Therapy for ocular neovascularization

Abstract

Provided are molecules and compositions for eye-targeted gene therapy and methods of using the same. The eye-targeted gene therapy alleviates ischemic retinopathy by reducing pathologic neovascularization, gliosis and neuronal apoptosis.

Description

[0001] THERAPY FOR OCULAR NEOVASCULARIZATION RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. § 119(e) of U.S. provisional 5 application number 63 / 659,633, filed June 13, 2024, the contents of which is incorporated by reference herein in their entirety. FEDERALLY SPONSORED RESEARCH This invention was made with government support under grant numbers 10 HL021796 and DK063275, awarded by the National Institutes of Health. The government has certain rights in the invention. REFERENCE TO AN ELECTRONIC SEQUENCE LISTING The contents of the electronic sequence listing (B066270109WO00-SEQ- 15 EAS.xml; Size: 54,864 bytes; and Date of Creation: June 10, 2025) is herein incorporated by reference in its entirety. BACKGROUND Proliferative retinopathies (PR), such as retinopathy of prematurity (ROP) and 20 diabetic proliferative retinopathy (DPR), are the leading causes of blindness worldwide. Despite differences in their etiologies, these conditions share common features including the loss of retinal endothelial cells (EC) and reactive retinal gliosis triggered by hypoxia, leading to the release of pro-inflammatory cytokines and angiogenic factors, primarily VEGF-A, referred to hereafter as VEGF. These factors promote the development of 25 aberrant compensatory retinal neovascularization consisting of immature and leaky vessels, causing hemorrhages and retinal scaring that worsen retinal ischemia and culminate in photoreceptor and neuronal death. Since their introduction in 2002 for treating wet AMD, five intravitreal anti-VEGF therapies have received FDA approval for AMD, PDR, diabetic macular edema (DME), 30 retinal vein occlusion, and ROP. Despite their revolutionary impact, benefits of these therapies are impeded by ocular and systemic side effects, as well as significant primary and secondary resistance. Several trials reported that 15% to 30% of neovascular AMD and DME patients experience primary non-response, with over 60% developing secondary resistance. Resistance to anti-VEGF therapies underscores the complex pathophysiology

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[0003] of PR, including the roles of inflammation and angiogenic molecules other than VEGF. Various therapies targeting these additional culprits through combined inhibition of VEGF-A and angiopietin-2 (Faricimab) and / or the use of anti-inflammatory steroids were only marginally successful due to suboptimal benefits and side effects. Thus, there 5 remains a critical need for the development of effective and safer alternatives informed by pathophysiology. SUMMARY The limited success of anti-VEGF therapies in treating ischemic proliferative 10 retinopathies (PR) underscores the complex interplay between the various pathogenic contributors to this disease. Mechanistic studies have emphasized the importance of aberrant / pathologic angiogenesis, reactive inflammatory gliosis, and heightened neuronal apoptosis as key determinants of proliferative retinopathy disease outcomes. Thus, there a critical need for the development of therapeutic strategies capable of targeting all three 15 components. As exemplified in the Examples and described throughout the disclosure, gain and loss-of-function studies of A20 / TNFAIP3, a ubiquitous inhibitor of NF-kB with established cytoprotective functions in endothelial and neuronal cells, were used to uncover a novel function for A20 as a key physiologic regulator of angiogenesis in human retinal endothelial cells (HREC). The technology disclosed herein, in vivo, intravitreal 20 delivery of A20 gene therapy, surprisingly yielded transgene expression throughout retinal layers, significantly reduced central vaso-obliteration and pathologic peripheral neovascular tuft formation in a mouse model of oxygen-induced PR. In contrast, mice with total or partial A20 knockdown exhibited worse lesions. Remarkably, A20-related benefits were associated with a significant reduction in inflammatory gliosis, Müller cell 25 activation, and apoptotic cell death rates across the retina, including neuronal cells. Conversely, A20 knockdown aggravated all these aspects. The A20 gene therapy technology described herein provides an innovative approach of concurrently reducing pathologic neovascularization, reactive gliosis, and neuronal apoptosis in the retina, and emerges as a unique therapeutic target with promising translational potential. 30 Aspects of the technology relate to a nucleic acid comprising a polynucleotide encoding a Tumor Necrosis Factor Alpha Inducible Protein-3 (TNFAIP-3) and an inverted terminal repeat (ITR) as described in FIG.10. In some embodiments, the TNFAIP-3 is human TNFAIP-3. In some embodiments, the ITR is an adeno-associated virus 2 (AAV2) ITR.

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[0005] In some embodiments, the nucleic acid further comprises a promoter. In some embodiments, the promoter comprises a CMV enhancer and a chicken β-actin promoter sequence. In some embodiments, the nucleic acid further comprises a polyadenylation (poly 5 A) sequence. In some embodiments, the poly A is a human growth hormone poly A. Further aspects relate to a vector comprising a nucleic acid of the technology. In some embodiments, the vector is selected from the group consisting of a plasmid and a recombinant viral genome. In some embodiments, the recombinant viral genome is selected from the group consisting of a recombinant AAV genome and / a recombinant 10 adenoviral genome. Further aspects relate to a viral particle comprising a nucleic acid of the technology or a vector of the technology. In some embodiments, the viral particle further comprises a viral capsid protein. In some embodiments, the viral capsid protein is selected from an AAV capsid protein and an adenoviral capsid protein. In some embodiments, the AAV 15 capsid protein is selected from an AAV1, AAV2, AAV4, AAV5, AAV7, AAV8, AAV9, AAV10 and AAV5 capsid protein. Further aspects relate to a composition comprising a nucleic acid of the technology, a vector of the technology, or a viral particle of the technology. In some embodiments, the composition further comprises a pharmaceutically acceptable excipient. 20 Further aspects relate to a method of treating a retinopathy in a subject in need thereof, the method comprising administering to the subject a nucleic acid of the technology, a vector of the technology, a viral particle of the technology, or a composition of the technology in an amount that is effective in treating the retinopathy, wherein the treating comprises prophylactic treatment and therapeutic treatment. In some 25 embodiments, the retinopathy is ischemic, inflammatory, or proliferative retinopathy. In some embodiments, the retinopathy is diabetic retinopathy, macular edema, age-related macular degeneration, or retinopathy of prematurity. In some embodiments, the administering is by local administration. In some embodiments, the local administration is by intraocular, intravitreal, or subretinal injection. 30 BRIEF DESCRIPTION OF THE FIGURES The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure, which can be better

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[0007] understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. FIGs.1A-1C show A20 overexpression in HREC reduces tube formation and migration. FIG.1A shows HREC were transduced with rAd.A20 or rAd.βgal at 100 MOI, 5 then cultured in reduced (rGF) or full growth factor (fGF) Matrigel for 48 hours. Tube-like structures quantified by the "Angiogenesis Analyzer" software showed that total MA and total MS were significantly reduced in rAd.A20 compared to rAd.βgal-transduced HREC in both rGF and fGF. Results are expressed as mean ± SEM of 3-5 independent experiments performed in triplicate, *p<0.05, **p<0.01. Representative phase-contrast10 micrographs are shown (40X magnification). FIG.1B shows the scratch assay in non- transduced (NT) and rAd.A20 or rAd.βgal-transduced HREC (MOI 100). The percentage of residual acellular area 9h post-wounding was significantly larger in rAd.A20 vs. NT and rAd.βgal-transduced HREC. Results are expressed as mean ± SEM of 3 independent experiments, *p<0.05, **p<0.01. Representative Images are shown (40X). FIG.1C shows 15 WB analysis of VEGF-R2 showed significantly higher expression in rAd.A20 compared to NT and rAd.βgal-transduced HREC. Membranes were probed with anti-βactin and anti- A20 antibodies to correct for loading and confirm transduction. Corrected densitometry, calculated as fold induction of NT HREC, represents mean ± SEM of 3 experiments, **p<0.01. 20 FIGs.2A-2C show A20 knockdown in HREC increases branch length and node numbers in the tube formation assay and promotes vascular sprouting of A20-deficient mouse aortae in the aortic ring assay. FIG.2A shows that HREC was transduced with rAd.shA20 or rAd.shScr at a MOI of 100 for 30h, then plated for 48 hours in reduced (rGF) or full (fGF) growth factor Matrigel. Tube-like structures were imaged and analyzed 25 using the "Angiogenesis Analyzer" software. A20 knockdown In HREC significantly increased total branch length under both rGF and fGF conditions, with a trend towards higher node numbers in A20-deficient HREC cultured in rGF medium (p=0.06). Results are expressed as mean ± SEM of 4-5 independent experiments performed in triplicate, *p<0.05. FIG.2B shows the percentage of residual acellular area 9 hours post-wounding, 30 analyzed by the TScratch software, was not affected in HREC transfected with A20.siRNA, compared to non-transfected (NT) and AllStars.siRNA-transfected controls. Results are expressed as mean ± SEM of 5 independent experiments. FIG.2C shows vessel sprouting areas (mm2) in the aortic ring assay were significantly greater in A20 knockout (KO) vs. wild-type (WT) and heterozygous (Het) aortic rings, with Het rings

