CD59 for inhibiting 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 of U.S. Provisional Patent Application No. 62 / 629,435, filed on February 12, 2018; and U.S. Provisional Patent Application No. 62 / 637,460, filed on March 2, 2018. Government Support

[0002] This invention was made with government support under grant number EY021805 awarded by the National Institutes of Health. The U.S. government has certain rights in this invention. BACKGROUND OF THE INVENTION

[0003] Complement is an important element of the innate immune system, and its role has been described, among other things, in various inflammatory diseases such as autoimmune diseases, cancer, and ischemia / reperfusion injury (Non - Patent Document 1). The role of complement has also been previously described in an experimental model of autoimmune uveitis (EAU) (Non - Patent Documents 2 - 5). Little is known about the mechanism linking complement activation and T - cell - mediated pathology in EAU. Complement activation culminates in the assembly of the membrane attack complex (MAC) at the cell membrane, resulting in pore formation. At sublytic levels, pore formation promotes ion exchange and cytokine release through the plasma membrane. As a result, MAC deposition leads to cell lysis. The role of MAC in the pathophysiology of EAU remains unclear. The entry of Ca 2+ into cells after MAC deposition is known to activate the NLRP3 inflammasome in primary human lung epithelial cells (Non - Patent Document 6).

[0004] The NLRP3 inflammasome is said to be involved in various inflammatory diseases such as age-related macular degeneration, cardiomyopathy, arthritis, chronic kidney disease, and neurodegenerative diseases (Non-Patent Document 7). Administration of lipopolysaccharide (LPS) in mice causes MAC-related IL-1β maturation (Non-Patent Document 8). The inflammasome is highly regulated by complement and is necessary for tissue damage and disease recovery, but a disordered inflammasome can lead to severe inflammation and damage to host tissues (Non-Patent Document 9). Activation of the NLRP3 inflammasome is controlled by a two-step process, which requires an initial priming signal accompanied by an increase in the expression of the protein NLRP3 and a subsequent activation signal necessary for the formation of an inflammasome protein complex that results in the cleavage of pro-IL-1β to mature IL-1β via caspase-1 (Non-Patent Document 10). Disordered IL-1β expression can lead to the onset of autoimmune and autoinflammatory diseases such as Behçet's disease, Vogt-Koyanagi-Harada disease, rheumatic diseases, autoimmune thyroid diseases, insulin-dependent diabetes, gout, familial Mediterranean fever, and cryopyrin-associated periodic syndrome (Non-Patent Documents 11 to 13). Furthermore, it has been established that IL-1β is actively secreted by myeloid cells in the retina, indicating a potential etiological role of IL-1R signaling in EAU (Non-Patent Document 14). However, detailed knowledge of the mechanism by which IL-1β is regulated in EAU remains unknown.

[0005] Uveitis is a chronic ocular inflammatory disease of the uveal and retinal layers of the eye and is responsible for approximately 10% to approximately 15% of total blindness in the United States (Non-Patent Document 15). Uveitis is classified as infectious when external biological factors induce an immune response and non-infectious when an autoimmune reaction is induced. Clinical treatment of uveitis has been carried out using corticosteroids, immunosuppressive drugs, or monoclonal antibodies, but these approaches have achieved only limited success and are accompanied by serious side effects (Non-Patent Documents 16 and 17). Therefore, there is an unmet clinical need to develop an effective treatment for uveitis.

Prior Art Documents

Non-Patent Documents

[0006]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Non-Patent Document 6

Non-Patent Document 7

Non-Patent Document 8

Non-Patent Document 9

Non-Patent Document 10

Non-Patent Document 11

Non-Patent Document 12

Non-Patent Document 13

Non-Patent Document 14

Non-Patent Document 15

Non-Patent Document 16

Non-Patent Document 17

Summary of the Invention

Means for Solving the Problems

[0007] Described herein are methods, compositions, and kits for treating individuals suffering from inflammasome-related disorders using membrane-independent (soluble) CD59 protein. The membrane-independent CD59 protein can be expressed from administered nucleic acid or can be from direct administration of the protein. Nucleic acids or compositions thereof encoding membrane-independent CD59 protein for use in treating inflammasome-related disorders are contemplated. Membrane-independent CD59 protein or compositions thereof for use in treating inflammasome-related disorders are also contemplated. In certain embodiments, the inflammasome-related disorder is in the eye of the subject. In certain embodiments, inflammasome-related 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, sarcoidosis. In certain embodiments, the inflammasome-related disorder includes uveitis. The protein or nucleic acid encoding soluble / membrane-independent CD59 can be administered to a subject that shows a positive result for the expression or activity of at least one inflammasome activity marker. In certain embodiments, the positive result is in the eye of the subject. 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, Th1 T cell marker or cytokine, Th17 T cell marker or cytokine, and CD4+.In certain embodiments, the inflammasome activation marker is selected from apoptosis-associated speck-like protein containing a CARD (PYCARD / ASC), or a nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain-containing protein (NALP). In certain embodiments, the NALP is nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain-containing protein 3 (NLRP3).

[0008] One aspect of the invention herein is a method of inhibiting the activation of an inflammasome in cells of an eye that has developed inflammation, the method comprising administering to a 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 cells of the inflamed eye, wherein the composition inhibits the activation of the inflammasome. 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 the cell membrane or cell membrane-associated structures (such as membrane-bound proteins). Embodiments of this method provide that membrane-independent CD59 inhibits the activation of the inflammasome independent of the function of the membrane attack complex (MAC).

[0009] In embodiments of the method, the composition inhibits the activation of the inflammasome independent of the function of the membrane attack complex (MAC). In embodiments of the method, 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, pterygoid episcleritis, Stevens-Johnson syndrome, Eales' disease, Behcet's disease, sarcoidosis, systemic lupus erythematosus, polyarteritis nodosa, Wegener's granulomatosis, Vogt-Koyanagi-Harada disease, psoriasis, immune 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 embodiments of the method, the uveitis is at least one selected from anterior uveitis, intermediate uveitis, posterior uveitis, and panuveitis. Embodiments of the method further include obtaining a sample from a subject. In embodiments of the method, the sample is at least one selected from tears, blood, urine, eye discharge, sputum, and mucus.

[0011] Embodiments of the method further include measuring in the eye at least one of retinal function and inflammasome activity markers before administration. Embodiments of the method further include measuring in the eye at least one of retinal function and inflammasome activity markers after administration.

[0012] In embodiments 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 protein containing a CARD (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 embodiments of the method, the inflammasome activity marker is selected from apoptosis-associated speck-like protein containing a CARD (PYCARD / ASC), or NACHT, LRR and PYD domain-containing protein (NALP). In embodiments of the method, NALP is NACHT, LRR and PYD domain-containing protein 3 (NLRP3).

[0013] In embodiments of the method, measuring the retinal function or inflammasome activity marker further includes performing at least one technique selected from eye 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 method embodiments, the viral vector is a genetically engineered genome of at least one virus selected from the group consisting of adenovirus, adeno-associated virus, herpes virus, and lentivirus. In method embodiments, the lentivirus is a retrovirus.

[0016] Method embodiments further include 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. Method embodiments of this further include manipulating the nucleotide sequence encoding the membrane-independent CD59 protein by deleting nucleotides encoding the region of the glycosylphosphatidylinositol (GPI) anchor domain.

[0017] In method embodiments, administration further includes injecting the composition into the eye. In method embodiments of this, administration further includes topically applying the composition. In one embodiment of this method, the eye injection is selected from the group consisting of subretinal injection, intravitreal injection, intraocular injection, subconjunctival injection, and sub-Tenon's injection. In method embodiments, the eye injection further includes administering to the outer layer of the eye.

[0018] Method embodiments further include administering an additional therapeutic agent to the eye. In some embodiments of the method, the additional therapeutic agent is at least one selected from the group consisting of an anti-tumor agent, an antiviral agent, an antibacterial agent, an anti-mycobacterial agent, an antifungal agent, an anti-proliferative agent, and an anti-apoptosis agent. In some embodiments of the method, the additional therapeutic agent is a growth factor, an anti-inflammatory agent, a hypertensive agent, 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, heparan sulfate, and hyaluronic acid, selected from the group consisting of.

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

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

[0021] One aspect of the invention herein provides a method of inhibiting the activation of the inflammasome in cells of an eye that has developed inflammation, the method comprising administering to a 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 cells of the inflamed eye, the composition inhibiting the activation of the inflammasome, and the subject showing a positive result for the expression or activity of at least one inflammasome activation marker in cells of the eye of the subject that has developed inflammation. In certain embodiments of this method, the inflammasome activation 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 certain embodiments of this method, the inflammasome activation marker is selected from apoptosis-associated speck-like card protein (PYCARD / ASC), or NACHT, LRR and PYD domain-containing protein (NALP). In certain embodiments of this method, the NALP is NACHT, LRR and PYD domain-containing protein 3 (NLRP3). In certain embodiments of this method, the method further comprises measuring the inflammasome activation marker in the eye after administration of the 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 certain embodiments of this method, the subject is diagnosed with or suspected of having uveitis, allergic conjunctivitis, blepharitis, chronic conjunctivitis, episcleritis, keratitis, retinitis, ocular cicatricial pemphigoid, mucous membrane pemphigoid, pterygoid episcleritis, Stevens-Johnson syndrome, Eales' disease, Behçet's disease, sarcoidosis, polyarteritis nodosa, Wegener's granulomatosis, Vogt-Koyanagi-Harada disease, sympathetic ophthalmia, or sarcoidosis. In certain embodiments, the subject is diagnosed with or suspected of having uveitis.Positive results regarding the expression or activity of at least one inflammasome activity marker in the eye of a subject with inflammation described herein indicate that the expression or activity of at least one inflammasome activity marker is elevated compared to the expression or activity of at least one inflammasome activity marker in the eyes of individuals not diagnosed with or suffering from uveitis, allergic conjunctivitis, blepharitis, chronic conjunctivitis, episcleritis, keratitis, retinitis, ocular cicatricial pemphigoid, mucous membrane pemphigoid, pterygoid episcleritis, 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 eye of a subject with inflammation is determined by histological score.

[0022] The specific embodiments described herein are methods for inhibiting the activation of the inflammasome in cells of a subject with inflammation, 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 the expression and secretion of the membrane-independent CD59 protein in cells of the subject with inflammation; or (b) administering to the subject a composition comprising a soluble CD59 protein, wherein the composition inhibits the activation of the inflammasome. In certain embodiments, the composition inhibits the activation of the inflammasome independent of the function of the membrane attack complex (MAC). In certain embodiments, the subject has at least one inflammasome-related condition selected from Alzheimer's disease, multiple sclerosis, myocardial infarction, atherosclerosis, microvascular disorders, thyroiditis, inflammatory bowel disease, organ transplant rejection, membranous nephritis, sympathetic ophthalmia, and sarcoidosis. In certain embodiments, the method further comprises obtaining a sample from the subject. In certain embodiments, the sample is at least one selected from blood, plasma, serum, peripheral blood mononuclear cells, cerebrospinal fluid, or urine. In certain embodiments, the method further comprises measuring an inflammasome activity marker in the subject prior to administration. In certain embodiments, the method further comprises measuring an inflammasome activity marker in the subject after administration. In certain embodiments, the inflammasome activity marker is at least one selected from caspase 1, caspase 5, IL-1β, IL-β17, IL-18, apoptosis-related speck-like protein containing a CARD (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 administering the composition at a dosage sufficient to treat the inflammasome-related condition.In certain embodiments, the nucleotide sequence encoding a membrane-independent CD59 protein operably linked to a promoter for the 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 certain embodiments, the lentivirus is a retrovirus. In certain embodiments, administration further comprises intravenous injection of the composition. In certain embodiments, the method further comprises topical application of the composition. In certain embodiments, the method further comprises administering an additional therapeutic agent. In certain embodiments, the additional therapeutic agent is at least one selected from the group consisting of an anti-tumor agent, an anti-viral agent, an antibacterial agent, an anti-mycobacterial agent, an anti-fungal agent, an anti-proliferative agent, and an anti-apoptotic agent. In certain embodiments, 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, heparan sulfate, and hyaluronic acid. In another aspect, described herein is a kit for inhibiting the activation of the inflammasome in cells that have caused inflammation in a subject, the kit comprising: (a) a pharmaceutical composition comprising (i) a membrane-independent CD59 protein and / or a nucleotide sequence encoding a CD59 protein; or (ii) a soluble CD59 protein; the pharmaceutical composition being in a dosage sufficient to inhibit the inflammasome in cells that have caused inflammation in the subject; (b) instructions for use; and (c) a container.

[0023] Another aspect described herein is a method of treating at least one condition selected from Alzheimer's disease, multiple sclerosis, myocardial infarction, atherosclerosis, microvascular disorders, thyroiditis, inflammatory bowel disease, organ transplant rejection, membranous nephritis, sympathetic ophthalmia, and sarcoidosis, the method comprising administering to a subject a composition comprising (a) (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; or (ii) a soluble CD59 protein; wherein the composition inhibits activation of the inflammasome. Another aspect described herein is a method for inhibiting activation of the inflammasome in cells of a subject having inflammation, the method comprising (a) measuring an inflammasome activity marker in the subject; and (b) if the inflammasome activity marker 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 subject having inflammation; or (ii) a soluble CD59 protein; wherein the composition inhibits activation of the inflammasome. 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 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 an inflammasome activity marker in the 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 the membrane-independent CD59 protein or a soluble CD59 protein.

