoter for expression and secretion of the membrane-independent CD59 protein in cells of an inflamed subject, the composition inhibiting inflammasome activation; A composition in which an inflammasome activity marker is measured in the subject and administered to the subject if the inflammasome activity marker is positive.

4. The composition of claim 3, wherein the inflammasome activity marker is measured in the subject's eye.

5. The composition of claim 3 or 4, wherein an inflammasome activity marker is measured in the subject after administration of the composition.

6. A gene therapy composition for treating uveitis in an inflamed subject suffering from autoimmune uveitis, comprising: a viral expression vector encoding a membrane-independent CD59 protein operably linked to a promoter for expression and secretion of the membrane-independent CD59 protein in cells of an inflamed subject, the composition inhibiting inflammasome activation; The composition, wherein the subject is a subject who has shown a positive result for expression or activity of at least one inflammasome activity marker in an inflamed subject.

7. The composition of claim 6, wherein the positive result for expression or activity of at least one inflammasome activity marker is in an inflamed eye of a subject.

8. A gene therapy composition for treating uveitis in an inflamed subject suffering from autoimmune uveitis, comprising: A composition comprising a viral expression vector encoding a membrane-independent CD59 protein operably linked to a promoter for expression and secretion of the membrane-independent CD59 protein in cells of an inflamed subject, which inhibits inflammasome activation.

9. The composition of claim 8, wherein the subject has a positive result for expression or activity of at least one inflammasome activity marker in the inflamed subject's eye.

10. The composition of any one of claims 3 to 7 and 9, wherein the positive result for the expression or activity of at least one inflammasome activity marker, or the positive inflammasome activity marker is an increase in the expression or activity of at least one inflammasome activity marker compared to the expression or activity of at least one inflammasome activity marker in a subject not diagnosed with or not suffering from autoimmune uveitis.

11. A gene therapy composition for treating uveitis in an inflamed subject suffering from autoimmune uveitis, comprising a viral expression vector encoding a membrane-independent CD59 protein operably linked to a promoter for expression and secretion of the membrane-independent CD59 protein in cells of the inflamed subject, wherein the composition inhibits inflammasome activation, and the subject has a positive result for expression or activity of at least one inflammasome activity marker.

12. The composition of claim 11, wherein the subject has a positive result for expression or activity of at least one inflammasome activity marker in the subject's eye.

13. The composition described in claim 11 or 12, wherein an inflammasome activity marker is measured in the subject after administration of the composition.

14. The composition of any one of claims 1, 2 and 11 to 13, wherein the positive result for the expression or activity of at least one inflammasome activity marker is an increase in the expression or activity of at least one inflammasome activity marker compared to the expression or activity of at least one inflammasome activity marker in a subject not diagnosed with or not suffering from autoimmune uveitis.

15. The composition of any one of claims 1 to 7 and 9 to 14, wherein a positive result for the expression or activity of at least one inflammasome activity marker, or the positive inflammasome activity marker, is determined by a histological score.

16. The composition of any one of claims 1 to 7 and 9 to 15, wherein the inflammasome activity marker is selected from caspase 1, caspase 5, IL-1β, IL-β17, IL-18, apoptosis-associated speck-like card protein (PYCARD / ASC), NACHT, LRR and PYD domain-containing protein (NALP), IFN-γ, a Th1 T cell marker or cytokine, a Th17 T cell marker or cytokine, and CD4+.

17. The composition of claim 16, wherein the NALP is NACHT, LRR and PYD domain-containing protein 3 (NLRP3).

18. 18. The composition of any one of claims 1 to 17, wherein the subject has been diagnosed with or is suspected of suffering from uveitis, allergic conjunctivitis, blepharitis, chronic conjunctivitis, episcleritis, keratitis, retinitis, ocular cicatricial pemphigoid, mucous membrane pemphigoid, pterygium scleritis, Stevens-Johnson syndrome, Eales disease, Behcet's disease, sarcoidosis, systemic lupus erythematosus, polyarteritis nodosa, Wegener's granulomatosis, Vogt-Koyanagi-Harada disease, sympathetic ophthalmia, or sarcoidosis.

19. The composition described in any one of claims 1 to 18, wherein the viral expression vector is at least one selected from the group consisting of adenovirus, lentivirus, adeno-associated virus (AAV), helper-dependent adenovirus, and herpes virus.

20. The composition of any one of claims 1 to 19, wherein the viral expression vector is AAV serotype 2 (AAV2).

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