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[0009] showing a trend towards larger sprouting areas than WT. Results are expressed as mean ± SEM of 3-4 aortae / group, performed in 6 replicates. ***p<0.001. Representative images in A and B are shown at 40X magnification. FIGs.3A-3C show gain and loss-of-function studies in the mouse model of OIR 5 uncover a role for A20 in protecting from ischemic retinopathy. FIG.3A shows temporal fluorescein-angiography images of P17 mouse pup retinae injected intravitreally at P12 with saline, rAd.A20 or rAd.βgal (1x108MOI / eye). Results indicate that the percentage of central avascular area over total retinal area per field, quantified by image J, is significantly smaller in rAd.A20 compared to saline and rAd.βgal-treated controls. Results 10 are expressed as mean ± SEM of 3-4 mice / group. FIGs.3B-3C show representative photomicrographs of flat-mounted FITC-stained whole retinae of P17 left pup eyes treated at P12 with intravitreal injection of saline, rAd.A20 or rAd.βgal, and their respective contralateral eyes; and of P17 WT, A20 Het and A20 KO pups’ retinae. Areas of retinal vaso-obliteration (VO) and neovascular tufts (NV) are presented as mean ± SEM of15 percent VO and NV areas over total retinal area (n=3-4 mice / group). In FIGs.3A-3C, age- matched littermates raised in normoxia served as controls. Statistical significance: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 for comparison between hyperoxia treatment groups, and #p<0.05, ##p<0.01, ###p<0.001, ####p<0.0001 for comparison with normoxia controls. Images in B and C are shown at 100X magnification. 20 Representative VO areas are delineated by dotted lines and NV tufts are marked. FIGs.4A-4C shows intravitreal A20 gene therapy prevents pathologic neovascularization in the OIR mouse model. FIG.4A shows ocular A20 therapy significantly decreases the number of CD31+vascular structures in the GCL, as gauged by IHC, and normalizes vessel morphology compared to saline and rAd.βgal controls and 25 non-treated contralateral eyes. Results represent mean ± SEM of 4-5 mice / group. *p<0.05, **p<0.01. Paired representative images are shown (200X magnification, 20 µm scale bar). FIG.4B shows expression of the A20 (cytoplasmic) and βgal (nuclear) transgenes in P17 mouse pups’ retinae, verified by IHC with methyl green counterstain, demonstrate mosaic but robust expression of A20 and βgal (black arrows) across all retinal layers. 30 Representative 400X magnification images are shown, 50 µm scale bar (n=4-5 mice / group). Boxed areas were amplified for clarity. Annotated retinal layers: NFL / GCL=Nerve fiber layer / ganglion cell layer, IPL=inner plexiform layer, INL=inner nuclear layer, OPL=outer plexiform layer, ONL=outer nuclear layer. FIG.4C shows robust mRNA levels of the human A20 (hA20) and LacZ transgenes were detected in

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[0011] rAd.A20 and rAd.βgal-treated, but not untreated (ND=not detected), P17 mouse pups’ retinae, respectively. Results are reported as 1 / ΔCTx102. Mouse 28S was used as housekeeping gene. FIGs.5A-5C show intravitreal A20 gene therapy mitigates angiogenic signaling in 5 the OIR mouse model downstream of VEGF-R2. FIG.5A shows rAd.A20 and rAd.βgal- treated P17 retinae exhibit comparable increase in VEGF and VEGF-R2 (IHC) compared to retinae from age-matched pups kept in normoxia. Results are expressed as mean ± SEM of integrated (integ) density x106AU (arbitrary units) / section, n= 3-6 / group; *p<0.05. FIG.5B shows IHC analysis of proliferating Ki67+nuclei and P-PKCβII staining density10 show a significantly lower number of Ki67+nuclei (mostly in the GCL) and lower P- PKCβII staining in rAd.A20 vs. rAd.βgal-treated P17 retinae. Results are presented as mean±SEM of Ki67+nuclei / HPF (n=4-6 / group,****p<0.0001) and as mean±SEM of Integ P-PKCβII staining density x 106AU / section (n=4-5 / group, **p<0.01). Representative images are displayed in A&B (200X magnification, 20 µm scale bar). 15 Annotated retinal layers: NFL / GCL= Nerve fiber layer / ganglion cell layer, IPL=inner plexiform layer, INL=inner nuclear layer, ONL=outer nuclear layer. FIG.5C shows the Western blot analysis of phosphorylated-ERK1 / 2 in P17 mouse pup retinae showed significantly lower P-ERK1 / 2 levels in rAd.A20 vs. saline and rAd.βgal-treated retinae. Results are presented as mean±SEM of P-ERK1 / 2 band density corrected by βactin and 20 expressed as fold of controls (n=3 / group, *p<0.05). FIGs.6A-6C show A20 deficiency aggravates pathologic angiogenesis in the OIR mouse model by promoting VEGF signaling. FIG.6A shows CD31 immunostaining of P17 mouse retinae show higher CD31+staining (GCL, INL), albeit not significant, in A20 KO and Het compared to WT pups, with all groups showing significantly higher CD31+25 staining compared to age-matched normoxia controls (#p<0.05 and ##p<0.01, n=4-11). Results are expressed as mean±SEM of pixels / HPF. There was also a trend, towards higher VEGF-R2 staining in P17 retinae of WT, Het, and KO mice compared to normoxia controls, albeit it was only significant for KO (n= 3-11 / group, #p<0.05). Results are reported as mean±SEM of Integ. Density x 106AU per retinal section. FIG.6B shows IHC 30 analysis of proliferating Ki67+nuclei and P-PKCβII shows a significantly higher number of Ki67+nuclei in retinae of P17 KO and Het compared to wild-type WT pups (n=4-8, **p<0.01, ***p<0.001 respectively). Results are reported as mean ± SEM of Ki67+nuclei / HPF. There was also significantly higher expression of P-PKCβII in retinae of P17 KO vs. WT mice, (n=3 and 7, *p<0.05). Staining in Het retinae faired in between. Results

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[0013] are reported as mean±SEM of Integ. Density x 106AU / section. #p<0.05, ##p<0.01 and ### p<0.001 indicate comparisons with normoxia controls (n= 4-6). Representative images in A&B (200X magnification, scale bar 20 µm) are shown. Annotated retinal layers: NFL / GCL= Nerve fiber layer / ganglion cell layer, IPL=inner plexiform layer, 5 INL=inner nuclear layer, OPL=outer plexiform layer, ONL=outer nuclear layer. FIG.6C shows the Western blot analysis of phosphorylated-ERK1 / 2 shows significantly higher levels in retinae of P17 A20 KO and Het vs. WT mice (n=2-4 / group, **p<0.01). Results are presented as mean±SEM of P-ERK1 / 2 band density corrected by βactin and expressed as fold of WT. 10 FIGs.7A-7C show A20 regulates reactive retinal gliosis in the ischemic retina. FIG.7A shows a significant increase in Iba-1+cell number (black arrows) was detected across all retinal layers of saline and rAd.βgal but not rAd.A20-treated P17 retinae compared to normoxia controls (n=3-6 / group). FIG.7B shows Co-IF staining with GFAP (red) and Vimentin (green), markers of Müller cell activation and fragmentation (white 15 arrows), was substantially increased in the IPL of saline and rAd.βgal-treated compared to rAd.A20-treated P17 retinae, which displayed reduced GFAP staining and no fragmentation. FIG.7C shows loss-of-function demonstrated an inverse correlation between A20 loss and gliosis severity, with P17 A20 KO retinae exhibiting significantly higher numbers of Iba-1+cells compared to WT (n=3 and 6). A20 Het retinae showed 20 intermediate numbers (n=5). Representative images are provided at 200X magnification (scale bar 20 µm). Results in A and C are expressed as mean±SEM of Iba-1+cells / mm2.*p<0.05, **p<0.01. Annotated retinal layers: NFL / GCL= Nerve fiber layer / ganglion cell layer, IPL=inner plexiform layer, INL=inner nuclear layer, OPL=outer plexiform layer, ONL=outer nuclear layer. 25 FIGs.8A-8B show intravitreal A20 gene therapy protects retinal cells, including neurons, from ischemia-induced apoptosis. FIG.8A shows TUNEL staining of P17 retinal sections shows a significantly lower number of TUNEL+nuclei / HPF in rAd.A20 vs. rAd.βgal-treated mice (n=6-7, black arrows). Fluoro-Jade C (FJC) IF, marking neuronal apoptosis (white arrows), is also significantly reduced in rAd.A20 vs. rAd.βgal-treated 30 retinae, including at the level of the optic nerve (n=6). FIG.8B shows IHC staining for Ser-473 phosphorylated-AKT (P-AKT) shows significantly higher P-AKT staining in P17 rAd.A20 compared to rAd.βgal-treated retinae, predominantly in the GCL and INL (black arrows) layers (n=6-7). Age-matched control mice kept in normoxia served as controls (n=4-5). Representative photomicrographs are provided at a magnification of 200X (scale

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[0015] bar 20 µm). Results are reported as mean ± SEM of number of TUNEL+nuclei / HPF and of Integ. Density x 106AU (arbitrary unit) per retinal section (FJC and P-AKT).*p<0.05, **p<0.01. Annotated retinal layers: NFL / GCL=Nerve fiber layer / ganglion cell layer, IPL=inner plexiform layer, INL=inner nuclear layer, OPL=outer plexiform layer, 5 ONL=outer nuclear layer. FIGs.9A-9B show A20 deficiency aggravates neuronal cell death in the OIR mouse model. FIG.9A shows the number of TUNEL+apoptotic nuclei / HPF, predominating in the INL and ONL (black arrows) of P17 mice subjected to hyperoxia was significantly higher than in age-matched WT mice kept in normoxia, regardless of 10 genotype: WT, Het or KO (n=4-10). #p<0.05 and ##p<0.01 vs. controls. However, neuronal death, gauged by FJC IF staining, was significantly higher in P17 retinal sections of A20 KO mice compared to Het and WT mice (n=3-9), including at the level of the optic nerve. Dotted lines delineate areas analyzed on retinal sections. FIG.9B shows there was significantly lower P-AKT staining in the retina of P17 A20 KO compared to WT mice 15 (black arrows), with A20 Het mice showing intermediate expression (n=3-10). Representative photomicrographs are shown in A&B at a magnification of 200X (scale bar 20 µm). Results are reported as mean ± SEM of number of TUNEL+nuclei / HPF and of Integ. Density x 106AU (arbitrary unit) per retinal section (FJC, P-AKT). *p<0.05, **p<0.01. Annotated retinal layers: NFL / GCL=Nerve fiber layer / ganglion cell layer, 20 IPL=inner plexiform layer, INL=inner nuclear layer, OPL=outer plexiform layer, ONL=outer nuclear layer. FIG.10 shows the sequence of pAAV-CAG2-hTNFAIP3-hGHpA-KanR.dna (SEQ ID NO: 22). Various features of the sequence are annotated below the nucleotide sequence: AAV2 IVR fragments, CAG2 promoter, CMV enhancer, chicken β-actin 25 promoter, TNFAIP-3, hGH poly(A) signal. FIG.11 shows the plasmid map of the pAAV-CAG2-hTNFAIP3-hGHpA- KanR.dna. FIG.12 shows a schematic of A20 / TNFAIP3 gene therapy on ischemic proliferative retinopathy. Inhibition of gliosis, neuronal damage, and pathologic 30 angiogenesis is depicted. FIGs.13A-13D show tA20 overexpression increases VEGF mRNA and VEGF-R2 protein levels in HREC subjected to hypoxia. FIGs.13A-13B show relative mRNA levels of VEGF and VEGF-R2, and FIG.13C shows protein levels of VEGF-R2 were assessed by qPCR and WB analysis, respectively, in non-transduced (NT), rAd.A20, and rAd.βgal-