[0024] Another aspect described herein is a method of inhibiting activation of the inflammasome in cells of a subject with inflammation, the method comprising administering to the 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 a subject with inflammation; or (b) a soluble CD59 protein; wherein the composition inhibits activation of the inflammasome, and the subject shows a positive result for the expression or activity of at least one inflammasome activation marker in the subject with inflammation. In certain embodiments, the inflammasome activation 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 domain-containing protein (NALP), IFN-γ, Th1 T cell marker or cytokine, Th17 T cell marker or cytokine, and CD4+. In certain embodiments, the inflammasome activation marker is selected from apoptosis-associated speck-like protein containing a CARD (PYCARD / ASC), or NACHT, LRR and PYD domain-containing protein (NALP). In certain embodiments, the NALP is NACHT, LRR and PYD domain-containing protein 3 (NLRP3). In certain embodiments, the method further comprises measuring an inflammasome activation marker in the subject after administration of the 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; or (b) a soluble CD59 protein. In certain embodiments, 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, pterygoid episcleritis, Stevens-Johnson syndrome, Eales' disease, Behçet's disease, sarcoidosis, systemic lupus erythematosus, polyarteritis nodosa, Wegener's granulomatosis, Vogt-Koyanagi-Harada disease, sympathetic ophthalmia, or sarcoidosis.In certain embodiments, the subject is diagnosed with or suspected of having uveitis. In certain embodiments, a positive result for the expression or activity of at least one inflammasome activation marker in an inflamed subject is an increase in the expression or activity of at least one inflammasome activation marker as compared to the expression or activity of at least one inflammasome activation marker in a subject not diagnosed with or having Alzheimer's disease, multiple sclerosis, myocardial infarction, atherosclerosis, microvascular disorders, thyroiditis, inflammatory bowel disease, organ transplant rejection, membranous nephritis, sympathetic ophthalmia, and sarcoidosis. In certain embodiments, a positive result for the expression or activity of at least one inflammasome activation marker in an inflamed subject is determined by histological score.

Brief Description of the Drawings

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Modes for Carrying Out the Invention

[0026] Described herein are methods, compositions, and kits for treating individuals suffering from inflammasome-related disorders using a membrane-independent (soluble) CD59 protein. The membrane-independent CD59 protein can be expressed from an administered nucleic acid or can be from direct administration of the protein. Nucleic acids or compositions thereof encoding a membrane-independent CD59 protein for use in treating inflammasome-related disorders are contemplated. Membrane-independent CD59 proteins or compositions thereof for use in treating inflammasome-related disorders are also contemplated. In certain embodiments, the inflammasome-related disorder is in the eye of the subject. In certain embodiments, inflammasome-related disorders include uveitis, allergic conjunctivitis, blepharitis, chronic conjunctivitis, episcleritis, keratitis, retinitis, ocular cicatricial pemphigoid, mucous membrane pemphigoid, pterygoid episcleritis, 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 certain embodiments, the inflammasome-related disorder includes uveitis. The protein or nucleic acid encoding soluble / membrane-independent CD59 can be administered to a subject that shows a positive result for the expression or activity of at least one inflammasome activity marker. In certain embodiments, the positive result is in the eye of the subject. 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 domain-containing protein (NALP), IFN-γ, Th1 T cell marker or cytokine, Th17 T cell marker or cytokine, and CD4+. In certain embodiments, the inflammasome activity marker is selected from apoptosis-associated speck-like protein containing a CARD (PYCARD / ASC), or NACHT, LRR and PYD domain-containing protein (NALP). In certain embodiments, NALP is NACHT, LRR and PYD domain-containing protein 3 (NLRP3).

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

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

[0029] MAC contains one molecule each of C5b, C6, C7, C8 and up to 12 molecules of C9. C9 - / - Since mice are unable to assemble a functional MAC, such mice may be partially protected from EAU. In the examples herein, control C57BL / 6J mice and C9 - / - mice were induced to develop EAU, and the course of EAU in the control mice was compared with that in C9 - / -The disease state of EAU was compared in mice. The therapeutic potential of inhibiting C9 in EAU was investigated by a gene therapy approach using an adeno-associated virus (AAV) expressing soluble CD59 (AAVCAGsCD59), a protein that prevents the incorporation of C9 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 the activation of the NLRP3 inflammasome was measured by Western blot, enzyme-linked immunosorbent assay (ELISA), and real-time polymerase chain reaction (RT-PCR). The results observed in the examples herein are the first to associate a direct role of MAC as an activator of the NLRP3 inflammasome and IL-1β production in the development of EAU. The data of the examples herein are the first to demonstrate that AAV-mediated expression of soluble CD59 is a potential gene therapy for the treatment of uveitis.

[0030] In the examples herein, the direct role of MAC in the activation of the NLRP3 inflammasome in EAU was investigated. The data from the examples herein demonstrate that MAC deposits in the EAU retina and that it results in the activation of the NLRP3 inflammasome and increased production of IL-1β. These examples show C9 - / - indicating that EAU mice were unable to form MAC on the retina, and concomitantly, the activation of the NLRP3 inflammasome occurred, which indicates the association between MAC deposition and NLRP3 inflammasome activation in EAU. C9 - / - Mice cannot rescue the pathological phenotype of EAU, but AAV-mediated delivery of sCD59, an inhibitor of C9 incorporation into MAC, unexpectedly reduces many aspects of the pathological phenotype of EAU, including the activation of the NLRP3 inflammasome.

[0031] The complement system is a major component of innate immunity and consists of a diverse group of plasma and membrane-bound proteins. These membrane-bound proteins play a central role in defense against pathogens and in the regulation of immune and inflammatory processes. Activation of complement against pathogens is required for host defense, but over-activated complement can damage host tissues (Morgan, B. P., et al. (2015) Nat. Rev. Drug Discov. 14, 857-877; Carroll, M. V., et al. (2011) Adv. Drug Deliv. Rev. 63, 965-975). Therefore, it is necessary to maintain a balance between complement activation and complement inhibition by complement regulatory proteins. An over-activated and disordered complement system culminates in the formation of MAC, and the formation of MAC has been shown to be involved in various eye diseases including EAU (An, F., et al. (2009) Invest. Ophthalmol. Vis. Sci. 50, 3778-3782; Copland, D. A., et al. (2010) Clin. Exp. Immunol. 159, 303-314; Read, R. W., et al. (2006) Exp. Eye Res. 82, 389-394; Gehrs, K. M., 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 etiology of EAU has remained uninvestigated. In the examples herein, it was observed that MAC was deposited in EAU and that it led to an increase in the production of IL-1β. However, C9, which lacks the ability to form MAC - / - EAU mice were observed to have reduced IL-1β levels as a result.

[0032] MAC deposition on the cell membrane ultimately leads to cell death by lysis, while sublytic MAC on the cell membrane is involved in the regulation of the cell cycle and proliferation, apoptosis, cytokine production, and the initiation of downstream signaling cascades (Morgan, B. P., et al. (2015) Nat. Rev. Drug Discov. 14, 857-877). The NLRP3 inflammasome complex is a group of cytoplasmic proteins consisting of the major regulatory subunit NLRP3, the adapter subunit ASC, and the effector subunit caspase-1 that 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 a model primed with LPS have shown that sublytic MAC-induced pore formation is intracellular Ca 2+resulted in its accumulation, which subsequently activated the NLRP3 inflammasome, as shown (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 associated with several eye diseases (Devi, T. S., et al. (2012) Exp. Diab. Res. 2012, 438238; Tseng, W. A., et al. (2013) Invest Ophthalmol Vis Sci 54, 110-120). The results in the examples herein demonstrated that attenuation of NLRP3 inflammasome activation is a new therapeutic approach for the treatment of uveitis. The examples herein are the first to demonstrate the direct role of MAC and NLRP3 inflammasome activation in the EAU mouse model. The data obtained in the examples herein demonstrate that MAC directly activated the NLRP3, caspase-1, and ASC subunits and increased their protein expression. These activated subunits form the NLRP3 inflammasome complex, which generates 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] Auto-reactive effector CD4+ T cells have been associated with the etiology 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, R. R., 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 able to treat EAU in mice (Wan, C. K., 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, are approved for the treatment of uveitis (Knickelbein, J. E., et al. (2017) Handb. Exp. Pharmacol. 242, 231-268). However, their use is limited due to various side effects and short duration of action. In the examples herein, an increase in IL-1β production was observed in the retinas of mice with EAU; however, in the retinas of C9 - / - mice with EAU, lower IL-1β levels were observed. Furthermore, enhanced differentiation of Th1 and Th17 cells was observed in the retinas of mice with EAU; however, in the retinas of C9 - / - mice with EAU, a decrease in Th1 and Th17 cells was observed, as shown 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 - / -In DLN from EAU mice, it was observed that the levels of Th1 and Th17 positive CD4 cells remained unchanged. Without being limited to a particular theory or mechanism of action, it is predicted that MAC-induced IL-1β production and the 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). A decrease or deficiency in the activity of these complement regulatory proteins can cause immunopathologies including EAU and experimental autoimmune anterior uveitis (Morgan, B. P., et al. (2015) Nat. Rev. Drug Discov. 14, 857-877; An, F., et al. (2009) Invest. Ophthalmol. Vis. Sci. 50, 3778-3782; Carroll, M. V., 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 diseases. Various studies have shown that inhibition of complement activation may help improve the EAU disease state in mice (An, F., et al. (2009) Invest. Ophthalmol. Vis. Sci. 50, 3778-3782; Copland, D. A., et al. (2010) Clin. Exp. Immunol. 159, 303-314; Read, R. W., et al. (2006) Exp. Eye Res. 82, 389-394). Embodiments of the methods described herein demonstrate a long-acting gene therapy approach that utilizes AAV to deliver sCD59 to EAU mice. It was first demonstrated that AAVCAGsCD59 inhibits MAC deposition in the EAU retina. The examples herein demonstrate that AAVCAGsCD59 inhibits the activation of the NLRP3 inflammasome and attenuates IL-1β production.

[0035] Furthermore, the examples in this specification show that single intravitreal injection of AAVCAGsCD59 inhibited the phenotypic symptoms in EAU mice. In these mice, clinical symptoms associated with EAU, such as reduction of inflammation, fewer immune cell infiltrates, and reduction of vasculitis, were significantly improved. The data obtained in the examples of this specification demonstrate that injection of AAVCAGsCD59 results in improvement of the loss of retinal function associated with EAU in both dark-adapted and light-adapted ERGs. In the examples of this specification, C9 - / - mice were observed to significantly inhibit the activation of the NLRP3 inflammasome and further observed to reduce the upregulation of IL-1β. A tendency for improvement in histological scores and improvement in retinal function was observed in C9 - / - EAU mice, but except for some data points of ERG, the differences were observed not to 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 been previously reported to bind to CD2 and transmit activation signals within T cells (Deckert, M. et al. (1995) Eur J Immunol. 25, 1815-22). CD59 crosslinking induces the T cell receptor zeta (ζ) / ZAP-70 signaling cascade and interleukin-2 (IL-2) synthesis. IL-2 has been found to properly 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), which supports the hypothesis that sCD59 can attenuate inflammation in a MAC-independent manner. The EAU model used in this specification to analyze uveitis has contradictions associated with the development of mild to moderate EAU. These minor changes may 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 the management of severe and advanced uveitis in patients with systemic autoimmune diseases (such as Behçet's disease, Vogt-Koyanagi disease, etc.) due to significant side effects and short-term efficacy. Treatments for managing uveitis have not advanced significantly over the past few decades (Knickelbein, J. E., et al. (2017) Handb. Exp. Pharmacol. 242, 231-268). Considering these concerns, it is practical to develop long-acting therapies such as continuous inhibition of the MAC-dependent activation of the inflammasome in uveitis patients. Therefore, single intravitreal injection of AAVsCD59 has great potential and advantages compared to the short-term effects of current treatments. AAV-dependent gene therapy has been shown to be successfully applied to the treatment of eye diseases in animal models (Adhi, M., et al. (2013) PLoS One 8, e79661; Ildefonso, C. J., et al. (2015) Hum. Gene Ther. 26, 59-68). Furthermore, AAV-mediated gene therapy is progressing well in human clinical trials for age-related macular degeneration (Mingozzi, F., et al. (2011) Nat. Rev.

[0038] Examples in this specification demonstrated that MAC is an important regulator of the activation of the NLRP3 inflammasome and IL-1β production in EAU. MAC plays an important role in the differentiation of Th1 and Th17 cells by increasing IL-1β production. It was observed that AAV-mediated expression of sCD59 effectively inhibits MAC deposition and subsequently inhibits the activation of the NLRP3 inflammasome. 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 associated with the membranes of cells such as human erythrocytes, lymphocytes, and vascular endothelial cells. The CD59 protein inhibits the assembly of functional MAC and thus protects cells from complement-mediated activation and / or lysis.

[0040] Without being limited to a particular theory or mechanism of action, the plasma membrane of cells is generally thought to be protected from the effects of complement by cell surface proteins such as CD59 that specifically inhibit the activation of C5b-9 pores by the binding of C9 complement protein to membrane-bound 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 C9 complement protein for binding to 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 effects membrane anchoring. Amino acid sequences of examples of precursor human CD59, mature human CD59, and CD59 sequences of other mammals (e.g., baboon, African green monkey, cynomolgus monkey, marmoset, HVS-15, pig, rabbit, rat, and mouse) are shown 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 (U.S. Patent No. 5,624,837, issued April 29, 1997 to Fodor et al., incorporated by reference). This gene is located on the short arm of human chromosome 11, specifically at chromosome 11p13 and 11p14 (accession number 107271 in Online Mendelian Inheritance in Man) and is composed of four exons spanning 20 kb (Petranka et al. 1992 Proc. Nat. Acad. Sci. 89: 7876-7879). Before the non-translated first exon, there is a G- and C-rich promoter region lacking a consensus TATA or CAAT motif. 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 remaining portion of the mature protein, including a hydrophobic sequence for attachment of the glycosylphosphatidylinositol (GPI) anchor 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 Schwann cells), astrocytes, epithelial cells, acinar cells of the salivary gland, 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. The cDNA encoding CD59 has been reported in Sawada, R. et al. 1989 Nucleic Acids Res 17(16): 6728. The cDNA encoding CD59 has also been cloned from human T cell leukemia (YT) and human erythroleukemia (K562) cell lines, and CD59 is transiently expressed in COS cells (Walsh, L.A. 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 the attachment of a glycosylphosphatidylinositol anchor (GPI anchor) at amino acid asparagine at position 77. The cDNA sequence of CD59 is shown in U.S. Patent No. 5,624,837 to Fodor et al., issued on April 29, 1997, which is incorporated herein by reference in its entirety.