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[0017] transduced HREC (at 100 MOI / cell) before and 6 hours post-hypoxia induction. VEGF and VEGF-R2 mRNA levels were normalized to the housekeeping gene Cyclophilin A (CyA) and expressed as fold (RQ) change relative to NT HREC cultured under normoxia. The findings demonstrate significantly elevated VEGF mRNA levels 6 hours post-hypoxia 5 in all groups compared to their respective baseline (#p<0.05), while VEGF-R2 mRNA levels remained comparable across groups before and after hypoxia. Notably, VEGF-R2 protein levels, assessed by WB using a rabbit anti-VEGF-R2 antibody from Cell Signaling (Beverly, MA), were substantially higher in rAd.A20-transduced compared to NT and rAd.βgal-transduced HREC both before and 6 hours post-hypoxia. Results are presented 10 as mean±SEM of 3 (VEGF) and 4 (VEGF-R2) independent experiments. FIG.13D shows NT, rAd.A20, and rAd.βgal-transduced HREC were pre-cultured for 2 hours in serum-free and growth factor-free medium prior to the addition of 50 ng / ml of VEGF(R&D SYSTEMS®). Cell lysates were collected 2- and 5-minutes later and assessed by WB for total and P-VEGF-R2 expression, using anti-phospho VEGF-R2 Tyr-1175 antibody (P- 15 VEGFR2) from Cell signaling. The results indicate similar levels of P-VEGFR2 in NT, rAd.A20, or rAd.βgal-transduced HREC. Immunoblotting with an anti-βactin antibody (SANTA CRUZ BIOTECHNOLOGY®) served a loading control. Representative blots are shown from 2-3 experiments. HREC passages 6-8 were used in all experiments following 48 hours transduction. 20 FIGs.14A-14B show A20 knockdown in HREC. FIG.14A shows transduction of HREC with GFP-tagged rAd.shA20 (Ad-GFP-U6-h-TNFAIP3-shRNA) from Vector Biolabs at a MOI of 100 resulted in >95% transduction efficiency at 48 hours, as visualized by GFP fluorescence, and led to a significant reduction in A20 protein levels. A20 knockdown was evaluated by WB analysis using a monoclonal rabbit anti-human 25 A20 antibody (Abcam). HREC transduced with scrambled shRNA Ad-GFP-U6-shRNA (rAd.shScr) served as control. FIG.14B shows HREC transfected with 40 nM of A20 siRNA (Hs_TNFAIP3_1 FlexiTube) using Hiperfect (Qiagen) exhibited a significant 65- 75% reduction in A20 protein levels, as assessed by WB. HREC transfected with AllStars fluorescent siRNA served as control. Representative WB shown are shown from 4 (FIG. 30 14A) and 6 (FIG.14B) experiments. Immunoblotting for GAPDH (EMD Chemicals) was performed to normalize loading. WB bands were quantified using the IMAGE STUDIOTMSoftware (LI-COR). Results are presented as mean ± SEM of A20 / GAPDH corrected density and expressed as a fold change relative to control. *p<0.05, **p<0.01.

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[0019] FIG.15 shows the immunohistochemistry analysis of A20 expression in P17 mouse retinae of A20 wild-type, heterozygous, and knockout mice. Expression of the A20 (cytoplasmic) transgene in P17 mouse pups’ retinae was assessed by IHC using rabbit anti-human A20 (Abcam). Nuclei were counterstained with methyl green. Mosaic 5 cytoplasmic A20 expression was observed across retinal layers (upper panel) and at the level of the optic nerve (lower panel) in wild-type (WT) and Het (heterozygous), as indicated by black arrows, while no expression was detected in knockout (KO) mice. Representative images of 3-4 mice / group are depicted at 400X magnification (50µm scale bar). Annotated retinal layers: NFL / GCL = Nerve fiber layer / ganglion cell layer, IPL = 10 Inner plexiform layer, INL = Inner nuclear layer, OPL = Outer plexiform layer, ONL = Outer nuclear layer. FIG.16 shows elevated D-Glucose concentrations blunt TNF-mediated upregulation of A20 in HREC cultures. HREC, cultured until confluence in media supplemented with glucose concentrations ranging from physiological (5mM / L) to supra- 15 physiological (15mM and 30 mM / L), were treated with 100U / ml of recombinant human TNF (R&D). Total protein lysates were collected before and 4 hours after TNF treatment and analyzed by WB for A20 expression, using a rabbit anti-human A20 antibody (Abcam). Baseline A20 expression levels were similar in all culture conditions. However, TNF-mediated upregulation of A20 protein levels was reduced by more than 30% when 20 HREC were cultured in 15mM / L and 30mM / L of D-glucose, as quantified by Image J- based densitometry of A20 / βactin bands and reported as fold change relative to non- treated HREC cultured in 5mM / L D-glucose. Immunoblotting for βactin (SANTA CRUZ BIOTECHNOLOGY®) was used to correct for loading. WB shown is representative of 2 independent experiments. 25 FIGs.17A-17D show A20 overexpression in retinal pigmented epithelial cells (RPEC) inhibits TNF-induced NF-kB activation and protects against oxidative-stress- induced cell death. ARPE-19, a human retinal pigment epithelial cell line CRL-2302™ (RPEC) derived from a healthy individual was purchased from ATCC® (Manassas, VA) and cultured according to the manufacturer’s recommendations. ARPE cells were30 transduced at 70-80% confluency with rAd.A20 or rAd.βgal at a MOI of 100. Non- transduced cells served as controls (NT Ctrl). Forty-eight hours following transduction, cells were treated with 200U / mL of recombinant human TNF (R&D SYSTEMS®) and: (FIG.17A) cell lysates were recovered 15 minutes later and subjected to WB analysis to assess IkBα expression, using a rabbit anti-human IkBα antibody (SANTA CRUZ

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[0021] BIOTECHNOLOGY®). Membranes were also probed with rabbit anti-human A20 monoclonal antibody (Abcam), rabbit anti-βgalactosidase polyclonal antibody (NB600- 305, Novus Biological), and mouse anti-human βactin antibody (SANTA CRUZ BIOTECHNOLOGY®) to confirm transgene expression and control for loading, 5 respectively. HRP-conjugated secondary antibodies were used (Thermo Scientific). Results show that TNF-induced degradation of IkBα, indicating NF-kB activation, was prevented in rAd.A20 compared to NT and rAd.βgal-transduced ARPE cells, (FIG.17B) RNA was extracted 1 hour, 3 hours, and 6 hours later (RNAse spin columns, Qiagen), and cDNA was synthesized (iScript cDNA synthesis kit, Biorad). Real-time quantitative PCR 10 (qPCR) was performed using iTaq Fast SYBR Green Supermix with ROX (BIO-RAD®) and ABI 7500 Fast Real-time PCR System (Applied Biosystems) to analyze mRNA levels of the NF-kB target genes Vascular cell adhesion molecule 1 (VCAM-1) and Intercellular adhesion molecule 1 (ICAM-1) using specific primers and the comparative threshold cycle (Ct) method. VCAM-1 and ICAM-1 mRNA levels were normalized to the housekeeping 15 gene 28S. Results indicate that TNF-induced up-regulation of VCAM-1 and ICAM-1 mRNA was totally blunted in A20-overexpressing ARPE cells, contrasting with a substantial increase in NT and rAd.βgal-transduced cells. Peak mRNA levels were detected 3 hours (ICAM-1) and 6 hours (VCAM-1) after TNF treatment. FIG.17C shows NT, rAd.A20, and rAd.βgal-transduced ARPE cells were exposed to 250 µM or 500 µM 20 H2O2in Hanks balanced solution salt (HBSS). H2O2-induced cytotoxicity was assessed 16 hours and 24 hours later using either the 3-(4,5-Dimethylthiazol-2-yl)-2,5- Diphenyltetrazolium Bromide (MTT) done in 8 replicates, or the lactic dehydrogenase (LDH) CYTOTOX 96C® assays (n=2), both from PROMEGA®. Overexpression of A20 in ARPE cells mitigated H2O2-mediated cytotoxicity in both assays compared to control 25 NT and rAd.βgal-transduced cells. *p<0.05, ***p<0.001. FIG.17D shows transduction efficiency in ARPE cells was evaluated by eosin and Xgal staining. Representative photomicrographs of ARPE cell cultures transduced for 48 hours with rAd.A20 or rAd.βgal at 100 MOI demonstrate robust transgene expression in >95% of cells, evidenced by eosin-positive red cytoplasmic punctuations corresponding to A20 overexpression in30 the lysosomes (black arrows), and blue nuclear staining marking expression of nuclear β- galactosidase. NT cells served as staining controls. DETAILED DESCRIPTION Proliferative retinopathies (PR), such as retinopathy of prematurity (ROP) and