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

[0046] Active surface-exposed amino acid residue side chains available for binding to C8 / C9, identified from the solution structure of mature human CD59 from published NMR data and knowledge of the active portion of the CD59 molecule, are 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 structure of CD59 is described in deposits by Kieffer et al., Human Complement Regulatory Protein CD59 (Extracellular Region, Residues 1 - 70; NMR, 10 Structures), MMDB Id:891, PDB Id:1ERH; Kieffer et al., Human Complement Regulatory Protein CD59 (Extracellular Region, Residues 1 - 70; NMR, Constrained), MMDB Id:890, PDB Id:1ERG; deposits by Fletcher et al., CD59 Complexed with Glcnac-Beta-1,4-(Fuc-Alpha-1,6)-Glcnac-Beta-1 (NMR, 10 Structures), MMDB Id:498, PDB Id:1CDS; deposits by Fletcher et al., CD59 Complexed with Glcnac-Beta-1,4-Glcnac-Beta-1 (NMR, 10 Structures), MMDB Id:497, PDB Id:1CDR. Deposits 1CDS and 1CDR by Fletcher et al. Amino acid sequences of CD59 presenting these side chains at 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 of this specification.

[0047] The relationship between complement activation and abnormal levels of autoantibodies has been analyzed with respect to eye diseases such as macular degeneration and other conditions. U.S. Patent Application Publication No. 2005 / 0287601, published on 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 the causative role of complement in Alzheimer's disease and age-related macular degeneration to prevention of complement system activation and MAC formation. U.S. Patent Application Publication No. 2007 / 0196367 by Dinu, published on August 23, 2007 (incorporated by reference), proposes preventing the formation of cellular debris by inhibiting complement as a therapeutic for Alzheimer's disease and AMD. U.S. Patent Application Publication No. 2007 / 0203190 by Patil et al., published on 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 on December 1, 2005 by Tomlinson et al. (incorporated by reference) uses an agent created by linking each of the complement inhibitors Crry and CD59 to the amino terminus of a single-chain antibody (scFv) that binds to rat glomerular epithelial cells and proximal tubular epithelial cells to inhibit complement-directed proteinuria in rats. In this reference, soluble CD59 is described as ineffective as an inhibitor. Bora et al. (Bora et al. 2007 J. Immunol 178: 1783-1790) used recombinant methods to create a membrane-targeting composition by fusing the binding arm Fc of immunoglobulin G (IgG1) to the CD59 protein (rsCD59a-Fc), and injected this fusion into mice via intravenous, intraocular (intravitreal), and intraperitoneal routes. The number of CNV positive spots decreased in subjects treated with the fusion protein by the intraperitoneal route compared to the intravitreal and intravenous routes. The functionality of Fc can result in immediate binding when the fusion protein contacts cells generally and non-specifically after administration. The 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) intravenously injected 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 into animals with CNV spots, reducing the size of the CNV spots in a mouse model.

[0050] The CD59 compositions provided herein lack the major amino acid sequence of the functional GPI anchor. Functionally equivalent proteins include the amino acid sequence of a modified GPI anchor domain that is functionally defective and lacks the ability to target the membrane. 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, e.g., synthesizing CD59 in vivo or in vitro such that the GPI anchor is absent. Methods for obtaining membrane-independent CD59 are shown in the examples herein. Further 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 proteins described herein are soluble CD59 proteins. The composition provides a CD59 protein and includes a full-length nucleic acid of CD59 modified to remove the signal sequence for attachment of the GPI anchor with a nucleotide encoding the amino acid asparagine at position 77. Alternatively, the nucleic acid sequence of CD59 is modified by point mutation, substitution, or deletion so that the protein cannot attach to the cell membrane, resulting in a nucleic acid sequence encoding an amino acid sequence having a modified amino acid sequence at the GPI anchor position.

[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 a cell membrane-related structure such as the cell membrane or a membrane-bound protein. The GPI anchor can be modified by recombinant DNA technology 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 multi-step pathway in the endoplasmic reticulum that includes the interaction of numerous gene products. Many proteins, including CD59, are expressed on the cell surface and require GPI for their effective function. The mechanism by which the structure of the protein signal encodes the attachment of the GPI anchor is outlined in Orlean et al. 2007 JLR 48: 993-1011. Attachment of GPI requires an amino acid sequence that includes a hydrophobic N-terminal secretory signal that targets the protein to the ER and a C-terminal GPI signal anchor sequence. In addition to the native CD59 secretory signal, which is located at the amino terminus of the protein and is cleaved in vivo, other secretory signals are also suitable for the CD59 protein and are within the scope of the methods described herein. Suitable general eukaryotic secretory signals for use in mammalian cells 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, which are hereby incorporated by reference in their entirety.

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

[0055] Other promoter sequences useful for regulating the transcription of the CD59 gene sequence are also within the scope of expression of the vectors of the present specification. These promoters are, 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, for example, in U.S. Patent No. 6,677,311 issued January 13, 2004 to Evans et al.; U.S. Patent No. 7,109,029 issued September 19, 2006 to Clark et al.; and U.S. Patent No. 5,998,205 issued December 7, 1999 to Hallenbeck et al., each of which is incorporated herein by reference in its entirety.

[0056] In the remaining amino acid sequence of the portion of the CD59 protein not involved in GPI anchoring, the scope of the CD59 protein herein is assumed 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 alter the properties of the CD59 protein or the membrane-independent CD59 containing its amino acid sequence, i.e., an amino acid modification in which the amino acid sequence of CD59 presenting their side chains at the same relative positions functions similarly to human CD59. Such conservative modifications include amino acid substitutions, additions, and deletions. Modifications of the amino acid sequence of CD59 are achieved using any known technique in the art, for example, 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., J. M. Walker et al., Humana Press, 2008, and Handbook of Molec. and Cellul. Methods in Biol. and Med., 3rd ed., L. J. Ceske et al., CRC Press, 2011.

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

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

[0059] As used herein, the term "about" refers to a number, measurement, or quantity that is close to the recited amount with a difference of less than or equal to 10%.

[0060] The CD59 polypeptide is useful for the treatment of inflammasome mediated disorder. The amino acid sequence of the human CD59 polypeptide useful in the methods described herein is shown by the following sequence: MGIQGGSVLFGLLLVLAVFCHSGHSLQCYNCPNPTADCKTAVNCSSDFDACLITKAGLQVYNKCWKFEHCNFNDVTTRLRENELTYYCCKKDLCNFNEQLENGGTSLSEKTVLLLVTPFLAAAWSLHP (SEQ ID NO: 1). The signal sequence of CD59 is cleaved prior to secretion from the cell and is not required for therapeutic utility. However, a nucleic acid encoding CD59 having a signal sequence enhances the secretion of the CD59 polypeptide and thus the signal sequence may be desirable to include in the CD59 polypeptide encoded by the nucleic acid. The signal sequence-cleaved human CD59 polypeptide useful in the methods described herein is shown by the following sequence: LQCYNCPNPTADCKTAVNCSSDFDACLITKAGLQVYNKCWKFEHCNFNDVTTRLRENELTYYCCKKDLCNFNEQLENGGTSLSEKTVLLLVTPFLAAAWSLHP (SEQ ID NO: 2). However, the exact signal sequence terminus may 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 in either the N-terminal or C-terminal direction from the first leucine of SEQ ID NO: 2. Further deletion of the 26 N-terminal amino acids removes the GPI anchor domain of human CD59. The signal sequence-cleaved soluble human CD59 polypeptide useful in the methods described herein is shown by the following sequence: LQCYNCPNPTADCKTAVNCSSDFDACLITKAGLQVYNKCWKFEHCNFNDVTTRLRENELTYYCCKKDLCNFNEQLEN (SEQ ID NO: 3).This soluble version can also vary at its exact N-terminus 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, and may result in further deletions from or repair of its N-terminus. In certain embodiments, the soluble version of CD59 comprises the amino acid sequence shown in SEQ ID NO: 2 having one or more mutations to the residues required for GPI attachment. In certain embodiments, the residue is either or both of the asparagines at amino acid 70 or 77 of SEQ ID NO: 2. In certain embodiments, the CD59 polypeptide useful in the methods described herein comprises deletions 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 the sequence of any of SEQ ID NO: 1, 2, or 3. The CD59 polypeptide used in the methods described herein may comprise or consist of an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the sequence shown in any of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. Further, the CD59 polypeptide used herein can include additional non-CD59-derived sequences such as a purification tag, the remainder 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 a polypeptide comprising or consisting of the CD59 polypeptide described herein are also useful for the treatment of inflammasome-mediated disorders. Accordingly, one or more nucleic acids encoding a polypeptide comprising or consisting of the CD59 polypeptide described herein are also contemplated. The nucleic acids can be suitably included in a recombinant vector such as a viral vector or plasmid vector suitable for administration to a subject.These vectors can be additionally included in a pharmaceutical composition for administration to an individual having an inflammasome-mediated disorder.

[0061] A polypeptide comprising or consisting of the amino acid sequence shown in any one of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3 can be used in a method for treating an inflammasome disorder in a subject described herein. In certain embodiments, a polypeptide comprising or consisting of the amino acid sequence shown in any one of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3 is used in a method for treating an inflammasome disorder in the eye of a subject. In certain embodiments, the inflammasome disorder is uveitis. In certain embodiments, the subject shows a positive result for the expression or activity of at least one inflammasome activity marker. 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 domain-containing protein (NALP), IFN-γ, Th1 T cell marker or cytokine, Th17 T cell marker or cytokine, and CD4+. In certain embodiments, the inflammasome activity marker is selected from apoptosis-associated speck-like protein containing a CARD (PYCARD / ASC), or NACHT, LRR and PYD domain-containing protein (NALP). In certain embodiments, NALP is NACHT, LRR and PYD domain-containing protein 3 (NLRP3). In certain embodiments, the method described herein comprises administering a human CD59 polypeptide to an individual who shows a positive result for the expression or activity of at least one inflammasome activity marker. The CD59 polypeptide can comprise an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the sequence shown in any one of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3.

[0062] One nucleic acid or a plurality of nucleic acids encoding a polypeptide comprising or consisting of the amino acid sequence shown in any one of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3 can be used in a method for treating inflammasome disorders in a subject. In certain embodiments, one nucleic acid or a plurality of nucleic acids encoding a polypeptide comprising or consisting of the amino acid sequence shown in any one of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3 are used in a method for treating inflammasome disorders in the eye of a subject. In certain embodiments, the inflammasome disorder is uveitis. In certain embodiments, the subject shows a positive result for the expression or activity of at least one inflammasome activity marker. In certain embodiments, the inflammasome activity marker is selected from caspase 1, caspase 5, IL-1β, IL-β17, IL-18, apoptosis-related speck-like protein containing a CARD (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 inflammasome activity marker is selected from apoptosis-related speck-like protein containing a CARD (PYCARD / ASC), or NACHT, LRR and PYD domain-containing protein (NALP). In certain embodiments, NALP is NACHT, LRR and PYD domain-containing protein 3 (NLRP3).

[0063] In certain embodiments, the methods described herein include administering a human CD59 polypeptide to an individual who has shown a positive result for the expression or activity of at least one inflammasome activity marker. The CD59 polypeptide can comprise or consist of an amino acid sequence that is 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 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 domain-containing protein (NALP), IFN-γ, Th1 T cell marker or cytokine, Th17 T cell marker or cytokine, and CD4+. In certain embodiments, the inflammasome activity marker is selected from apoptosis-associated speck-like protein containing a CARD (PYCARD / ASC), or NACHT, LRR and PYD domain-containing protein (NALP). In certain embodiments, NALP is NACHT, LRR and PYD domain-containing protein 3 (NLRP3). In certain embodiments, the positive result is obtained from the eye of the individual.

[0064] In certain embodiments, the methods described herein include administering a human CD59 polypeptide to the eye of an individual who has shown a positive result for the expression or activity of at least one inflammasome activation marker. The CD59 polypeptide can comprise or consist of an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the sequence shown in any one of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. In certain embodiments, the inflammasome activation 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 domain-containing protein (NALP), IFN-γ, Th1 T cell marker or cytokine, Th17 T cell marker or cytokine, and CD4+. In certain embodiments, the inflammasome activation marker is selected from apoptosis-associated speck-like protein containing a CARD (PYCARD / ASC), or NACHT, LRR and PYD domain-containing protein (NALP). In certain embodiments, NALP is NACHT, LRR and PYD domain-containing protein 3 (NLRP3). In certain embodiments, the positive result is obtained from the eye of the individual.

[0065] In certain embodiments, the methods described herein include administering a human CD59 polypeptide to an individual suffering from uveitis. The CD59 polypeptide can comprise or consist of an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the sequence shown 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 certain embodiments, the methods described herein include administering to an individual who has shown a positive result for the expression or activity of at least one inflammasome activity marker, a nucleic acid encoding a human CD59 polypeptide. The nucleic acid can encode a CD59 polypeptide comprising or consisting of an amino acid sequence that is 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 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 domain-containing protein (NALP), IFN-γ, Th1 T cell markers or cytokines, Th17 T cell markers or cytokines, and CD4+. In certain embodiments, the inflammasome activity marker is selected from apoptosis-associated speck-like protein containing a CARD (PYCARD / ASC), or NACHT, LRR and PYD domain-containing protein (NALP). In certain embodiments, NALP is NACHT, LRR and PYD domain-containing protein 3 (NLRP3). In certain embodiments, the positive result is obtained from the eye of the individual.

[0067] In certain embodiments, the methods described herein include administering to the eye of an individual who has shown a positive result for the expression or activity of at least one inflammasome activation marker, a nucleic acid encoding a human CD59 polypeptide. The nucleic acid can encode a CD59 polypeptide comprising or consisting of an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to the sequence shown in SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. In certain embodiments, the inflammasome activation 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 domain-containing protein (NALP), IFN-γ, Th1 T cell markers or cytokines, Th17 T cell markers or cytokines, and CD4+. In certain embodiments, the inflammasome activation marker is selected from apoptosis-associated speck-like protein containing a CARD (PYCARD / ASC), or NACHT, LRR and PYD domain-containing protein (NALP). In certain embodiments, NALP is NACHT, LRR and PYD domain-containing protein 3 (NLRP3). In certain embodiments, the positive result is obtained from the eye of the individual.

[0068] In certain embodiments, the methods described herein include 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 that is at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to the sequence shown 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] The calculation of sequence identity between arrays is performed as follows. To determine the percentage of identity between 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). Next, the amino acid residues at the corresponding amino acid positions or nucleotide positions are compared. If the 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 percentage of 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 the determination of the percentage of identity (%) between two sequences are performed using a mathematical algorithm. The percentage of identity (%) between two amino acid sequences is determined using an alignment software program with 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, the source of CD59 includes, for example, a polynucleotide sequence encoding a CD59 protein designed in a recombinant DNA molecule to direct the expression of the CD59 protein in a suitable host cell. To express a biologically active CD59 protein, a nucleotide sequence encoding the CD59 protein or a functional equivalent is inserted into a suitable expression vector (i.e., a vector containing the nucleic acid necessary to encode elements that regulate the transcription and translation of the inserted coding sequence, operably linked to the nucleotide sequence encoding the amino acid sequence of the CD59 protein).

[0073] Using methods well known to those skilled in the art, an expression vector containing a sequence encoding a CD59 protein operably linked to appropriate transcriptional and translational regulatory elements is constructed. These methods include in vitro recombinant DNA techniques, synthetic techniques, 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., J. M. Walker et al., Humana Press, 2008, and Handbook of Molec. and Cellul. Methods in Biol. and Med., 3rd ed., L. J. Ceske et al., CRC Press, 2011.

[0074] A variety of commercially available expression vector / host systems are useful for expressing including sequences encoding the CD59 protein. These include microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell lines 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 plasmid); or animal cell systems, but are not limited thereto.

[0075] Viral vectors include, but are not limited to, adenoviral vectors, lentiviral vectors, adeno-associated virus (AAV) vectors, and helper-dependent adenoviral vectors. Viral vectors deliver nucleic acid sequences encoding CD59 protein that interfere with the deleterious effects of MAC in the pathogenesis of AMD, as shown herein. Adenoviral packaging vectors are commercially available from the American Type Tissue Culture Collection (Manassas, VA). Methods of constructing adenoviral vectors and methods of using adenoviral vectors are shown 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). Furthermore, recombinant adenoviral vectors are used in gene therapy (U.S. Patent No. 7,235,391, issued June 26, 2007 to Wu et al., which is hereby incorporated by reference in its entirety).

[0077] Recombinant adenoviral vectors are produced, for example, by homologous recombination between a shuttle vector and a proviral vector (U.S. Patent No. 7,235,391, issued June 26, 2007 to Wu et al.; incorporated by reference). The adenoviral vectors herein are replication-deficient, for example, conditionally defective, lacking the adenoviral E1 region, and a polynucleotide encoding CD59 is introduced at a position where the E1 coding sequence has been removed. Alternatively, the 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 simian embryonic mesenchymal or epithelial cells. The production and propagation of these replication-deficient adenoviral vectors using helper cell lines are described in Graham et al 1977 J. Gen. Virol. 36: 59-72.

[0079] The lentiviral vector packaging vector is 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] For packaging using a system based on the third-generation lentiviral SIN vector backbone, many vector constructs are available (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 the green fluorescent protein (GFP) of Aequorea victoria driven by the CMV promoter; and a woodchuck hepatitis virus PRE element that is thought to enhance nuclear export. The GFP marker gene enables quantification of the efficiency of transfection or transduction by direct observation using 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] The manipulation of retroviral nucleic acids to construct retroviral vectors and packaging cells containing genes encoding CD59 protein is accomplished using techniques known in the art. See 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, N.Y.; Miller, et al., Biotechniques. 7:981-990, 1989; Molecular Biomethods Handbook, 2nd ed., J. M. Walker et al., Humana Press, 2008; Handbook of Molec. and Cellul. Methods in Biol. and Med., 3rd ed., L. J. 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 International Publications Nos. 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).

[0082] A retroviral vector is constructed and packaged into non-infectious transduction viral particles (virions) using a two amphotropic packaging system. 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. Patent Nos. 4,650,764, 4,861,719, 4,980,289, 5,122,767, and 5,124,263, and International Publication Nos. 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 production 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 International Publication Nos. WO 85 / 05629, WO 90 / 02797, and WO 92 / 07943, each of which is incorporated herein by reference in its entirety.

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

[0085] Herpes virus DNA vectors can be constructed using techniques well known to those of skill in the art. For example, a DNA fragment encoding the entire genome of a herpes virus is divided into a number of 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, N.Y., 1989), or Escherichia coli F-factor plasmids (O'Conner et al. 1989 Science 244:1307-1313).

[0086] For example, a set of cosmids containing overlapping clones representing the entire genomes of various herpesviruses, including Epstein-Barr virus, varicella-zoster virus, pseudorabies virus, and HSV-1, has been isolated. See M. van Zijl et al. 1988 J. Virol. 62: 2191; Cohen et al. 1993 Proc. Nat'l Acad. Sci. U.S.A. 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 depends on co-infection with another virus (adenovirus or a herpesvirus family virus) to grow and infect in cultured cells (Muzyczka 1992 Curr Top Microbiol Immunol, 158: 97 129). For example, recombinant AAV (rAAV) virus is produced by co-transfecting 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 terminal repeats. Also, cells are contacted with or transfected with an adenovirus or plasmid carrying the adenovirus genes necessary for AAV helper functions. Recombinant AAV virus stocks produced in such a way contain adenovirus and must be physically separated from the recombinant AAV particles (e.g., 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 is useful for gene delivery to mammalian cells in tissue culture because it infects non-dividing cells (Muzyczka 1992 Curr Top Microbiol Immunol 158: 97-129). AAV has a broad host range of infectivity (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 of constructing AAV vectors and methods of using AAV vectors are described, for example, in U.S. Patent No. 5,139,941 (Wu et al.) issued on June 26, 2007 and U.S. Patent No. 4,797,368 (Carter et al.) issued on January 10, 1989, which are hereby incorporated by reference in their entirety. 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 have been successfully 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 transduction of genes involved in human diseases (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 certain embodiments, the vector is a non-viral vector, e.g., a synthetic gene delivery vehicle or vector that specifically delivers CD59 gene-encoding material, unrelated to viral particles, to target cells or tissues. Examples of non-viral vectors include liposomes, peptides, nanoparticles, emulsions, or encapsulated two or more phase systems or other suitable preparations. Thus, in certain embodiments, the method, kit, or composition includes a non-viral vector that carries and contacts a nucleic acid to a tissue or cell. For example, naked DNA encoding a membrane-independent CD59 protein having 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 a tissue or cell so that the nucleic acid is effectively delivered to the tissue or cell for the treatment of complement-related diseases.

[0092] antibody As used herein, the term "antibody" includes whole antibodies and any antigen-binding fragment (i.e., "antigen-binding portion") or single chains thereof. A naturally occurring "antibody" is a glycoprotein 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" binds to human MAC with a K -9 of 5×10 -9 M or less, 2×10 -10 M or less, or 1×10 D M or less. For example, the antibody can be monoclonal or polyclonal. As used herein, the term "monoclonal antibody" or "monoclonal antibody composition" refers to a preparation of antibody molecules of a single molecular composition. A monoclonal antibody composition exhibits a single binding specificity and affinity for MAC or a specific epitope of MAC. The antibody can be IgM, IgE, IgG (such as IgG1 or IgG4).

[0094] Also useful in the MAC assay 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 the 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, which are described in Urlaub and Chasin 1980 Proc. Natl. Acad. Sci. USA 77: 4216-4220 and used with a DH FR selectable marker as described, for example, in R.J. Kaufman and P.A. 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 International Publication No. 87 / 04462, International Publication No. 89 / 01036, and European Patent Application Publication No. 338,841. To produce an antibody, an expression vector encoding the intact target protein or a portion of the target protein is introduced into a mammalian host cell, and the host cell is cultured for a time sufficient to allow expression of the antibody in the host cell or secretion of the antibody into the culture medium in which the host cell has been grown. The antibody can be recovered from the culture medium using standard protein purification methods.

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

[0096] General methodologies for antibody production, including criteria to consider when selecting animals for antiserum production, are described in Harlow et al. (1988 Antibodies, Cold Spring Harbor Laboratory, pp. 93-117). For example, animals of appropriate size, such as goats, dogs, sheep, mice, or camels, are immunized by administering an immunogen, such as a native protein or a portion thereof, containing an epitope derived from human MAC that is effective in eliciting an immune response. An exemplary protocol is as follows. 100 μg to 100 mg of antigen is subcutaneously injected into the back of the animal, depending on the size 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 intraperitoneally injected. The adjuvant (e.g., complete Freund's adjuvant) is further intraperitoneally injected every two weeks until the antibodies in the animal's blood reach an appropriate titer. Exemplary titers include a titer of at least about 1:5000 or a titer of 1:100,000 or higher (i.e., a dilution rate with detectable activity). The 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. See, for example, 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. Using these techniques, antigen-reactive monoclonal antibodies, such as antigen-specific IgG and IgM monoclonal antibodies, can be produced. Any antibody or fragment thereof having affinity for and specific to human MAC is within the scope of the MAC deposition assays provided herein.

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

[0099] In the examples herein, cell lysis is measured by 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 is generally excluded from viable cells, and thus PI is generally used to identify and / or determine the amount of non-viable cells in a mixed population.

[0100] In the examples and certain embodiments of this specification, detectable proteins are fluorescent proteins, 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 compositions isolated from Aequorea victoria. When calcium ions bind to aequorin, the complex decomposes into apoaequorin and the luminescent composition, emitting blue light. Synthetic aequorin is commercially available from Sealite, Sciences (Bogart, GA) as AQUALITE™. GFP emits light in the green portion of the visible spectrum, and synthetic GFP is commercially available from Clontech (Mountain View, CA).

[0101] Mutations were made to the amino acid sequence of GFP to generate derived amino acid sequences of GFP that emit different colored 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 each commercially available from Clontech (Mountain View, CA).

[0102] In another embodiment, the detectable substance is a fluorescent substance other than a fluorescent protein, such as indocyanine green, doxorubicin, riboflavin, chlorophyll, and porphyrin.

[0103] Indocyanine green (ICG) is a tricarbocyanine dye that emits light at approximately 800 nm, approximately 820 nm, approximately 840 nm, or approximately 860 nm upon excitation. ICG is commercially available from H.W. Sands Corp. (Jupiter, Florida). Doxorubicin is fluorescent and emits light, for example, at wavelengths of approximately 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), is fluorescent, and emits light, for example, at wavelengths of approximately 450 nm, approximately 550 nm, approximately 650 nm, or approximately 750 nm. Chlorophyll A is a green photosynthetic pigment that emits light, for example, at wavelengths of approximately 600 nm, approximately 700 nm, or approximately 800 nm. Chlorophyll A is commercially available from suppliers such as Sigma Chemical (St. Louis, Missouri) and Turner Designs (Sunnyvale, California). Porphyrin is a heterocyclic macrocyclic molecule made up of four pyrrole subunits linked on both sides via four methine bridges (=CH-). Due to the large conjugated structure of porphyrin, the compound is colored and, that is, fluoresces, for example, at wavelengths of approximately 600 nm, or approximately 650 nm, or approximately 700 nm. Porphyrin is commercially available from Sigma-Aldrich (St. Louis, Missouri).

[0104] In other alternative embodiments, the detectable substance is an enzyme agent, which is a protein such as, for example, β-galactosidase or alkaline phosphatase and can be expressed with a nucleotide vector.

[0105] β-Galactosidase is a hydrolase enzyme that catalyzes the hydrolysis of β-galactosides into monosaccharides. The Luminescent β-Galactosidase Detection Kit is commercially available from Clontech (Mountain View, California). Alkaline phosphatase is a hydrolase enzyme for removing phosphate groups from various types of molecules such as nucleotides, proteins, and alkaloids. The Luminescent Alkaline Phosphatase Detection Kit is commercially available from Sigma-Aldrich (St. Louis, Missouri).

[0106] pharmaceutical composition One aspect of the present invention provides a method of using a nucleic acid encoding CD59 protein or a soluble CD59 protein to inhibit the activation of inflammasome in cells of an eye that has developed inflammation in a subject. In various embodiments, the CD59 protein includes 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 preparation for administration to the eye and is formulated to enhance delivery to the fundus to provide local sustained release in the retina, or may be formulated to provide effective treatment of blood vessels and / or tissues involved in eye diseases including macular degeneration. In related embodiments, the pharmaceutical composition is formulated at a purity sufficient for administration to a human subject, e.g., into 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 agents include, but are not limited to, growth factors, anti-inflammatory agents, vasopressors including 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), nerve differentiation factor (NDF), hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), heparin-binding EGF (HBEGF), thrombospondin, von Willebrand factor C, heparin and heparan sulfate, and hyaluronic acid, and are selected from the group consisting of.

[0107] In other embodiments, the additional agent is a compound, composition, biological agent, etc. that enhances, stabilizes or synergistically effects 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 concurrently 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, anti-tumor agents, anti-viral agents, antibacterial agents, anti-mycobacterial agents, anti-fungal agents, anti-proliferative agents or anti-apoptotic agents. The drugs included in the compositions of the present invention are well known in the art. See, for example, 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, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants, and the like that are suitable 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 that can serve as pharmaceutically acceptable carriers include sugars such as glucose and sucrose; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil, soybean oil; glycols such as propylene glycol; esters such as ethyl oleate, ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer solutions, and other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, but are not limited thereto, and further, colorants, release 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 by the methods provided herein involves contacting the tissue or cells with a pharmaceutical composition, for example, administering a therapeutically effective amount of a pharmaceutical composition having a nucleic acid encoding a CD59 protein or a source of expression of the CD59 protein as an active agent to a subject in need thereof in an amount and for a time necessary to achieve the desired result. The method includes treating uveitis, for example, by contacting the eye tissue or cells with a CD59 protein or a vector encoding the CD59 protein.

[0110] The compositions according to the method 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. Thus, as used herein, the expression "an amount effective to treat uveitis" refers to an amount of the composition sufficient to beneficially prevent or ameliorate the symptoms of uveitis.