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[0023] diabetic proliferative retinopathy (DPR), are the leading causes of blindness worldwide. Despite differences in their etiologies, these conditions share common features including the loss of retinal endothelial cells (EC) and reactive retinal gliosis triggered by hypoxia, leading to the release of pro-inflammatory cytokines and angiogenic factors, primarily 5 VEGF-A, referred to hereafter as VEGF. These factors promote the development of aberrant compensatory retinal neovascularization by way immature and leaky vessels, causing hemorrhages and retinal scaring that worsen retinal ischemia and culminate in photoreceptor and neuronal death. VEGF, primarily produced by glial cells and retinal EC in response to hypoxia, 10 binds to VEGFR-2 / Fetal liver kinase-1 (VEGFR-2 / Flk-1), leading to downstream activation of extracellular signal-regulated kinase (ERK1 / 2) and PKC, mainly the βII isoform, respectively promoting EC proliferation and increased permeability. Strategies to treat PR by inhibiting PKCβ or directly blocking VEGF were developed. While the PKCβ inhibitor Ruboxistaurin failed to prevent pathologic angiogenesis and was discontinued,15 anti-VEGF therapies fared better yet had limitations due to primary or secondary non- response and side effects. Refractoriness to anti-VEGF may be in part imputed to the compensatory effect of other angiogenic contributors to PR like FGF. Concerns about prolonged VEGF blockade stem from its protective functions as a key survival factor for EC and neurons through an AKT-dependent mechanism. VEGF also supports the function 20 and integrity of retinal glial cells, including astrocytes, Müller cells, and microglia. The latter highlights the current appreciation that reactive gliosis and associated production of cytokines and chemokines are central pathogenic determinants of PR and their severity. Experimental data showing that blockade / modulation of pro-inflammatory TNF signaling or reduction of microglia / Müller cell activation significantly protects from PR in the 25 oxygen-induced retinopathy (OIR) mouse model underscore the need to redirect therapeutic targets beyond VEGF blockade. It was surmised that therapies comprehensively targeting the spiraling interplay between ischemic and inflammatory insults, reducing pathologic angiogenesis, and protecting neuronal death in retinae, would be ideal to address this unmet clinical need. 30 Herein, it was investigated whether “A20”, referred to interchangeably herein as Tumor Necrosis Factor Alpha Inducible Protein-3 (TNFAIP3), a protein that has been identified as a potent and ubiquitous inhibitor of NF-kB activation, can positively impact PR development and severity. The choice of A20 was informed by its effects, albeit in other vascular beds and anatomical contexts. Indeed, A20 blocks NF-κB activation in EC

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[0025] in response to inflammatory stimuli relevant to PR, such as TNF, oxidative stress, and PKC activation. A20 also reduces microglial activation in the brain and exerts potent anti- apoptotic functions in EC and neuronal cells. However, concerns about its potential pro- angiogenic effect need prior resolution. 5 The gain and loss-of-function studies described herein, using primary human retinal EC and the OIR mouse model of ROP, uncover a novel anti-angiogenic or, more accurately, angiogenic modulatory function for A20. Surprisingly, intravitreal A20 gene therapy protects against OIR by reducing pathologic retinal neovascularization, reactive gliosis, and EC / neuronal rate of apoptosis. Conversely, lesions of OIR were significantly 10 aggravated in mice with partial (Heterozygous / Het) or total (knockout / KO) loss of A20 (34), highlighting A20’s pivotal role as a physiologic modulator of the severity of ischemic PR and validating its promise as a therapy. Aspects of the disclosure provide, in part, a novel gene therapy comprising a nucleic acid comprising a polynucleotide encoding A20 (e.g., Tumor Necrosis Factor 15 Alpha Inducible Protein-3 (TNFAIP-3)). In some embodiments, a novel gene therapy comprising a nucleic acid comprising a polynucleotide encoding A20 and an inverted terminal repeat (see FIG.10). Tumor Necrosis Factor Alpha Inducible Protein-3 20 Tumor Necrosis Factor Alpha Inducible Protein-3 (TNFAIP3), referred to interchangeably herein as “A20”, is a protein that has been identified as a potent and ubiquitous inhibitor of NF-kB activation. In some embodiments, the technology relates to a nucleic acid comprising a polynucleotide encoding a Tumor Necrosis Factor Alpha Inducible Protein-3 (TNFAIP-3). In some embodiments, TNFAIP-3 is a human TNFAIP- 25 3. In some embodiments, the TNFAIP-3 comprises a polynucleotide sequence of SEQ ID NO: 19 or a polynucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to the amino acid sequence of SEQ ID NO: 19. 30 Exemplary TNFAIP-3 sequence: AGTATTATCAGCCATCAGCACCCTGGGGTGAACTGATTATCAGGACAACTAAA AACACACCTAATAAAGCCTTGGAGACACATGGCAAGCACCGTGATGGACTAG AAGCGATTTCTAAGCTGAAATGCTGCTTTGAATGCAATGTCAGCTGGATGCTT GGTGAGCAGAAATAGACAAGGGGAAATGGAGCAAGCGCGGCATAGGGGTGA

[0026] 13 / 57

[0027] GTGTTGTTCTGATTCCTAAGTTTACCTAAGACTGGGGCCACGGGGTCGGGTGT CCCCTGGGACCTACGTCTCATGCAGCCTGGGGTCCAGAGCTGGCAGGAACAAT GGAGATGGGGACCAAAGGCTGTCACCGCTGCTTTTTCTCGAAATGCCCGCCCG GGTCCTGTCTGGGGCTCCCGCCTACGCGCTCATCACGTGCACAGCCCAAACTT 5 TTCAGAGCCGGCCCGAGGCCTGCGGGCCCGGGCGCCAGGGTGGTTTTTAGGGT TTTCTTTCTTTCTTATTTCCCTTCTTCTTCTCCACAGTTGATGATTTTCTGCAGA AAAACAACTGCGAAAAAGAACCTATTTCATTTCCAGTTCCCATCACCGCGATT TCCACATGGATGTGACGTGACCCCAGCTTCCGAAATGCCCAGGTGACTCACGC GGGGACACCCCGGGGCGGGGCGAGGGAGTTTCTCCGGGCGCCTGCAGGGACC 10 GGGCGGGGCGGGGCAGCGGGGCGGGGCAGGGAAAGGGGGCGGGGCGGGGCC CGCAGGCCCGGTCGGGCGGAGGCCGCGCGCGCCCCTCGCCCCCTGCGCCCTCT GGCGGCCGGCTGGACGCACTTCGCAGCCCGACCCAGAGAGTCACGTGACTTTG GAAAGTCCCGTGGAAATCCCCGGGCCTACAACCCGCATACAACTGAAACGGG GCAAAGCAGACTGCGCAGTCTGCAGTCTTCGTGGCGGGCCAAGCGAGCTTGG 15 AGCCCGCGGGGGCGGAGCGGTGAGAGCGGCCGCCAAGAGAGATCACACCCCC AGCCGACCCTGCCAGCGAGCGAGCCCGACCCCAGGCGTCCATGGAGCGTCGC CTCCGCCCGGTCCCTGCCCCGACCCCCGCCTGCGGCGCGCTCCTGCCTTGACC AGGACTTGGGACTTTGCGAAAGGATCGCGGGGCCCGGAGAGGTAACCGCCGC GCCTCCCGGAGAGGTAACCGCCGCGCCTCCCGGAGAGGTAACCGCCGCGCCT 20 CCCCGTGCCGTCTGCCTCGGGCTCACCGCAGTCAAGGGCTGCGGGTGCATGTT GGGCAGCGACCCCCGGGCTGGCACCGTCTACCTGGCGTTGGTTTTGCAGCGCT CCTGGACTGGGAGTTTGTTGGACGTTTCTGTCTGGGTTTTGAAGTGCTGGGAGT TGAGGTCACTGCTGCAGAGGTTGGAGTGACAGCCCTGGATACCTCCAGCCAGC TGTTCCTTTCAGATAATGCACGGAACTTAAGCTGCAGCTGACTGGTGAAGAGG 25 CGCCCCTCGGGCCCTCCGATCGGGTGGTCGTTGGCGCTTTGCTCCTTTCGTGTC TTTTTTGGAGCCAAGCGTTGCATGCTCATGGCCACGTAGACCTTGCAGAAGCG CTGCGGCTTTTTCCCAAATTGTGCAGGACCAGCCCGACGGGGCGTAGGGTACC GCAGTGGCCGCCAGCCCGGCGGAGGTGGGGGGCTTCAGTCCCAGAGACAGCT TCCCAGACGCTCACGGGTCCTTGCAGGGGGCTGGGGTCCTGGCGCGGGGTTCA 30 GCTTTTCCAGTAGCAGCAGTGTAAGAGAAGTATGCATGTAGACGTGGGTCGCT GGCCCCTGCTCCAGCCCCTACCCCACGGTTAGACTGTCTTTGTCAGCTGGAGTC GTCCCACATCCTGCCACCTGGCCTCTCAGAAAAGCAGTGGCATTAAGTAAGGA AGGAGCTTAACTCTTTTTGAGCCCGCTTTGTTTTCGGTTCTCCTCGCCTCAGAT CTTCTTGCCTTGGAAAGCCAGTCTGGTCAAGTGTCTCAGGGTAGCATTTTGGC

[0028] 14 / 57

[0029] AGAACCATTGTTAATTAAAGGGACTTCTGGACCGCAACCTTAATGTACCAGAT TATTGAGCAGCCAATGAATGCTTCATTCTCATTGTTTAAGGTGCTGCTTTGATT TTTTTTTCAATTCTTTGTACTATTTTTGATTTTTTGGAGAGGCACATCCCCAAAT TTGGATGAGGTATTTGTTGATAAATAATTCATCAATTTCCACAATGCAGACAA 5 AAATGTCTGCCCAGAGTGGAAAAATAAAACAAGGGGGAGAAGAGTTTGAGTA ACGGAGAAGTTCTGTGGAATCCTAGTGACAAAAGTTGAGAAACTACCTTTAAA TAAGACAGTGAGGTAACAAATGTTAAAAAAAAAAAAGTTAAGGGTAATATTG CATTGTTTGCCACTTATAACGTATCTGAAACATTTGTTCTTTAATCTTAAGCGT AGAGTGGTCCAAGACCTGGAAATCTGGGCAGTGGGTTTTGATTTCTAAGGAGG 10 CAGTTTTACAGGCTGTGCAAGGCTAACCCTACCAATGAGATCACACTGGCGTA TCCGATATTGACATTTGCATGTCTTGCCTATGGTTTACAGCTCTCACTATTTTA GATCTTTGGTGAAAAATCTTTAGATTCAGAAGAGAAAAATAAAAGAGTCAGG CAAGCAAAAAGAGATAACACCTCATTTGTTTAAAATCTTCCTACTGCCCATCT CTTTCCATCTGATTGTGGCAGGCACTGTAGCCACAGGTTTGGTGTTGTGGGATT 15 GCAAGGGGCAGGTATTGAAAGACCTGGCTCCTAGGCAAAACTTTGCCCCTGA ATGGCTGTGGGATCTGAGCAGTACCTGTAACTCCATTGCCTTAGCTGCAGACT AAGGTGGTAGTGTAATTTCAGGGAAGGGAATAAGGTCATCTTTGACTTTTAGT CCCATCATCAAGCATGGTGATTGGCCTTCAGTAGGTGGTCAGGTAGTGCTTGT TGGGTGGATGGGTGGATGGATGGATAGATGGATGGATGGATGGATGGATGGA 20 TGGATGGATGGATGGACGGACGGACGGACGGACGGATAGATGATATTGTGAA AGAGACTTTTGCTGTGCCACAAAGGAAAGGTATATGTGTTATTTTTATCCTGCT ATGATTTTGCCCAATTCCACCTGTAGAGGACGTCTGACATCAAAAGAGAGCAC CATCTGATCCAGAGAATGACTCCCACTATGCCTTTCCCAGCAATCAGATTAAG AATCCCAATCTAAAACTATCACCAGAAAACTCCTCAGGAGAAACCATGGGTC 25 ATCTAGTCCAACCTCTCTCCTTTGAGTGGGCCACAAGAATTATCTCAACACCTT ATCAATGGTGTTGAGAGTCTAGACCCACACAGAAGTAGCTACCCTAAAATCTA GGTTTCTTCTCCTCCCTCTTCCATAGGTTTAACATTTTTATTAACTGGAAAGTTG CCCTGGCAAATAAGATGTTGCAGGAATGGAATTAACAATCACTGTGAAATTTC CTAGGGAATGTCTTTGTGCTCTGAAAACTTCTTCATCTTCTTGGAAATTAGGAA 30 GTGTGGCCAGACACCCTACTAAGGGCTAGTAAATCACAGCTGGCTTTTTACAG TGTTCCACACTGGAGAGATCCTACATCATGTTAATTTGCATCATCCTGCGCTGT TGGCATGGGTTGTAGATGCTGGTCTTTGCCACCTTTCCTCCTTCATTTGCCAGA GTGGCCTCCTGGTGGAAACTTCTTTCCTTGTCTACTTCTAGATTTTCATTCCTTC TACTAGTTGATTATGCTTAAAGATTTCAGGGTGAAAAGCCAGGCAGACTTGCC

[0030] 15 / 57

[0031] TCTGTGTTGGTACCTGGAGGGTGATGATAAAATTTCATTGGCAGTTGAGTTAA AATGATTGAGCTCCAATATCTGTTCTTAGAAGTATAGAATTTATTGTTCTATTA TTATTTTGCGGTTTTTCATTTTTTGTTTTCGTAGAGACCTAGGTCTCACCGTGTT GCCCAGGCTAGTCTCAAACTCCTGAGCTTAAGTGATCCTCCTCCCTCGACCTCC 5 CAAAGTGCCAGGATTAAAGGCATGAGCCACCATGCCTAGCCTATTGTTAGTCT GTTTCTTATTCTGATATGAAGTCCGGTGCGGAGGTTCTGAAGGAATGCTGACT GCTCTTAAAATTGCAAAGCATCTTGCTGGAAATGGACTTTTTTATTGATTAGAA AACATTCGGTTTTAACTCTTTATAAACATCAGAATAAACTTCATGATTAAACA CTTTATAAATACTTCTCTTTGGGGCTAAAGAGGAAACACCAACCAGCAGCCTA 10 AGTGACAGCATAAAATTGCAAGATTTTTCATAAAGTATTTTACTGTCCTTCCAG CATAGGGAGGGAGTGATAACTCAAGAGTTTGCCCAAGAGCAGGAGTGCTTGG TGGTGGTCTGAGGGGAGGAGCTACAGACTTGTCAGGGCTACTTTGAGCCCTTA ACTGGCATCCAGTACTCCCCAAATCCAGTGCTGTTCAGGGTGGCCTCAGAATG AAACGGGAGCTTAAGTTGGTTTTGAGGAGCAGTACCTGTTGCTGACAGACAGG 15 GTTGGCTCCCTGACAAACATTACTGAAAACACGGGTTATTCCCCTCCTCCTCA GTACCCACTCTCTGCCTTCCTTCTCTTGTTTAGGCTAATCTTTTAAAGTGATGTA ACTTAAAACAGTCCAGTGTGAGTTAATCTCTGGGGCCAGCTAGTATCCCGGGA GTAGAGGTGCTAAGATCTTTTGCCTACAGATCAGGGTAATGACAAGATCAAAC ACTGGGGTTTCCTGCAGGCAGCTATAGAGGAGTCGTATTAAAGTCAGGCTAAT 20 AGAATGGCTTTTTTTTTTTCCTTTCCTTTTCAGGTGTTGGAGAGCACAATGGCT GAACAAGTCCTTCCTCAGGCTTTGTATTTGAGCAATATGCGGAAAGCTGTGAA GATACGGGAGAGAACTCCAGAAGACATTTTTAAACCTACTAATGGGATCATTC ATCATTTTAAAACCATGCACCGATACACACTGGAAATGTTCAGAACTTGCCAG TTTTGTCCTCAGTTTCGGGAGATCATCCACAAAGCCCTCATCGACAGAAACAT 25 CCAGGCCACCCTGGAAAGCCAGAAGAAACTCAACTGGTGTCGAGAAGTCCGG AAGCTTGTGGCGCTGAAAACGAACGGTAAGACTTGTTCTGTTGTGTTTCTTTTG CCTGGGTGATAGCTCCCGCCTGCTGGATCCCCATTCATGAAGCTTTAATAGGA CAAGCCCAAACTCAAATCAATCTTGAGATTTAGTATTGAGACCTTTATATAGA ATCTCTATTCGGGGTATGTGATAATAGCAGACTTGTTTTGTGAATCTATTTATT 30 AAGGATATTTTCTGATTGTGTATATTGCAGAGCACATTGAGGCTTGGAGAATG CATTTGTAAACACCATACCTCCATTAGGTGATTTCTTTCTAATTCTTGCAAATG TCTAATCATCTGTAAAATACCCTTCTCTGGCCCGTAATGGAAATGTTAAAAGC CTTTAAGTTCAAAAGGAAAGAATGGTGTGAAAACTGGTAGTACATTTGTTTCC CACTGTCATTAGAAAATGCTTGTGATGTTCTTACACTTCTTGAATTGGTAGCAT

[0032] 16 / 57

[0033] TTTATGGGTTAACCAGTGAATGGCTGTTGAAGACATGAAGAACTTCTTTTGTCT CCTCCTCCTCTCTGACCTAGGTTCTATTGCCTATGCCCTGCAAGGTGAAATAGA CAGATAGCTGAGATTTTTAGCATGAATTTTAGACTTCACCTTCATTATTTTTAG GGGTTCTTAGTCACTGAATTCCTTCCTGTCTGTGCTGTTCTGCCAATGGCCTTT 5 ACCACTAGTAGCTATTTCCTATTCCAAGCTTTACTGTTTTGTTTTTTAATCATTT CCCTTGGGTAAGAATATGTGCATCAACAGGAATGTCAGAGCATACTGTTTCAT GCAGGCACAAAATTCTGCTCTATGGATGTGCATAATTATGACAATAAGGAAGT TGACTGTACAAGCTCAATTAAATGCTCATAGTCTACATAAGAGCAGGAATCTC AGGTCCTTATATCTGTGACAGTCCCAAACCATCCTAATAGAAATAATATGTCC 10 AAGGGCAGAAAATCCTTGTTGACAAGGATTACGATAAGAACACTAACTTAATT AACTTCATTCAAAAGCCAAGTACATTAACTTTGGCTTTTTTTGACTTGGGTAAC TTAGAGTGATTGGTGGACATGGGTTTTTAAATTTTGATTCTGGGGGCTCTTGTA CATTACCCCAACGTCTTGCCTAGATTTATGAAACACTGATTCTTAGAAACATA GATAGAAAGAAAACTGGGGATAGCTACTATCTGCATTGTTCTTGGCATTAAAT 15 TAAGTGATACTTAGAGGTAGGTAATTATGTGAAGTCATCATCACGGGGTTTAT GTGCCTGACAATTTGTGGTTTTAATCATCTCCCATTTGTTACCAAAAAGAGATG GAGTCAACTTTGGGGGAGTTGGAAACAAAGATTCTGCTGAATTAAAAATGTG GTAGAAAGAGGAACACCTTATTAAGAGAATTAACTGACTTTTAGCTTTATTTC CTTAAGAAAGGAAATGCTCTATTTCCACTAGAAAAAAAAAAGGAATATAAAT 20 AGGAAACACTTATTTTGTAAACATGTCATTTAAATCAACAAGGAGCCACACTA CAAATAAAACTACAAAAGCAGGTGCATTTTGGTAACATTTTCTGGGAAATCAT TTTTAGACTCAGAGTCCTTTTGTTTCTTCACTTTCAAGAAAACTCCTTAAGATT CTAAAAAAGGAAGAAAAGATATTTAGGGAGGGAGGAAAACAAATGACTTTTT CTTTGATTTTAATTAGCACCTTTTAAAAAAAAATTAATCCTTCCCCTGATAGGA 25 GGGTTCTTCAAAGGCAATATCTGTTTGGGGAGGGGGTGATTCACAAGTTTGCA TTTGAGGATATGAGACTGCGGTGAATGAGGAAGGGAGTAGGTGGCATAAAAT TGAAACTTCATGTGAAAAATTCCAGCGTCTATTGGCTGCAGCGATTTCACTTGT ATAATGAGTAGCCCTATTCCAGCTCACCCTGACCACACCCACTTGGAAAGTCC AGGGTGCACTTCGCAGTTTAAATGTCTACACACCAGAACAAAAAGTACAATA 30 GCTGTTGCTCAATTGCTAGTCAAATAACTTAGCACTGGGGAATTCCAGATGTT ACTTAGGGAATTTTATACTGGTGCATCTCAATAAAGAACTGAAAGTAAGCACA AGAAGAAAAAAAGCCTTATCTTTGCTCTAGATTTTGCAAAGGGGAAATTTCAA CAGAACGCAATCATTGCTACACGTCTGCCAAGACACAAGGCTTGGGCGATCTT TTTTGTTCATTTGTTTGAATACTTAGCTAGTTTTTCTAAATGTATACATTGAAG