[0111] The exact dosage is selected by the individual physician taking into account the patient being treated. The dosage and administration are adjusted to provide a sufficient level of the active substance or to maintain the desired effect. Additional factors that may be considered include, for example, the severity of the medical condition such as the intermediate or advanced stage of uveitis; the age, weight, and sex of the patient; diet, time and frequency of administration; route of administration; combination of drugs; responsiveness; and tolerance / response to treatment. Extended-release pharmaceutical compositions can 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 physically discrete units of the active substance appropriate to the patient to be treated. However, it will be understood that the total daily usage 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 dosage can first be estimated in either cell culture assays or animal models (usually mice, as provided herein, but rats, rabbits, dogs, or pigs). The animal cell models provided herein are also used to achieve the desired concentration and total dosage range as well as the route of administration. Using such information, the dosage and route useful for administration to humans can be determined.

[0113] A therapeutically effective amount refers to the amount of an active substance that improves a symptom or condition or prevents the progression of uveitis. The therapeutic effects and toxicity of the active substance can be determined by standard pharmaceutical procedures in cell culture or experimental animals, such as, for example, 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 LD50 / ED50 ratio. Pharmaceutical compositions showing a large therapeutic index are preferred. Data obtained from cell culture assays and animal experiments are used in formulating the dosage range for human use.

[0114] The daily dosage of the product can vary widely, such as from 0.001 to 100 mg / day per adult. 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 adjustment of the dosage according to the symptoms of the patient being treated.

[0115] The dosage of the soluble CD59 protein administered intravenously can be in the range of 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. The dosing protocol includes frequencies of daily, twice a week, once a week, once every two weeks, once every three weeks, or once every four weeks.

[0116] The unit dosage typically contains from about 0.001 μg to about 500 μg of the active ingredient, preferably from about 0.1 μg to about 100 μg of the active ingredient, more preferably from about 1.0 μg to about 10 μg of the active ingredient. The effective amount of the drug is usually supplied at a dosage level of from about 0.0001 mg / kg body weight / day to about 25 mg / kg body weight / day. For example, the range is from about 0.001 to 10 mg / kg body weight / day, or from about 0.001 mg / kg body weight / day to 1 mg / kg body weight / day. The composition can be administered, for example, in a regimen of one to four or more times per day. The unit dosage may be divided, for example, by administering in divided dosages more than once.

[0117] Administration as a source of CD59 protein is administration of a dose of viral vector or nucleic acid vector such that the dose contains at least about 50, 100, 500, 1000, or at least about 5000 particles per cell to be treated. The number of cells can be calculated from the retinal area in need of treatment by methods known to those of skill in the art of treating uveitis or eye disease.

[0118] administration of the pharmaceutical composition When formulated with a pharmaceutically acceptable carrier at the desired dosage, the pharmaceutical compositions provided herein are administered locally to humans and other mammals, such as in the eye (such as in the form of a solution, ointment, or eye drops), intranasally, intraorally, orally, rectally, parenterally, intracisternally, vaginally, or intraperitoneally.

[0119] Intraocular injection includes injection into the aqueous humor or vitreous humor of the eye, or injection into the outer layer of the eye, such as by subconjunctival injection or sub-Tenon's capsule injection.

[0120] Liquid dosage forms for ocular, 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, the liquid dosage forms can include inert diluents commonly used in the art, such as water or other solvents, solubilizing agents, and emulsifying agents, 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 the inert diluent, ophthalmic compositions, oral compositions, or other systemically delivered compositions can also include adjuvants such as wetting agents, emulsifying agents, 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 aseptic conditions with a pharmaceutically acceptable carrier and, optionally, any necessary preservative or buffer. For example, the route of administration to the eye or skin is achieved with drops, mists, emulsions, or creams. The administration can be either therapeutic or prophylactic. The present invention includes ophthalmic devices, surgical devices, audiological devices or products (such as gauze dressings or strips) containing the disclosed compositions, and methods of making or using such devices or products. These devices can be coated, impregnated, bound, or otherwise treated with the compositions described herein.

[0122] Transdermal patches have the additional advantage of providing controlled delivery of the active ingredient 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 flow of the compound through the skin. The rate can be controlled by providing a rate-controlling membrane or by dispersing the compound in a polymer matrix or gel.

[0123] Injectable formulations, for example, sterile injectable aqueous or oily suspensions, can be formulated according to known techniques using suitable dispersing or wetting agents and suspending agents. Sterile injectable formulations can be sterile injectable solutions, suspensions or emulsions in a non-toxic parenterally acceptable diluent or solvent, such as, for example, a solution in 1,3-butanediol. Acceptable vehicles and solvents that can be used include water, Ringer's solution, U.S.P., and isotonic sodium chloride solution. In addition, sterile fixed oils are conventionally used as a solvent or suspending medium. For this purpose, any bland fixed oil containing synthetic monoglycerides or diglycerides can be used. Further, fatty acids such as oleic acid are used in the preparation of injectables. Injectable formulations 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 before use. To prolong the effect of the active substance, it is often desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. Delayed absorption of the parenterally administered active substance can be achieved by dissolving or suspending the drug in an oily vehicle. Injectable depot forms are made by forming a microcapsule matrix of the drug in a biodegradable polymer such as polylactide-polyglycolide. Depending on the ratio of the active substance to the polymer and the nature of the particular polymer used, the release rate of the active substance can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Injectable depot formulations are also prepared by encapsulating 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 agent of the present invention with a suitable non-irritating excipient or carrier such as cocoa butter, polyethylene glycol or suppository wax, which are solid at room temperature but liquid at body temperature and thus melt in the rectal or vaginal cavity to release the active substance.

[0125] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active substance 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 starch, 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) disintegrants such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, e) solution retarding agents such as paraffin, f) absorption promoters such as quaternary ammonium compounds, g) wetting agents 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 glycol, sodium lauryl sulfate, and mixtures thereof.

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

[0127] A portion of this work, attached here as Appendix A, was 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, the entire contents of which are hereby incorporated by reference.

[0128] selection of patients for disease monitoring and treatment The CD59 nucleic acid and protein compositions described herein are useful for the treatment of inflammatory and inflammasome-related diseases. The described compositions can be administered to an individual selected for treatment based on positive results in a test for 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, the measurement of cytokines, chemokines, cell surface markers of inflamed or diseased tissue, 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 increase in response to inflammasome activation. Thus, the selected individual may show an increase in the levels of these cytokines in plasma, serum, blood, tissue sections, mRNA, or cell extracts of diseased tissue, or an increase in the expression or secretion levels by immune cells isolated from blood or diseased tissue. The selection criteria for treatment with the CD59 nucleic acid or protein composition may 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 protein containing a CARD (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 evaluated, for example, by ELISA, AlphaLISA® (registered trademark), 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 caspases and inflammasome components such as caspase 1, caspase 5, apoptosis-related speck-like protein containing a CARD (PYCARD / ASC), NLR family pyrin domain containing 1 protein (NALP). Th1 and Th17 cells can be identified using flow cytometry for cell surface markers CXCR3 or CCR5, or in the case of Th1, the transcription factor T-bet, or in the case of Th17, intracellular staining for the transcription factor RORγt.

[0130] The described CD59 nucleic acid and protein compositions are administered to individuals selected for treatment based on the diagnosis of inflammasome-related disorders such as Alzheimer's disease, multiple sclerosis, myocardial infarction, atherosclerotic vascular disease, microvascular disorders, thyroiditis, inflammatory bowel disease, organ transplant rejection, membranous nephritis, sympathetic ophthalmia, sarcoidosis, etc. Inflammasome-related eye disorders include uveitis, allergic conjunctivitis, blepharitis, chronic conjunctivitis, episcleritis, keratitis, retinitis, ocular cicatricial pemphigoid, mucous membrane pemphigoid, pterygium episcleritis, 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.

[0131] In certain embodiments, the CD59 nucleic acid or protein composition can be administered after monitoring an individual for a response to at least one administration of such a composition for reducing inflammation or resolving an inflammasome-related disease or for a response to another treatment. This cycle of administration and monitoring can be repeated 1, 2, 3, 4, 5, 6, 7, 8, 9 times or more, or until an appropriate clinical response is achieved, or until inflammasome activity is reduced. In certain embodiments, the CD59 nucleic acid and protein composition is administered to 1) an individual selected as having an inflammasome-related disease or having a positive result for inflammasome activity; 2) and then the individual is monitored and based on a test for inflammasome activity or a worsening, no change, or sub-optimal response as determined by diagnostic criteria for the inflammasome-related disease, the individual can be given at least one or multiple continued administrations of the composition. In certain embodiments, the CD59 nucleic acid and protein composition is administered to 1) an individual; 2) and then the individual is monitored and based on a test for inflammasome activity or a worsening, no change, or sub-optimal or greater response as determined by diagnostic criteria for the inflammasome-related disease, the individual can be given at least one or multiple continued administrations of the composition.

[0132] The following numbered embodiments are contemplated herein, including methods and compositions of CD59 polypeptide / nucleic acid:

[0133] 1. A method of inhibiting activation of an inflammasome in cells of an inflamed eye 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 cells of the inflamed eye, the composition inhibiting activation of the inflammasome.

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

[0135] 3. The method according to 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, pterygoid episcleritis, 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.

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

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

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

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

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

[0141] 9. The method according to 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-related speck-like protein containing a CARD (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+.

[0142] 10. The method according to embodiment 7 or 8, further comprising performing at least one technique selected from eye examination, optical coherence tomography (OCT), conjunctival impression cytology, RTPCR, ELISA, and PCR to measure retinal function or inflammasome activity marker.

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

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

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

[0146] 14. The method according to 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, herpes virus, and lentivirus.

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

[0148] 16. The method according to 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 according to embodiment 1, further comprising engineering the nucleotide sequence encoding the membrane-independent CD59 protein by deleting the nucleotides encoding the region of the glycosylphosphatidylinositol (GPI) anchor domain.

[0150] 18. The method according to embodiment 1, wherein administration further comprises injecting the composition into the eye.

[0151] 19. The method according to embodiment 1, wherein administration further comprises topically applying the composition.

[0152] 20. The method according to embodiment 18, wherein the eye injection is selected from the group consisting of subretinal injection, intravitreal injection, intraocular injection, subconjunctival injection, and sub-Tenon's injection.

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

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

[0155] 23. The method according to embodiment 22, wherein the additional therapeutic agent is at least one selected from the group consisting of an anti-tumor agent, an anti-viral agent, an antibacterial agent, an anti-mycobacterial agent, an anti-fungal agent, an anti-proliferative agent, and an anti-apoptosis agent.

[0156] 24. The method according to embodiment 22, wherein the additional therapeutic agent is selected from the group consisting of growth factors, anti-inflammatory agents, antihypertensive agents, collagenase inhibitors, steroids, matrix metalloproteinase inhibitors, ascorbates, angiotensins, calreticulins, tetracyclines, fibronectins, collagens, thrombospondins, 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, heparan sulfate, and hyaluronic acid.

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

[0158] 26. A kit for treating uveitis of a subject, a. 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 of a subject; b. Instructions for use, and c. A container The kit comprising.

[0159] 27. A method for treating at least one condition selected from uveitis, allergic conjunctivitis, blepharitis, chronic conjunctivitis, episcleritis, keratitis, retinitis, ocular cicatricial pemphigoid, mucous membrane pemphigoid, pterygium, Stevens-Johnson syndrome, Eales' disease, Behçet'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 the expression and secretion of the membrane-independent CD59 protein in cells of the inflamed eye; or b) Administering to a subject a composition comprising a membrane-independent CD59 protein wherein the composition inhibits the activation of the inflammasome.

[0160] 28. A method for inhibiting the activation of the inflammasome in cells of an inflamed eye of a subject, comprising: a) Measuring an inflammasome activity marker in the eye; and b) When the inflammasome activity marker is positive, 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 cells of the inflamed eye wherein the composition inhibits the activation of the inflammasome.

[0161] 29. The method according to embodiment 28, wherein 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 domain-containing protein (NALP), IFN-γ, Th1 T cell marker or cytokine, Th17 T cell marker or cytokine, and CD4+.

[0162] After administration of 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, further comprising measuring an inflammasome activity marker in the eye, the method according to embodiment 28 or 29.

[0163] 31. A method of inhibiting the activation of an inflammasome in cells of a subject with inflammation, a. 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 cells of the subject with inflammation; or b. administering to the subject a composition comprising a soluble CD59 protein comprising, wherein the composition inhibits the activation of the inflammasome, method.

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

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

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

[0167] 35. The method according to 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 according to any one of embodiments 31 to 35, further comprising measuring an inflammasome activity marker in the subject before administration.

[0169] The method according to any one of embodiments 31 to 35, further comprising measuring an inflammasome activity marker in a subject after administration.

[0170] The method according to embodiment 36 or 37, wherein the inflammasome activity marker is selected from caspase 1, caspase 5, IL-1β, IL-β17, IL-18, apoptosis-related speck-like 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+.

[0171] The method according to embodiment 38, further comprising administering the composition in a dosage sufficient to treat an inflammasome-related condition.

[0172] The method according to embodiment 39, wherein the nucleotide sequence encoding a membrane-independent CD59 protein operably linked to a promoter for the expression and secretion of the membrane-independent CD59 protein is carried by 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] The method according to embodiment 40, wherein the lentivirus is a retrovirus.

[0174] The method according to embodiment 31, wherein the administration further comprises intravenous injection of the composition.

[0175] The method according to embodiment 31, wherein the administration further comprises topical application of the composition.

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

[0177] 45. The method according to 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 anti-apoptosis agent.

[0178] 46. The method according to embodiment 44, wherein the additional therapeutic agent is selected from the group consisting of a growth factor, an anti-inflammatory agent, a hypertensive agent, a collagenase inhibitor, a steroid, a matrix metalloproteinase inhibitor, an ascorbate, an 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, heparan sulfate, and hyaluronic acid.

[0179] 47. A kit for inhibiting the activation of the inflammasome in cells that have caused inflammation in a subject, c. (i) a membrane-independent CD59 protein and / or a nucleotide sequence encoding the CD59 protein; or (ii) a soluble CD59 protein; a pharmaceutical composition comprising a dosage sufficient to inhibit the inflammasome in cells that have caused inflammation in a subject; d. instructions for use; and e. a container A kit comprising.