[0034] 17 / 57

[0035] GAATACTGGCTGTGGAAGTATATTTGAGGGTTTTTTGGTTTCTTTACAGTTTGA CTGTAATTACAATTTGATAAAATTTCAAATAGCTCTATTGCCCTACTTATAGAG TAATGTAAGAAACAATGTAACTTTTCATTATAAAAATTATTTTGCACTTGCCAA AGGAGATTAAGGAGTTAACTTTTTTTGTTTTAAACATTCCCCCAAAATATTTAT 5 CGTTTGGGGGTTGAAAAAAATGCAGCCCATTGAATTGTGAGTTTTAAACTGGA AAGGTCACAGATTAATGGCATACAATTAAACCTGTTTTTTTGTTTGTTTTTCTT CTCAAAATGTTAGCCCTAAACACTGAATTATTTTCAAATTTAGACACCCAGGT GCTTTTTAACTTTTGTACTTTACAGTTACTAACAAGCAGGTTTGCATAAGGCAT TTTTGAGAGCCGGACTAGCAGGCCATAGGTAATTCCGTGTTGTCAATACGACT 10 TTCCACACCAGGAGAGGAAACTCCCAGGGCCCAAAGGCTAAAGTGGAATGGT CAAGTCATTTGCCATTGAGTCATAGCCAGACCATCCCTATCTAGCAAGCCATG CCTGCTTCCTGGTTTGTGCATGTTGCCCTGTGTGCTCCTCCTTAGAACTAGAGA GCAGCAATGCCAGTGCCTTCACCAGCAAATCAAGGGATGGTAGGAAGGGATA GGGCTTTAGAGATCATCACTTACTGCCCTCATTTCACAAGAGGAAAAATGCCC 15 AGTGAACTTAAGGAATTTCCTCCAGGTCACCTAAACTAGTTAGGAGCAGACTT AAGCTAGAACCAAGGTCCCCTGGCTCCTTTGCAGTTGGTGTCATTCATTAAAA AGAAGAATAAAAAGAACTCTTTTTTTCTTAAAGCTGTCATCATCTTGTGAAAT ATCAGTTTGCCCTTGACTAGGAAATTACATCAAATTAAACCATTCAGTCCCCT AGAATAGCAGTAGGGCTGGTTTATTCTGAAAACCTTTGCTGGGTCTTACATGC 20 AGATAACTTGACTTTCCTTCTCTTCTCCTCCTTTCTGTCCTCAGGTGACGGCAA TTGCCTCATGCATGCCACTTCTCAGTACATGTGGGGCGTTCAGGACACAGACT TGGTACTGAGGAAGGCGCTGTTCAGCACGCTCAAGGAAACAGACACACGCAA CTTTAAATTCCGCTGGCAACTGGAGTCTCTCAAATCTCAGGAATTTGTTGAAA CGGGGCTTTGCTATGATACTCGGGTAGGTTTTTCCCCCTAATTATCTACTAACA 25 GAGCTCCATGGTGGGCATAGGGTACCCCTGGGCGAGTCCCTGCCCTCTGGTAG CATCTGATGGACTAGGTCACATGAATTTGGCTAAGCGAAGCATACTCAATGGA AAACACCAGAAACCTCCAGTGGGACTCACCCAAGGCTTTTGCCCTGCTTTTGG TTAGCAGATTCCCAGCTGTGGATCCGTTCCTGTAGCAGCGAGTCTTTTGATCAT GGCCTTTTCCACTGGTGTTTTGTTGTTTTTCAGTTTGATTGCCCTTAGCTTTGCT 30 GAGCCAGCCAGTAAGATTAAGACACGTGCCTCACCATCCTCAGTCACAAGTTT GGAATTTTTGTGAACATTTGGATAAGGGGCTGCAATATGATACCTCATCAGGG ATTTTTTTTTAAAGCAGGTGCTGTCCCCGTCCTCCCCAACTTTTGAGTTTGCCTT GTAGTGGAAAAAAGTAAAGGAAGAAAACCGAAATGGGGAAAAAAGGGTGAT CATTTGAATGATGGTTTCATGAAAGGGGAAAAAATAAGCTGAGTTATATAAAT

[0036] 18 / 57

[0037] GAATAATTGTAGAGTGATGTCAGAATGACTTTTTAGTACAGGGAGTACAGGAT ACATTCAAGCTTTTTTTTCACCCCGCTCCCCTTAGAACTGGAATGATGAATGGG ACAATCTTATCAAAATGGCTTCCACAGACACACCCATGGCCCGAAGTGGACTT CAGTACAACTCACTGGAAGAAATACACATATTTGTCCTTTGCAACATCCTCAG 5 AAGGCCAATCATTGTCATTTCAGGTGAGATGCCTGCAGATCACGGATCTGTAC TTAAATGCTTTCAGCCTTATGCCTTGGCTCCTGGAGAAAACCACACTGCCAAA GTTCAGGTAACAGAGTTCAATGGAATTTGATGAAAGTCACCTAAGGGCCTCAT TTTCCTTTCTCTTTCTTTGAACAGACAAAATGCTAAGAAGTTTGGAATCAGGTT CCAATTTCGCCCCTTTGAAAGTGGGTGGAATTTACTTGCCTCTCCACTGGCCTG 10 CCCAGGAATGCTACAGATACCCCATTGTTCTCGGCTATGACAGCCATCATTTT GTACCCTTGGTGACCCTGAAGGACAGTGGGCCTGGTGAGAAAACTGCATTAAT TCACATCTATAACTAGACACTGAAACATCAGGGTTTTCCTTTTTGCTTCTTATT AAAACAGTCTTATTTAAGTATATTATTTTTATTTAAATATATTGTTCTGTGACTT GCTTGGTTAGTAAGAGTAATGCAGTAGCAGTAATCATAATACCCTCATATCTT 15 TTTGAATATGACTTGGAAAGTACTTTCATACATGATAAATTAAGGCGCACAGT GCTCTTAATACAGTGCCTGGTATGGAGCAAGTAAACGCCTGTCAGGTTAGTTT TTTTCTTTTTGAGACAGGGTTTCCCTCCATTGTCCAGGCTGGAGTGCAGTGGCA CAATCATGGCTCATTGCAGCCTCCACCCCCTGGGCTCAAGCAGTCTCTCGCTTT GGCCTCTCAAATTTACAGGCACGAGCCACCATGCCTGGCCTGTTAGCTGTTAT 20 TTTAATAGCCTCATGTGGAATAAGCACTGTGCTTTGGAAATATTATTTTACTGC ATTTTTATCCTTTTAGCACTCTTGTGAGATGTAGGCCTTGGTAGCCTTATTTTAC ATATTAGACAGTTAAAGCTCAGAGAGGTCAACTAACCTGCCTAATGCCACACA GCTTGATAGTGACAAAGCCAGGATTCAAACTCAGGTCAGTCTGATTCTAAAGC CTGGGACTTTAACCAATAAGATCCACCACCTCCAGGCTGGTTAATGTTATGTG 25 AGTTTCCTGGAAACAGTGCATTTTTATATTAGATTGTAGCACATTTTGTTTTCC CATTTGGGTTTAACTGTGGCTAAGAATACTTTTCTATTCTGTAAGTTATATCTT TTATACATTTTCAAAATGAGATCTACTTACCTATGGCCTTGTTTAGTAGAATAC TGTTTTACTTATGTATTATTTTTTTCCTTAGAAATCCGAGCTGTTCCACTTGTTA ACAGAGACCGGGGAAGATTTGAAGACTTAAAAGTTCACTTTTTGACAGATCCT 30 GAAAATGAGATGAAGGAGAAGCTCTTAAAAGAGTACTTAATGGTGATAGAAA TCCCCGTCCAAGGCTGGGACCATGGCACAACTCATCTCATCAATGCCGCAAAG TAAGCAGTTTATGTTCAGCTCTCTCCTGTGTCATCTGTAACTGTCTTTAACCTC AGCCACCTGAGTTGCTGCCACCCTGAAGCTCAACAGTAGAGTGATTTGCGGCC AGTTTCTGCCAATGGGAAACAAATTCAGAGTCTTGTGCATGGTTAGAGAGGAT