[0180] 48. A method for treating at least one condition selected from Alzheimer's disease, multiple sclerosis, myocardial infarction, atherosclerosis, microvascular disorders, thyroiditis, inflammatory bowel disease, organ transplant rejection, membranous nephritis, sympathetic ophthalmia, and sarcoidosis, A method comprising administering to a 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 from cells; or a soluble CD59 protein; wherein the composition inhibits the activation of the inflammasome.

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

Examples

[0182] Example 1: Mouse C57BL / 6J, and C9 on a C57BL / 6J background - / - Mice were purchased from the Jackson Laboratory (Bar Harbor, Maine) and maintained in the animal facility of Tufts University School of Medicine in Boston. All animal test protocols complied with the Association for Research in Vision and Ophthalmology's resolution on the use of animals in vision research and the recommendations of the National Institutes of Health (NIH) Guidelines for the Care and Use of Laboratory Animals.

[0183] Example 2: AAV construct and intravitreal injection An AAV serotype 2 vector (AAVCAGsCD59) expressing a truncated form of human CD59 or protectin lacking the glycosylphosphatidylinositol (GPI) - anchoring signal was constructed by the protocol described in Cashman, S. M., et al. (2011) PLoS One 6, e19078. Soluble CD59 (sCD59) is 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 given 3.5×10 9Mice were injected with 1 μl of AAVCAGsCD59 or AAVCAGGFP at a genomic copy number of 1 genome copy / μl, or PBS (1 μl), and 1 week later, EAU was induced in the mice as described in the examples herein.

[0184] Example 3: Induction of EAU by active immunization Six-week-old C57BL / 6J and C9 mice with the same genetic background - / - 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 a 1:1 (v / v) mixture of complete Freund's adjuvant (CFA) containing Mycobacterium tuberculosis strain H37RA (2.5 mg / mL). At the same time, 1.5 μg of Bordetella pertussis toxin diluted in 100 μl of PBS was administered to the mice by intraperitoneal injection (Agarwal, R. K., 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% goat normal 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% goat normal serum). Subsequently, the sections were washed and incubated in anti-rabbit secondary antibody conjugated to Cy3 (Molecular Probes, Eugene, OR) to identify the location of C5b-9 in the retinal sections. The slides were mounted with an anti-fade agent containing DAPI (mounting medium for anti-fading) (Vectashield-DAPI; Vector Laboratories, Burlingame, CA) to counterstain the nuclei, and images were acquired with a Leica confocal microscope. The intensity of C5b-9 staining throughout 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 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, Nebraska, USA) mixed with 0.1% Tween-20, immunoblotting was 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, California; diluted 1:500) as primary antibodies. After incubation with appropriate secondary antibodies, immunoreactive bands were visualized using a LI-COR-Odyssey infrared scanner (LI-COR). The blot was re-probed with β-actin as a loading control.

[0187] Example 6: Enzyme-linked immunosorbent assay Using 20 μg of retinal protein supernatant, cytokines were quantified by sandwich ELISA for mouse IL-1β, IFN-γ, and IL-17 (PeproTech, Rocky Hills, NJ) as per the manufacturer's instructions. The supernatant was added in duplicate, and the cytokine being measured was revealed using a monoclonal antibody conjugated to horseradish peroxidase. Cytokine concentrations were expressed in pg / mL.

[0188] Example 7: Gene expression Total RNA was isolated from the mouse retina using the RNeasy Mini Kit (QIAGEN, Valencia, CA) according to the manufacturer's protocol. RNA was quantified by absorbance at 260 nm using 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 TaqMan primers pre-designed for β-actin (Mm02619580_g1), IL-1β (Mm00434228_m1), IL-17 (Mm00439619_m1), and IFN-γ (Mm01168134_m1). Denaturation was carried out at 95°C for 10 minutes, followed by 40 cycles of 15 seconds at 95°C, and annealing and extension were carried out at 60°C for 60 seconds. The final PCR products were electrophoresed on a 2% agarose gel to confirm PCR specificity. The Ct values obtained from RT-PCR were normalized to the Ct values from β-actin of the same samples using the ddCt method to obtain fold change data for gene expression. The gene expression of IL-17 was semi-quantitatively quantified.

[0190] Example 8: Flow cytometry Inguinal lymph node cells were isolated from mice, and a single cell suspension was obtained by passing the cells through a 40-μm nylon mesh. Prior to intracellular cytokine staining, inguinal 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 the data were analyzed with FlowJo software (Tree Star, Ashland, OR). Gates were set based on appropriate isotype controls. When shown, the percentage of positive cells represents the percentage of the gated population compared to normal controls.

[0191] Example 9: Electroretinogram The loss of rod and cone function was measured using scotopic and photopic electroretinogram (ERG) analysis. Three weeks after EAU induction, ERG recordings were made using a UTAS system equipped with a BigShot ganzfeld (LKC Technologies; Gaithersburg, Maryland). After overnight dark adaptation, mice were anesthetized with an intraperitoneal injection of ketamine (100 mg / kg) / xylazine (10 mg / kg) under dark conditions. The pupils were dilated with 1% tropicamide and 2.5% phenylephrine hydrochloride. The ERG active contact lens gold electrode was gently placed in the center of the cornea with a drop of lubricant (GenTeal, Alcon, Inc., Fort Worth, Texas) to maintain corneal hydration and good conductivity. The reference electrode and ground electrode were subcutaneously inserted near the back of the neck and the base of the tail, respectively. The scotopic ERG was elicited with a 10-ms flash of white light at 0 dB (5 cds / m 2 ), -10 dB (25 cds / m 2 ), and -20 dB (25 cds / m 2 ). After 2 minutes of white light bleach, simultaneous photopic ERGs were examined. The photopic responses were elicited with flashes of white light at intensities of 0 dB and 1 dB (3.15 cds / m 2 ). Ten responses were averaged at each flash intensity. The amplitude of the a-wave was measured from the 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 their pupils were dilated by instillation of 1% tropicamide and 2.5% phenylephrine. The corneas were kept moist by topical application of an eye lubricant (GenTeal, Alcon, Inc., Fort Worth, Texas). Optical coherence tomography (OCT) images were acquired using a Bioptigen Spectral Domain Ophthalmic Imaging System (Bioptigen Envisu R2300, Morrisville, North Carolina). As described in Chen, J., et al. (2013) PLoS One 8, e63904, averaged single B-scans and volume scans were acquired in images centered on the optic nerve head.

[0193] Twenty-four days after EAU, fundus imaging was performed, and images were acquired using a Micron III Retinal Imaging Microscope and StreamPix software (Phoenix Research Labs, Pleasanton, California). Specifically, the eyes were examined for physiological and pathological signs such as infiltrates, perivascular inflammatory cell infiltration (cuffing), white linear lesions, retinal dystrophy, subretinal hemorrhage, and retinal detachment. Individual scores were assigned by two independent observers in a blinded fashion on a 0 - 4 scale for each of the individual parameters: retinal infiltrates, changes in the optic nerve head, vascular distribution, 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 of all groups were harvested on day 24 after immunization and fixed in 10% buffered formalin. After 2 days of fixation, the specimens were dehydrated by a graded alcohol step and embedded in paraffin blocks. Six vertical sections (5 μm) were cut in six different planes including the optic nerve region and stained with hematoxylin and eosin. The detailed severity of EAU was evaluated on a scale of 0 - 4 for photoreceptor damage, infiltration, and vasculitis as previously described by Caspi, R. R., 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 was composed of a combination of granulomas, hemorrhages, DF nodules and infiltrates. The vasculitis score represents perivasculitis and perivascular CD4 cell infiltration, thrombus formation, and the extent of the affected vascular system in the retina.

[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 comparison between three or more groups, one - way ANOVA was performed. A p - value of 0.05 or less was considered statistically significant.

[0196] Example 13: MAC deposition in EAU The final step of complement activation results in the deposition of MAC on the cell surface. The recruitment of multiple C9 molecules to the pre - formed C5b - 8 complex is the last necessary step in the assembly of MAC (C5b - 9). Thus, C9 - deficient mice are unable to form a functional MAC complex.

[0197] To determine whether MAC is formed on the surface of retinal cells in EAU, frozen retinal sections were examined by staining with an antibody against C5b - 9 on day 24 after induction of EAU in C57BL / 6J mice. It was observed that the deposition of MAC on the retina of EAU mice was more than 70% compared to the MAC deposition on the retina of normal control mice. C9- / - MAC deposition on the surface of the EAU retina was observed to be insignificant (Figs. 1A and 9). The data obtained showed that complement was activated in EAU and the reaction was completed to form MAC on the retinal tissue. The data also showed that C9 - / - also indicated that EAU mice were unable to form MAC on the retina.

[0198] Example 14: Activation of inflammasome in EAU MAC deposition on the cell surface leads to pore formation, which results in an increase in intracellular calcium. In the examples herein, it was investigated whether MAC deposition causes 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 correspondingly 45-fold more IL-1β mRNA than normal mouse controls. In contrast, C9 - / - In EAU mice, 37% more IL-1β protein and correspondingly 3.8-fold more IL-1β mRNA were observed (Figs. 1B and C). Western blot analysis showed 200% more NLRP3 protein in EAU mice, and C9 - / - EAU mice had 8% more NLRP3 protein (Fig. 1D).

[0200] An important component of the inflammasome complex is caspase-1, which generally occurs in the form of an inactive precursor (zymogen), is activated by the NLRP3 complex, and is proteolytically processed into active p10 and p20 subunits to form the final multimeric inflammasome complex. Western blot analysis showed 80% higher activation (p20) of caspase-1 in the retina of EAU mice than normal; however, C9 - / -In the retina of EAU mice, 25% higher activation of caspase-1 (p20) was observed compared to normal (Figure 1E). The expression of the inflammasome adapter protein ASC protein in the retina of EAU mice was measured by Western blot. Western blot analysis showed that 44% more ASC protein was observed in EAU mice than in normal, C9 - / - It was shown that 18% more ASC protein was observed in EAU mice than in normal (Figure 1F).

[0201] Therefore, it was observed that each of IL-1β, NLRP3, caspase 1, and ASC increased in EAU, C9 - / - EAU mice were partially protected from such increases, indicating that MAC deposition is an important player in inflammasome activation in EAU.

[0202] Example 15: Role of MAC in retinal function in EAU EAU mice and C9 - / - The retinal function of EAU mice was measured using electroretinogram (ERG). The retinal function of EAU mice was observed to have a 33%, 50%, and 41% decrease in the amplitude of the a-wave during dark adaptation at flash intensities of -20 dB, -10 dB, and 0 dB, respectively, compared to control C57BL / 6J mice. C9 - / - The retinal function of EAU mice was observed to have an a-wave amplitude that 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 amplitude of the b-wave during dark adaptation of EAU mice was observed to have a 47%, 52%, and 49% decrease at flash intensities of -20 dB, -10 dB, and 0 dB, respectively, compared to control C57BL / 6J mice. C9 - / -In EAU mice, it was observed that the amplitude of the b-wave during dark adaptation was specifically 32%, 33%, and 17% less 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, and 0 dB was observed to be statistically significant (p<0.05) (Figures 2A and 2B).

[0204] In EAU mice, it was observed that the amplitude of the b-wave during light adaptation decreased by 44% and 37% at flash intensities of 0 dB and 1 dB, respectively, compared to control C57BL / 6J mice. C9 - / - In EAU mice, it was observed that the amplitude of the b-wave decreased by 5% and 15% at flash intensities of 0 dB and 1 dB, respectively, compared to control C57BL / 6J mice. Differences were observed in the amplitude of the b-wave during light adaptation, and 0 dB was observed to be statistically significant (p<0.05) (Figures 2A and 2B).

[0205] Example 16: C9 - / - There is no significant protection of retinal structure in EAU EAU and C9 - / - The retinal structure and pathological severity of the disease in EAU mice were quantified using fundus imaging, OCT, and histological examination on day 24 after induction of EAU. Based on the clinical scoring criteria developed by Xu.H. et al. (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 times higher than that of the control group, which included 38-fold higher vascular inflammation, 8-fold more infiltration of immune cells, 13-fold greater damage to the optic nerve head, and 34-fold greater structural damage compared to control C57BL / 6J mice. Unexpectedly, C9 mice with uveitis - / - were observed to have statistically equivalent pathological results to EAU mice (Figure 3).

[0206] Twenty-four days after immunization, as observed by OCT imaging and histopathological examination, EAU mice showed severe inflammatory cell infiltration into the vitreous and choroid, retinal vasculitis, retinal edema, and moderate to severe retinal folds and infiltrates, compared to C57BL / 6J control mice (Figures 4A and 4B). Control mice, EAU mice, and C9 - / - Detailed histological analysis was performed on paraffin-embedded sections from the retinas of EAU mice and scored based on the criteria described in Caspi, R. R., et al. (1988) J. Immunol 140, 1490-1495. EAU mice were observed to have at least approximately 27-fold more infiltrates (Figure 4C), increased vasculitis (Figure 4D), and 78-fold greater photoreceptor damage (Figure 4D) than C57BL / 6J control mice. C9 - / - The histological scores of EAU mice showed a 28% decrease in infiltration, a 35% decrease in photoreceptor damage, and a 31% decrease in vasculitis compared to EAU mice, but no statistically significant differences were observed (Figures 4B and C).

[0207] The photoreceptor damage score was calculated based on the criteria described in Caspi, R. R., et al. (1988) J. Immunol 140, 1490-1495 as a composite score of photoreceptor loss, retinal folds, and retinal detachment. Similarly, the infiltration score was composed of a combination of granulomas, hemorrhages, DF nodules, and infiltrates. The vasculitis score represents perivasculitis and perivascular CD4 cell infiltration, thrombus formation, and the extent of the affected vascular system in the retina.

[0208] Example 17: Soluble CD59-mediated inhibition of MAC deposition in EAU The data obtained in the examples herein indicate that due to the genetic deficiency of C9, C9 - / - mice are unable to form MAC and are also unable to activate the inflammasome, protecting against several of the features of uveitis. However, C9 - / - the retinas of EAU mice were observed to be not protected from the histological pathologies associated with EAU.

[0209] CD59 is a GPI-anchored protein present on the membranes of most nucleated cells. The main function of CD59 is to prevent the recruitment of C9 to pre-formed C5b-8 complexes. A recombinant adeno-associated virus vector (AAVCAGsCD59) expressing a truncated form of CD59 in which the GPI anchor signal is deleted and which is secreted and able to diffuse throughout the retina is described in Cashman, S. M., et al. (2011) PLoS One 6, e19078.

[0210] Mice were intravitreally injected with AAVCAGsCD59, and one week later (to allow for optimal levels of the transgene), EAU was induced in the mice as described in the examples herein. For the negative control, mice were intravitreally injected with AAVCAGGFP (a virus expressing green fluorescent protein (GFP)). On day 24 after induction of EAU, frozen retinal sections from both groups of mice were examined by staining with a C5b-9 antibody. In mice intravitreally injected with AAVCAGGFP and then induced with EAU, the transgene was expressed in the ganglion cell layer, inner plexiform layer, and inner nuclear layer (Figure 14B). In mice with more severe EAU phenotypically, transgene expression was also observed in the RPE and photoreceptors (Figure 14B, lower panel). It was observed that MAC deposition was approximately 45% less in EAU mice injected with AAVCAGsCD59 compared to EAU mice injected with AAVCAGGFP (Figure 5A and Figure 12). Thus, intravitreally injected AAVCAGsCD59 was observed to inhibit MAC formation in the retina of EAU mice.

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

[0212] Measured by ELISA and RT-PCR, it was observed that in AAVCAGsCD59-injected EAU mice, the IL-1β protein was 40% less and the IL-1β mRNA was 70% less compared to AAVAGsCD59-injected EAU mice (Figure 5B and Figure 5C). Similarly, the levels of NLRP3 protein and caspase 1 p20 were decreased by 60% in AAVCAGsCD59-injected EAU mice compared to control AAVCAGGFP-injected EAU mice (Figure 5D and Figure 5E). No substantial difference in the amount of ASC was observed in AAVCAGsCD59-injected EAU mice compared to control AAVCAGGFP-injected EAU mice (Figure 5F). Therefore, the obtained data indicate that sCD59 inhibits NLRP3-mediated IL-1β production by inhibiting MAC deposition in uveitis.

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

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

[0215] Control C57BL / 6J mice, EAU mice, and C9 - / - In EAU mice, 24 days after EAU, the levels of Th1 CD4+ cells and Th17 CD4+ cells were measured in the draining lymph node (DLN). A greater number of IL-17- and IFN-γ-positive CD4+ cells were observed in the DLN of EAU mice compared to control C57BL / 6J mice (Figures 11A–11C). The EAU mice and C9 - / - No statistical significant difference was observed in the levels of IFN-γ- and IL-17-positive CD4+ cells in the DLN among EAU mice (Figures 11A–11C). These data indicate that MAC plays an important role in T cell differentiation in the retina of EAU 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 effect on T cell differentiation in the eyes expressing sCD59 affected by EAU, the protein and mRNA levels of IFN-γ and IL-17 were measured in AAVCAGsCD59-injected EAU mice and AAVCAGGFP-injected EAU mice.

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

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

[0219] Example 22: Soluble CD59-mediated protection of retinal structure and pathological conditions in EAU To determine whether sCD59 can protect the retinal structure and reduce the conditions associated with uveitis, fundus imaging was performed on AAVCAGsCD59-injected EAU mice 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 the control AAVCAGGFP-injected EAU mice. The overall clinical score included 24% fewer inflammatory infiltrates, 27% less structural damage, 22% reduced vasculitis and vascular inflammatory cell infiltration (cuffing), and 13% less damage to the optic nerve head in AAVCAGsCD59-injected EAU mice compared to AAVCAGGFP-injected EAU mice (Figs. 7A-7F). No significant difference was observed between the PBS vehicle control and the AAVCAGGFP-injected EAU retina. Each score for vessels, infiltration, and structural damage was observed to be statistically significantly different (p<0.05), but the optic nerve head score did not reach statistical significance (p = 0.1). Therefore, the data obtained indicate that the expression of sCD59 reduced retinal inflammation and immune cell infiltration in uveitis.

[0220] The progression and severity of EAU were recorded and observed by OCT scans, and it was observed that EAU progressed in AAVCAGGFP-injected EAU mice compared to AAVCAGsCD59 (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 in Caspi, R. R., et al. (1988) J. Immunol 140, 1490-1495, it was observed that AAVCAGsCD59-injected EAU mice had approximately 76% less infiltration, 69% less photoreceptor damage, and 78% less vasculitis compared to control AAVCAGGFP-injected EAU mice (Figures 8B-8E). The differences in photoreceptor damage and infiltration were observed to be statistically significant (p = 0.03 and p = 0.01, respectively), while the vasculitis score was not statistically significant (p = 0.2).

[0221] Example 23: Treatment and subsequent monitoring of individuals with atherosclerosis using a composition of soluble CD59 protein Individuals are diagnosed with atherosclerosis based on ultrasound examinations showing an increase in intima-media complex thickness (IMT), and subsequently the individuals are administered a composition containing soluble CD59 protein by intravenous injection. Patients receive three consecutive administrations at a frequency of once a week. After administration, the individuals are re-evaluated by ultrasound examination, and if it is shown that there is no change in the IMT, the individuals receive four additional administrations of CD59 protein by intravenous injection at a frequency of once a week and are then re-evaluated. At follow-up, the thickness of the IMT has decreased to an acceptable range and no further CD59 treatment is performed.

[0222] Example 24: Treatment and subsequent monitoring of individuals with sarcoidosis using a composition of soluble CD59 protein An individual presents to a primary care physician with symptoms consistent with sarcoidosis, the chest x-ray shows growth consistent with such a diagnosis, a biopsy sample is taken, and an increase in the level of activated caspase 1 is identified by comparison with a healthy tissue control. Next, a composition containing soluble CD59 protein is administered to the individual by intravenous injection. After administration, the individual is re-evaluated by x-ray and shows partial reduction of sarcoidosis. The patient is re-administered the soluble CD59 protein composition, and re-evaluation by x-ray shows complete efficacy, and no further CD59 treatment is done.

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

[0224] Preferred embodiments of the invention have been shown and described herein, but it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, modifications, and substitutions will occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention.

[0225] All publications, patent applications, issued patents, and other documents mentioned in this specification are hereby incorporated by reference as if each individual publication, patent application, issued patent, or other document was specifically and individually indicated to be incorporated by reference. The definitions contained in the incorporated text are excluded only in the case of a conflict with the definitions in the present disclosure. Finally, the preferred embodiments of the invention are described item by item.

[0226] [Embodiment 1] A method for inhibiting the activation of the inflammasome in cells of an eye that has developed inflammation, the method comprising administering to a 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 cells of the inflamed eye, wherein the composition inhibits the activation of the inflammasome, and wherein the subject shows a positive result for the expression or activity of at least one inflammasome activity marker in the eye of the subject that has developed inflammation.

[0227] [Embodiment 2] The method according to embodiment 1, wherein the inflammasome activity marker is selected from caspase 1, caspase 5, IL-1β, IL-β17, IL-18, apoptosis-related 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+.

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

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

[0230] [Embodiment 5] The method according to any one of embodiments 1 to 4, further comprising measuring an inflammasome activity marker in the eye after administration of the 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.

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

[0232] [Embodiment 7] The method according to Embodiment 6, wherein the subject is diagnosed with or suspected of suffering from uveitis.

[0233] [Embodiment 8] A positive result regarding the expression or activity of at least one inflammasome activity marker in the eye that caused the inflammation in the subject is an increase in the expression or activity of at least one inflammasome activity marker as compared to the expression or activity of at least one inflammasome activity marker in the eye of an individual who is not diagnosed with or suffering from uveitis, allergic conjunctivitis, blepharitis, chronic conjunctivitis, episcleritis, keratitis, retinitis, ocular cicatricial pemphigoid, mucous membrane pemphigoid, pterygoid episcleritis, Stevens-Johnson syndrome, Eales' disease, Behçet's disease, sarcoidosis, systemic lupus erythematosus, polyarteritis nodosa, Wegener's granulomatosis, Vogt-Koyanagi-Harada disease, sympathetic ophthalmia, or sarcoidosis. The method according to any one of Embodiments 1 to 7.

[0234] [Embodiment 9] A method according to embodiment 8, wherein a positive result for the expression or activity of at least one inflammasome activity marker in the eye that caused the inflammation in the subject is an increase in the expression or activity of at least one inflammasome activity marker as compared to the expression or activity of at least one inflammasome activity marker in the eyes of individuals who have not been diagnosed with or are 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 eye that caused the inflammation in the subject is determined by a histological score.

[0236] [Embodiment 11] A method for inhibiting the activation of an inflammasome in cells of a subject with inflammation, a. measuring an inflammasome activity marker in the subject; and b. when the inflammasome activity marker is positive, (i) 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 cells of the subject with inflammation; or (ii) a soluble CD59 protein administering to the subject a composition comprising wherein the composition inhibits the activation of the inflammasome.

[0237] [Embodiment 12] The method according to embodiment 11, wherein the inflammasome activity marker is selected from caspase 1, caspase 5, IL-1β, IL-β17, IL-18, apoptosis-related 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+.

[0238] [Embodiment 13] The method according to embodiment 11, wherein the inflammasome activity marker is selected from apoptosis-related speck-like protein containing a CARD (PYCARD / ASC) or a protein containing NACHT, LRR and PYD domains (NALP).

[0239] [Embodiment 14] The method according to embodiment 13, wherein NALP is a protein containing NACHT, LRR and PYD domains 3 (NLRP3).

[0240] [Embodiment 15] The 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] The method according to any one of embodiments 11 to 15, further comprising measuring an inflammasome activity marker 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.

[0242] [Embodiment 17] The method according to any one of embodiments 11 to 16, wherein the subject is diagnosed with or suspected of having uveitis, allergic conjunctivitis, blepharitis, chronic conjunctivitis, episcleritis, keratitis, retinitis, ocular cicatricial pemphigoid, mucous membrane pemphigoid, pterygoid episcleritis, 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] The method according to embodiment 17, wherein the subject is diagnosed with or suspected of having uveitis.

[0244] [Embodiment 19] The method according to any one of Embodiments 11-18, wherein the expression or activity of at least one inflammasome activity marker is increased as compared to the expression or activity of at least one inflammasome activity marker in a subject not diagnosed with or suffering from Alzheimer's disease, multiple sclerosis, myocardial infarction, atherosclerosis, microvascular disorder, thyroiditis, inflammatory bowel disease, organ transplant rejection, membranous nephritis, sympathetic ophthalmia, uveitis, or sarcoidosis.

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

[0246] [Embodiment 21] The method according to any one of Embodiments 11-20, wherein the positive inflammasome activity marker in the subject with inflammation 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 the expression and secretion of the membrane-independent CD59 protein in cells of a subject with inflammation; or b. Soluble CD59 protein A method for inhibiting the activation of an inflammasome in cells of a subject with inflammation by administering to the subject a composition comprising: The composition inhibits the activation of the inflammasome, The subject shows a positive result for the expression or activity of at least one inflammasome activity marker in the subject with inflammation.

[0248] [Embodiment 23] The method according to embodiment 22, wherein the inflammasome activity marker is selected from caspase 1, caspase 5, IL-1β, IL-β17, IL-18, apoptosis-related speck-like 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+.

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

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

[0251] [Embodiment 26] The 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 subject with inflammation is in the eye of the subject with inflammation.

[0252] [Embodiment 27] The method according to any one of embodiments 22 to 26, wherein the subject is diagnosed with or suspected of having uveitis, allergic conjunctivitis, blepharitis, chronic conjunctivitis, episcleritis, keratitis, retinitis, ocular cicatricial pemphigoid, mucous membrane pemphigoid, pterygium episcleritis, 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] The method according to embodiment 27, wherein the subject is diagnosed with or suspected of having uveitis.

[0254] [Embodiment 29] A method according to any one of embodiments 22-28, wherein a positive result for the expression or activity of at least one inflammasome activity marker in the subject with the inflammation 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 suffering from Alzheimer's disease, multiple sclerosis, myocardial infarction, atherosclerosis, microvascular disorders, thyroiditis, inflammatory bowel disease, organ transplant rejection, membranous nephritis, sympathetic ophthalmia, uveitis, or sarcoidosis.

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

[0256] [Embodiment 31] A method according to any one of embodiments 22-30, wherein a positive result for the expression or activity of at least one inflammasome activity marker in the subject with the inflammation 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 the expression and secretion of the membrane-independent CD59 protein in cells of a subject with inflammation; or b. A soluble CD59 protein A method for inhibiting the activation of the inflammasome in cells of a subject suffering from uveitis by administering a composition comprising, wherein the composition inhibits the activation of the inflammasome.

[0258] [Embodiment 33] The method according to embodiment 32, wherein the subject shows a positive result for the expression or activity of at least one inflammasome activation marker in the eye of the subject with inflammation.

[0259] [Embodiment 34] The method according to embodiment 33, wherein the inflammasome activation marker is selected from caspase 1, caspase 5, IL-1β, IL-β17, IL-18, apoptosis-related 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+.

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

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

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

[0263] [Embodiment 38] The method according to embodiment 37, wherein the subject is diagnosed with or suspected of suffering from uveitis.

[0264] [Embodiment 39] A positive result for the expression or activity of at least one inflammasome activity marker in the subject with the inflammation is an increase in the expression or activity of at least one inflammasome activity marker as compared to the expression or activity of at least one inflammasome activity marker in a subject not diagnosed with or suffering from Alzheimer's disease, multiple sclerosis, myocardial infarction, atherosclerosis, microvascular disorder, thyroiditis, inflammatory bowel disease, organ transplant rejection, membranous nephritis, sympathetic ophthalmia, uveitis, or sarcoidosis. The method according to any one of embodiments 33 to 38.

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

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

[0267] [Embodiment 42] A method for inhibiting the activation of an inflammasome in cells of a subject with inflammation, 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 cells of the subject with inflammation, wherein the composition inhibits the activation of the inflammasome, and the subject shows a positive result regarding the expression or activity of at least one inflammasome activity marker.

[0268] [Embodiment 43] The method according to 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 marker or cytokine, Th17 T cell marker or cytokine, and CD4+.

[0269] [Embodiment 44] The method according to 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 according to embodiment 44, wherein NALP is NACHT, LRR and PYD domain-containing protein 3 (NLRP3).