[0038] 19 / 57

[0039] TATGTAAGGCAGTACTAACTTGGGTTTCAAATCAAAACATGTCATTTATCCAT AATGATTTTCCTGGATATCTGGATCCCAAAAGTCAGAATTGCAGAATTATGGG TTTTCAAAGTGCATTTACATCAAGGATAGTTGTTGTTTCTTGTTTTTGTATTTTA ATTACTCTGGTACATATTAAAAAGACTAGTGAAAACACTTGGAAACAAAAGC 5 CTTTCTCTCTTGGCTTCTTGGTAAGAACAGGCACATCAGACATAGATGGAGCT GTAGAATTGTTTATGTTGCAGCTCATGATTCTGCCACATGGAAATGGGAGGAA AGCCCCAGAATACCCATTGTGCAGGTATCCGGTCTTTAAACATTGGCTTTATGT TAAAGAGATGCCTGTTTCTGAGCACTTGAAACGCACAACAGATGACAAGGAA GCCGTAAGAGAATCTGTCTCTCAATGTGGAGATTAGCAAATTACAGAGTCAAC 10 AGTAAATAGACAAGCGCCTTTGAGTGTGTCTGCGTCTTAGTTACTCATGGCTG CTAATGCTGGTGGAGACTAGACACAAGTTTAAAGTTAGTAACAGCAAAATAC CCAACCTGAGGTTCCAAAGTAGTTTGTACTATAAAGTAGACTAGTCCCAAACC AATGGCCTCAGTAATATTTTACTCAGAATTTGATCTCTGAGAAATTGGAAAGT CAACTGATTTTTCTTAGCCCTTGTTTTAGAACTATCTATAATTTTTGGATGGAC 15 CAAGATTGATTAAATGAAACTGTGATTTTAAAAATAAGGAAGTCTTAGCAAAC TAACTGACATCATTCTTGACTCGAACATGTTGGGAAATGTGTCAGATCATGTT GCGTGAAAAGTGTGAGCTCTTCATCACAGGCCTGCATTTCAGTGAATGGTTCT ACAATTCTTGCCATAATCCACATTCTAAAACTTTGTTCTATGAGCTAATGATGT AAAATCTTGTGTGTGATTTTGTGTATTCTCATACATATTTTTTCCTTTTGGTCTT 20 CAGGTTGGATGAAGCTAACTTACCAAAAGAAATCAATCTGGTAGATGATTACT TTGAACTTGTTCAGCATGAGTACAAGAAATGGCAGGAAAACAGCGAGCAGGG GAGGAGAGAGGGGCACGCCCAGAATCCCATGGAACCTTCCGTGCCCCAGCTT TCTCTCATGGATGTAAAATGTGAAACGCCCAACTGCCCCTTCTTCATGTCTGTG AACACCCAGCCTTTATGCCATGAGTGCTCAGAGAGGCGGCAAAAGAATCAAA 25 ACAAACTCCCAAAGCTGAACTCCAAGCCGGGCCCTGAGGGGCTCCCTGGCAT GGCGCTCGGGGCCTCTCGGGGAGAAGCCTATGAGCCCTTGGCGTGGAACCCTG AGGAGTCCACTGGGGGGCCTCATTCGGCCCCACCGACAGCACCCAGCCCTTTT CTGTTCAGTGAGACCACTGCCATGAAGTGCAGGAGCCCCGGCTGCCCCTTCAC ACTGAATGTGCAGCACAACGGATTTTGTGAACGTTGCCACAACGCCCGGCAAC 30 TTCACGCCAGCCACGCCCCAGACCACACAAGGCACTTGGATCCCGGGAAGTG CCAAGCCTGCCTCCAGGATGTTACCAGGACATTTAATGGGATCTGCAGTACTT GCTTCAAAAGGACTACAGCAGAGGCCTCCTCCAGCCTCAGCACCAGCCTCCCT CCTTCCTGTCACCAGCGTTCCAAGTCAGATCCCTCGCGGCTCGTCCGGAGCCC CTCCCCGCATTCTTGCCACAGAGCTGGAAACGACGCCCCTGCTGGCTGCCTGT

[0040] 20 / 57

[0041] CTCAAGCTGCACGGACTCCTGGGGACAGGACGGGGACGAGCAAGTGCAGAAA AGCCGGCTGCGTGTATTTTGGGACTCCAGAAAACAAGGGCTTTTGCACACTGT GTTTCATCGAGTACAGAGAAAACAAACGTGAGTGAAGTGGTTGACTTCCTAAC ACAGCGGCTGCTGTCCAGAAGGGGTTTTGTTCCTGACCTTTAAGAAAAAAAGC 5 CCATGTAGGCAGTCAGGCAGAACTGTCAGGACCTACCGTGCTTTTCTACCAGC TTGTGCAGGGGACAGTCACGGTGGCCCTGCCAGCCGCCCATGGAGGCAAAAG GCACTGCGTGATTACAGCAGCTCCTAATATCACATTCCAAAAACCAGAGTGCC CTGTAGACAGAGCTCATGGGATGTGCTTTTACTAGACCATTCAGGTCTGGCAT ATCTAAGAGAGTCTAAGTTTAAACTAGACTTATTTTGCAACTTAGATATAACA 10 GAACAATCTTAAAATCTGAAGAGTAGTAACTGGAGAGAAGGAAGTCTCTGTT GACACTGTAAGGAAACAGATGTGGTTTGGAAATCTCTCATTGGAAAGAAGGG TGATCAGGTAATGCCTGCTCCTTGCCTGTTCCTGTGAGCAATCAGTGCTTCAAG TTCAAAATGAGAGATTGGTAAAGCCAAAGATGTTTCCATAATAGGGATGTTAT TTCTTTTGCTGAATTTATAGACTTAAACATATACATATATATATTTCTATACAT 15 TTAAGGCTGGCCTAATCTGTATTTGGAACCCATTTATTTCTCTACTGTCAGCAT CTCTGTATCGGTGGGGTGACCCCTATGTGGTACTAACTAGCATCCATTCTCATG TAGATTTTGCTGCTGCCTCAGGGAAAGTCAGTCCCACAGCGTCCAGGTTCCAG AACACCATTCCGTGCCTGGGGAGGGAATGCGGCACCCTTGGAAGCACCATGTT TGAAGGATACTGCCAGAAGTGTTTCATTGAAGCTCAGAATCAGAGATTTCATG 20 AGGCCAAAAGGACAGAAGAGCAACTGGTGAGACACTTGGAGGAGCTTTCCCT CCCTCCCGTGTGTTCGATGCTTTTTGCTGGAGCGTTTTCTACCGACAGTGACAA GCAGGCTTTCCTCTCTGCCAGGTTCTGTCTCCTCATGATAAAGGATTAATTCAG AACCTGAAGGGGAATGTCCATGTCACTGTGGCTCCCCAGGTCCGAAATGCTAC CTCCAATTTTCATGCTCCAAGTATGCTTTCAAATTTATTTTAAAAACAAAGAAA 25 CACAGCCATTGCATAAAACTTCTTCAAGCCTCCAAAAAGCCAGATGGTGATCA GAGGCTTCTTTGATTTGTAAGAGAAGCACAGACATCTAAAAAGCAGCCCTTCT TAAATAATCCTTTATCATAAGGAGCGAACAGACAGAGCTTCCCAGGGGCCTGC CTCCACCTAATGGCGCGTCAGAAAAGCCAGCTTTGACAGATAGGTGTGTCGCC TGTTGCTCACAGAATGGTAGGGAAGCTATGTAAGAACAGTGGTTTTTGGTCCC 30 AACAGAAGAGAGCCAGGGAAAGTTTGCAGGCCAACTTAGGCTTGGCGGTTTT CCTCAGCCTCCCTGGGGGCCAAGTCAGCTTTCCTTGAGACTGGAAAGAAACAT CCTTCGAGAGCTCTGGCTCATAGACTGCAATGCTCTCCTGTTCCCTTGCTCAGG CAGGTAAAGCTCTTTGTAGACTCCACACTCTCCAATGAGATTTCATTGTGCTCT CCCTAAGAAATGTGAGCAATAGTTTCCTGACTTTTTAATGATCTGCCTGTTCTT

[0042] 21 / 57

[0043] TCCACTCAGAGATCGAGCCAGCGCAGAGATGTGCCTCGAACCACACAAAGCA CCTCAAGGCCCAAGTGCGCCCGGGCCTCCTGCAAGAACATCCTGGCCTGCCGC AGCGAGGAGCTCTGCATGGAGTGTCAGCATCCCAACCAGAGGATGGGCCCTG GGGCCCACCGGGGTGAGCCTGCCCCCGAAGACCCCCCCAAGCAGCGTTGCCG 5 GGCCCCCGCCTGTGATCATTTTGGCAATGCCAAGTGCAACGGCTACTGCAACG AATGCTTTCAGTTCAAGCAGATGTATGGCTAACCGGAAACAGGTGGGTCACCT CCTGCAAGAAGTGGGGCCTCGAGCTGTCAGTCATCATGGTGCTATCCTCTGAA CCCCTCAGCTGCCACTGCAACAGTGGGCTTAAGGGTGTCTGAGCAGGAGAGG AAAGATAAGCTCTTCGTGGTGCCCACGATGCTCAGGTTTGGTAACCCGGGAGT 10 GTTCCCAGGTGGCCTTAGAAAGCAAAGCTTGTAACTGGCAAGGGATGATGTCA GATTCAGCCCAAGGTTCCTCCTCTCCTACCAAGCAGGAGGCCAGGAACTTCTT TGGACTTGGAAGGTGTGCGGGGACTGGCCGAGGCCCCTGCACCCTGCGCATCA GGACTGCTTCATCGTCTTGGCTGAGAAAGGGAAAAGACACACAAGTCGCGTG GGTTGGAGAAGCCAGAGCCATTCCACCTCCCCTCCCCCAGCATCTCTCAGAGA 15 TGTGAAGCCAGATCCTCATGGCAGCGAGGCCCTCTGCAAGAAGCTCAAGGAA GCTCAGGGAAAATGGACGTATTCAGAGAGTGTTTGTAGTTCATGGTTTTTCCC TACCTGCCCGGTTCCTTTCCTGAGGACCCGGCAGAAATGCAGAACCATCCATG GACTGTGATTCTGAGGCTGCTGAGACTGAACATGTTCACATTGACAGAAAAAC AAGCTGCTCTTTATAATATGCACCTTTTAAAAAATTAGAATATTTTACTGGGAA 20 GACGTGTAACTCTTTGGGTTATTACTGTCTTTACTTCTAAAGAAGTTAGCTTGA ACTGAGGAGTAAAAGTGTGTACATATATAATATACCCTTACATTATGTATGAG GGATTTTTTTAAATTATATTGAAATGCTGCCCTAGAAGTACAATAGGAAGGCT AAATAATAATAACCTGTTTTCTGGTTGTTGTTGGGGCATGAGCTTGTGTATACA CTGCTTGCATAAACTCAACCAGCTGCCTTTTTAAAGGGAGCTCTAGTCCTTTTT 25 GTGTAATTCACTTTATTTATTTTATTACAAACTTCAAGATTATTTAAGTGAAGA TATTTCTTCAGCTCTGGGGAAAATGCCACAGTGTTCTCCTGAGAGAACATCCTT GCTTTGAGTCAGGCTGTGGGCAAGTTCCTGACCACAGGGAGTAAATTGGCCTC TTTGATACACTTTTGCTTGCCTCCCCAGGAAAGAAGGAATTGCATCCAAGGTA TACATACATATTCATCGATGTTTCGTGCTTCTCCTTATGAAACTCCAGCTATGT 30 AATAAAAAACTATACTCTGTGTTCTGTTAATGCCTCTGAGTGTCCTACCTCCTT GGAGATGAGATAGGGAAGGAGCAGGGATGAGACTGGCAATGGTCACAGGGA AAGATGTGGCCTTTTGTGATGGTTTTATTTTCTGTTAACACTGTGTCCTGGGGG GGCTGGGAAGTCCCCTGCATCCCATGGTACCCTGGTATTGGGACAGCAAAAGC CAGTAACCATGAGTATGAGGAAATCTCTTTCTGTTGCTGGCTTACAGTTTCTCT