[0271] [Embodiment 46] The method according to any one of embodiments 42 to 45, wherein the subject shows a positive result for the expression or activity of at least one inflammasome activity marker in the eye of the subject.

[0272] [Embodiment 47] The method according to any one of embodiments 42 to 46, further comprising measuring an inflammasome activity marker in a subject with inflammation after administration of 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.

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

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

[0275] [Embodiment 50] A positive result regarding the expression or activity of at least one inflammasome activity marker in the subject with the inflammation, as compared to the expression or activity of at least one inflammasome activity marker in a subject not diagnosed with or not suffering from uveitis, allergic conjunctivitis, blepharitis, chronic conjunctivitis, episcleritis, keratitis, retinitis, ocular cicatricial pemphigoid, mucous membrane pemphigoid, pterygoid episcleritis, Stevens-Johnson syndrome, Eales' disease, Behçet's disease, sarcoidosis, systemic lupus erythematosus, polyarteritis nodosa, Wegener's granulomatosis, Vogt-Koyanagi-Harada disease, sympathetic ophthalmia, or sarcoidosis, is an increase in the expression or activity of at least one inflammasome activity marker, according to the method of any one of embodiments 42 to 49.

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

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

Claims

**Claim 1** A method of inhibiting the activation of the inflammasome in cells of an eye that has developed inflammation, comprising administering to a 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 cells of the inflamed eye, wherein the composition inhibits the activation of the inflammasome, and wherein the subject shows a positive result for the expression or activity of at least one inflammasome activation marker in the eye of the subject that has developed inflammation. **Claim 2** The method according to claim 1, wherein the inflammasome activation 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 domain-containing protein (NALP), IFN-γ, Th1 T cell marker or cytokine, Th17 T cell marker or cytokine, and CD4+. **Claim 3** The method according to claim 1, wherein the inflammasome activation marker is selected from apoptosis-associated speck-like protein containing a CARD (PYCARD / ASC), or NACHT, LRR and PYD domain-containing protein (NALP). **Claim 4** The method according to claim 3, wherein the NALP is NACHT, LRR and PYD domain-containing protein 3 (NLRP3). **Claim 5** The method according to any one of claims 1 to 4, further comprising measuring the inflammasome activation marker in the eye after administration of the 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. **Claim 6** The method according to any one of claims 1 to 5, wherein the subject is diagnosed with or suspected of having uveitis, allergic conjunctivitis, blepharitis, chronic conjunctivitis, episcleritis, keratitis, retinitis, ocular cicatricial pemphigoid, mucous membrane pemphigoid, pterygoid episcleritis, Stevens-Johnson syndrome, Eales' disease, Behçet's disease, sarcoidosis, systemic lupus erythematosus, polyarteritis nodosa, Wegener's granulomatosis, Vogt-Koyanagi-Harada disease, sympathetic ophthalmia, or sarcoidosis. **Claim 7** The method according to claim 6, wherein the subject is diagnosed with or suspected of having uveitis. **Claim 8** A positive result for the expression or activity of at least one inflammasome activity marker in the eye of the subject that caused the inflammation, compared to the expression or activity of at least one inflammasome activity marker in the eye of an individual not diagnosed with or suffering from uveitis, allergic conjunctivitis, blepharitis, chronic conjunctivitis, episcleritis, keratitis, retinitis, ocular cicatricial pemphigoid, mucous membrane pemphigoid, pterygoid episcleritis, Stevens-Johnson syndrome, Eales' disease, Behçet's disease, sarcoidosis, systemic lupus erythematosus, polyarteritis nodosa, Wegener's granulomatosis, Vogt-Koyanagi-Harada disease, sympathetic ophthalmia, or sarcoidosis, is an increase in the expression or activity of at least one inflammasome activity marker, the method according to any one of claims 1 to 7. **Claim 9** A positive result for the expression or activity of at least one inflammasome activity marker in the eye of the subject that caused the inflammation, compared to the expression or activity of at least one inflammasome activity marker in the eye of an individual not diagnosed with or suffering from uveitis, is an increase in the expression or activity of at least one inflammasome activity marker, the method according to claim 8. **Claim 10** A positive result for the expression or activity of at least one inflammasome activity marker in the eye of the subject that caused the inflammation, as determined by a histological score, the method according to any one of claims 1 to 9. **Claim 11** A method of inhibiting the activation of an inflammasome in cells of a subject having inflammation, comprising: a. measuring an inflammasome activity marker in the subject; and b. when the inflammasome activity marker is positive, (i) 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 cells of the subject having inflammation; or (ii) a soluble CD59 protein administering to the subject a composition comprising wherein the composition inhibits the activation of the inflammasome. **Claim 12** The method according to claim 11, wherein the inflammasome activity marker is selected from caspase 1, caspase 5, IL-1β, IL-β17, IL-18, apoptosis-related speck-like 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+.

13. The method according to claim 11, wherein the inflammasome activity marker is selected from apoptosis-related speck-like protein (PYCARD / ASC) or NACHT, LRR and PYD domain-containing protein (NALP).

14. The method according to claim 13, wherein NALP is NACHT, LRR and PYD domain-containing protein 3 (NLRP3).

15. The method according to any one of claims 11 to 14, wherein the inflammasome activity marker is measured in the eye of the subject.

16. The method according to any one of claims 11 to 15, further comprising measuring an inflammasome activity marker 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 the expression and secretion of the membrane-independent CD59 protein.

17. The method according to any one of claims 11 to 16, wherein the subject is diagnosed with or suspected of having uveitis, allergic conjunctivitis, blepharitis, chronic conjunctivitis, episcleritis, keratitis, retinitis, ocular cicatricial pemphigoid, mucous membrane pemphigoid, pterygoid episcleritis, Stevens-Johnson syndrome, Eales' disease, Behçet's disease, sarcoidosis, systemic lupus erythematosus, polyarteritis nodosa, Wegener's granulomatosis, Vogt-Koyanagi-Harada disease, sympathetic ophthalmia, or sarcoidosis.

18. The method according to claim 17, wherein the subject is diagnosed with or suspected of having uveitis.

19. The method according to any one of claims 11 to 18, wherein the expression or activity of at least one inflammasome activity marker is increased as compared to the expression or activity of at least one inflammasome activity marker in a subject not diagnosed with or suffering from Alzheimer's disease, multiple sclerosis, myocardial infarction, atherosclerosis, microvascular disorder, thyroiditis, inflammatory bowel disease, organ transplant rejection, membranous nephritis, sympathetic ophthalmia, uveitis, or sarcoidosis.

20. The method according to claim 19, wherein the expression or activity of at least one inflammasome activity marker is increased as compared to the expression or activity of at least one inflammasome activity marker in a subject not diagnosed with uveitis.

21. The method according to any one of claims 11 to 20, wherein the positive inflammasome activity marker in the subject with inflammation is determined by a histological score.

22. 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 in cells of a subject with inflammation; or b. A soluble CD59 protein A method for inhibiting the activation of inflammasomes in cells of a subject with inflammation by administering to the subject a composition comprising: The composition inhibits the activation of inflammasomes, The subject shows a positive result for the expression or activity of at least one inflammasome activity marker in the subject with inflammation.

23. The method according to claim 22, wherein the inflammasome activity marker is selected from caspase 1, caspase 5, IL-1β, IL-β17, IL-18, apoptosis-related 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+.

24. The method according to claim 22, wherein the inflammasome activity marker is selected from apoptotic speck-like protein containing a caspase activation and recruitment domain (PYCARD / ASC), or a protein containing NACHT, LRR and PYD domains (NALP).

25. The method according to claim 24, wherein NALP is NACHT, LRR and PYD domain-containing protein 3 (NLRP3).

26. The method according to any one of claims 22 to 25, wherein the positive result regarding the expression or activity of at least one inflammasome activity marker in the subject with inflammation is in the eye of the subject with inflammation.

27. The method according to any one of claims 22 to 26, wherein the subject is diagnosed with or suspected of suffering from uveitis, allergic conjunctivitis, blepharitis, chronic conjunctivitis, episcleritis, keratitis, retinitis, ocular cicatricial pemphigoid, mucous membrane pemphigoid, pterygoid episcleritis, 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.

28. The method according to claim 27, wherein the subject is diagnosed with or suspected of suffering from uveitis.

29. The method according to any one of claims 22 to 28, wherein the positive result regarding the expression or activity of at least one inflammasome activity marker in the subject with inflammation is an increase in the expression or activity of at least one inflammasome activity marker as compared to the expression or activity of at least one inflammasome activity marker in a subject not diagnosed with or suffering from Alzheimer's disease, multiple sclerosis, myocardial infarction, atherosclerosis, microvascular disorder, thyroiditis, inflammatory bowel disease, organ transplant rejection, membranous nephritis, sympathetic ophthalmia, uveitis, or sarcoidosis.

30. The method according to claim 29, wherein the positive result regarding the expression or activity of at least one inflammasome activity marker in the subject with inflammation is an increase in the expression or activity of at least one inflammasome activity marker as compared to the expression or activity of an inflammasome activity marker in a subject not diagnosed with uveitis.

31. The method according to any one of claims 22 to 30, wherein a positive result for the expression or activity of at least one inflammasome activity marker in the subject with the inflammation is determined by a histological score.

32. 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 in cells of a subject with inflammation; or b. A soluble CD59 protein A method of inhibiting activation of an inflammasome in cells of a subject suffering from uveitis by administering to the subject a composition comprising the same, wherein the composition inhibits activation of the inflammasome.

33. The method according to claim 32, wherein the subject shows a positive result for the expression or activity of at least one inflammasome activity marker in at least one eye of the subject with the inflammation.

34. The method according to claim 33, wherein the inflammasome activity marker is selected from caspase 1, caspase 5, IL-1β, IL-β17, IL-18, apoptosis-associated speck-like 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+.

35. The method according to claim 33, wherein the inflammasome activity marker is selected from apoptosis-associated speck-like protein (PYCARD / ASC), or NACHT, LRR and PYD domain-containing protein (NALP).

36. The method according to claim 35, wherein NALP is NACHT, LRR and PYD domain-containing protein 3 (NLRP3).

37. The method according to any one of claims 33 to 36, wherein the subject is diagnosed with or suspected of suffering from uveitis, allergic conjunctivitis, blepharitis, chronic conjunctivitis, episcleritis, keratitis, retinitis, ocular cicatricial pemphigoid, mucous membrane pemphigoid, pterygoid episcleritis, 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. Claim 38 The method according to claim 37, wherein the subject is diagnosed with or suspected of having uveitis. Claim 39 A positive result for the expression or activity of at least one inflammasome activity marker in the subject with the inflammation, compared to the expression or activity of at least one inflammasome activity marker in a subject not diagnosed with or suffering from Alzheimer's disease, multiple sclerosis, myocardial infarction, atherosclerosis, microvascular disorders, thyroiditis, inflammatory bowel disease, organ transplant rejection, membranous nephritis, sympathetic ophthalmia, uveitis, or sarcoidosis, is an increase in the expression or activity of at least one inflammasome activity marker. The method according to any one of claims 33 to 38. Claim 40 A positive result for the expression or activity of at least one inflammasome activity marker in the subject with the inflammation, compared to the expression or activity of an inflammasome activity marker in a subject not diagnosed with uveitis, is an increase in the expression or activity of at least one inflammasome activity marker. The method according to claim 39. Claim 41 The method according to any one of claims 33 to 40, wherein a positive result for the expression or activity of at least one inflammasome activity marker in the subject with the inflammation is determined by a histological score. Claim 42 A method of inhibiting the activation of an inflammasome in a cell of a subject with inflammation, 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 the cell of the subject with inflammation, wherein the composition inhibits the activation of the inflammasome and the subject shows a positive result for the expression or activity of at least one inflammasome activity marker. Claim 43 The method according to claim 42, wherein the inflammasome activity marker is selected from caspase 1, caspase 5, IL-1β, IL-β17, IL-18, apoptosis-related speck-like 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+.

44. The method according to claim 42, wherein the inflammasome activity marker is selected from apoptosis-related speck-like protein (PYCARD / ASC) or NACHT, LRR and PYD domain-containing protein (NALP).

45. The method according to claim 44, wherein NALP is NACHT, LRR and PYD domain-containing protein 3 (NLRP3).

46. The method according to any one of claims 42 to 45, wherein the subject shows a positive result for the expression or activity of at least one inflammasome activity marker in the eye of the subject.

47. The method according to any one of claims 42 to 46, further comprising measuring an inflammasome activity marker in a subject with inflammation after administration of 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.

48. The method according to any one of claims 42 to 47, wherein the subject is diagnosed with or suspected of having uveitis, allergic conjunctivitis, blepharitis, chronic conjunctivitis, episcleritis, keratitis, retinitis, ocular cicatricial pemphigoid, mucous membrane pemphigoid, pterygium episcleritis, 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.

49. The method according to claim 48, wherein the subject is diagnosed with or suspected of having uveitis.

50. A positive result for the expression or activity of at least one inflammasome activity marker in the subject with the inflammation, compared to the expression or activity of at least one inflammasome activity marker in a subject not diagnosed with or suffering from uveitis, allergic conjunctivitis, blepharitis, chronic conjunctivitis, episcleritis, keratitis, retinitis, ocular cicatricial pemphigoid, mucous membrane pemphigoid, pterygoid episcleritis, Stevens-Johnson syndrome, Eales' disease, Behçet's disease, sarcoidosis, systemic lupus erythematosus, polyarteritis nodosa, Wegener's granulomatosis, Vogt-Koyanagi-Harada disease, sympathetic ophthalmia, or sarcoidosis, is an increase in the expression or activity of at least one inflammasome activity marker, the method according to any one of claims 42 to 49.

51. A positive result for the expression or activity of at least one inflammasome activity marker in the cells of the subject with the inflammation, compared to the expression or activity of at least one inflammasome activity marker in a subject not diagnosed with or suffering from uveitis, is an increase in the expression or activity of at least one inflammasome activity marker, the method according to claim 50.

52. A positive result for the expression or activity of at least one inflammasome activity marker in the subject with the inflammation is determined by a histological score, the method according to any one of claims 42 to 51.

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