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[0045] GTGTGCTTTGTGGTTGCTGTCATATTTGCTCTAGAAGAAAAAAAAAAAAGGAG GGGAAATGCATTTTCCCCAGAGATAAAGGCTGCCATTTTGGGGGTCTGTACTT ATGGCCTGAAAATATTTGTGATCCATAACTCTACACAGCCTTTACTCATACTAT TAGGCACACTTTCCCCTTAGAGCCCCCTAAGTTTTTCCCAGACGAATCTTTATA 5 ATTTCTTTCCAAAGATACCAAATAAACTTCAGTGTTTTCATCTAATTCTCTTAA AGTTGATATCTTAATATTTTGTGTTGATCATTATTTCCATTCTTAATGTGAAAA AAAGTAATTATTTATACTTATTATAAAAAGTATTTGAAATTTGCACATTTAATT GTCCCTAATAGAAAGCCACCTATTCTTTGTTGGATTTCTTCAAGTTTTTCTAAA TAAATGTAACTTTTCACAAGAGTCAACATTAAAAAATAAATTATTTAAGAACA 10 (SEQ ID NO: 19) In some embodiments, the TNFAIP-3 polynucleotide sequence encodes an amino acid sequence of SEQ ID NO: 24 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to the amino acid sequence of SEQ ID NO: 24. 15 Exemplary TNFAIP-3 amino acid sequence: MAEQVLPQALYLSNMRKAVKIRERTPEDIFKPTNGIIHHFKTMHRYTLEMFRTCQ FCPQFREIIHKALIDRNIQATLESQKKLNWCREVRKLVALKTNGDGNCLMHATSQ YMWGVQDTDLVLRKALFSTLKETDTRNFKFRWQLESLKSQEFVETGLCYDTRN WNDEWDNLIKMASTDTPMARSGLQYNSLEEIHIFVLCNILRRPIIVISDKMLRSLES 20 GSNFAPLKVGGIYLPLHWPAQECYRYPIVLGYDSHHFVPLVTLKDSGPEIRAVPLV NRDRGRFEDLKVHFLTDPENEMKEKLLKEYLMVIEIPVQGWDHGTTHLINAAKL DEANLPKEINLVDDYFELVQHEYKKWQENSEQGRREGHAQNPMEPSVPQLSLMD VKCETPNCPFFMSVNTQPLCHECSERRQKNQNKLPKLNSKPGPEGLPGMALGASR GEAYEPLAWNPEESTGGPHSAPPTAPSPFLFSETTAMKCRSPGCPFTLNVQHNGFC 25 ERCHNARQLHASHAPDHTRHLDPGKCQACLQDVTRTFNGICSTCFKRTTAEASSS LSTSLPPSCHQRSKSDPSRLVRSPSPHSCHRAGNDAPAGCLSQAARTPGDRTGTSK CRKAGCVYFGTPENKGFCTLCFIEYRENKHFAAASGKVSPTASRFQNTIPCLGREC GTLGSTMFEGYCQKCFIEAQNQRFHEAKRTEEQLRSSQRRDVPRTTQSTSRPKCA RASCKNILACRSEELCMECQHPNQRMGPGAHRGEPAPEDPPKQRCRAPACDHFG 30 NAKCNGYCNKCFQFKQMYG In some embodiments, expression of a nucleic acid of the technology is driven by a promoter. In some embodiments, a nucleic acid of the technology is operably linked to a promoter. Thus, in some embodiments, TNFAIP-3 expression is driven by a promoter. In some embodiments, the promoter comprises a CMV enhancer and a chicken β-actin

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[0047] promoter sequence. In some embodiments, TNFAIP-3 is operably linked to a promoter. As used herein, the term "operably linked" refers to a first polynucleotide molecule, such as a promoter, connected with a second transcribable polynucleotide molecule, such as a TNFAIP-3, where the polynucleotide molecules are so arranged that 5 the promoter can direct an RNA polymerase to transcribe the second polynucleotide molecule. The two polynucleotide molecules may or may not be part of a single contiguous polynucleotide molecule and may or may not be adjacent. For example, a promoter is operably linked to a gene of interest if the promoter regulates or mediates transcription of the gene of interest in a cell. 10 In some embodiments, the promoter is a constitutively active promoter. In some embodiments, the promoter is from a non-human species. In some embodiments, the promoter is from a human species. In some embodiments, the promoter is selected from the group consisting of a CMV promoter, an EF1α promoter, an EF1α-short promoter, a chicken β-actin promoter, a CAG promoter, a PGK promoter, H1 promoter, or a U6 15 promoter. In some embodiments, the U6 promoter is from a human U6 promoter. In some embodiments, the U6 promoter is from cow, mice, rat, pig, yeast, dog, cat, drosophila, or C. elegans. In some embodiments, the promoter is a H1 promoter. In some embodiments, the promoter is a tissue-specific promoter (e.g., the HP1, CD14, CD43, CD45, C68, elastase, endoglin, fibronectin, Flt, GFAP, GPIIb, ICAM-2, mIFN-beta, Mb, 20 NphsI, OG-2, SP-B, SYN1, or WASP gene promoter). In some embodiments, the promoter is an inducible promoter (e.g., a tet or lac promoter). In some embodiments, the promoter is chicken β-actin promoter. In some embodiments, a chicken β-actin promoter comprises a polynucleotide sequence of SEQ ID NO: 20 or a polynucleotide sequence having at least 80%, at least 85%, at least 90%, at 25 least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to the amino acid sequence of SEQ ID NO: 20. Exemplary chicken β-actin promoter: TCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCAC CCCCAATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGG 30 GGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGG GCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCG AAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGA AGCGCGCGGCGGGCG (SEQ ID NO: 20). In some embodiments, the promoter is a hybrid promoter that includes the

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[0049] cytomegalovirus (CMV) enhancer and the chicken β-actin promoter. In some embodiments, a cytomegalovirus (CMV) enhancer comprises a polynucleotide sequence of SEQ ID NO: 21 or a polynucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to the 5 amino acid sequence of SEQ ID NO: 21. Exemplary CMV enhancer sequence: CGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCC GCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACT TTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGT 10 ACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTA AATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTT GGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGC AGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTC CACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTT 15 TCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTG TACGGTGGGAGGTCTATATAAGCAGAGCT (SEQ ID NO: 21) In some embodiments, a nucleic acid of the technology e.g., a polynucleotide encoding a TNFAIP-3) is flanked by AAV inverted terminal repeat (ITR) sequences. For example, in some embodiments, two AAV inverted terminal repeat (ITR) sequences that 20 flank the promoter and the TNFAIP-3 (one left ITR and one right ITR). In some embodiments, the AAV ITR sequences are AAV2 ITR sequences. In some embodiments, the AAV2 ITR comprise the AAV2 ITR sequences shown in FIG.10. In some embodiments, a nucleic acid of the technology (e.g., a polynucleotide encoding a TNFAIP-3) further comprises a polyadenylation (poly A) sequence. In some 25 embodiments, the poly A is a human growth hormone poly A. In some embodiments, a nucleic acid of the technology (e.g., a polynucleotide encoding a TNFAIP-3) is encoded by the sequence shown in FIG.10 (SEQ ID NO: 22). Retinopathies 30 Aspects of the technology relate to methods of treating a retinopathy in a subject in need thereof. Retinopathy refers to any disease or disorder of the retina, the light-sensitive tissue at the back of the eye. Retinopathy can affect vision and, in severe cases, lead to blindness. Retinopathies can be ischemic, inflammatory, or proliferative. Excessive and pathological vascular growth is the main cause of retinal pathologies.

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[0051] Ischemic retinopathy, also known as ischemic optic neuropathy (ION), is a disease or disorder where the optic nerve the eye to the brain is damaged due to a lack of blood flow and oxygen. This can lead to vision loss. In some embodiments, a retinopathy is ischemic. 5 Retinal inflammatory disease, also known as posterior uveitis, includes a number of conditions that can cause swelling and damage to the structures of the retina. For example, retinal inflammatory disease may be caused by an autoimmune disorder that affects multiple systems within the body, or by an infection or trauma to the eye. In some embodiments, a retinopathy is inflammatory. 10 Proliferative retinopathy is a disease of vasoproliferative origin. For example, proliferative retinopathy may be classified by neovasucalarization on the surface of the retina. In some embodiments, a retinopathy is a proliferative retinopathy. In some embodiments, the retinopathy is diabetic retinopathy. In some embodiments, the retinopathy is macular edema. In some embodiments, the retinopathy is 15 age-related macular degeneration. In some embodiments, the retinopathy is retinopathy of prematurity (ROP). In some embodiments, the retinopathy is proliferative retinopathy (DPR). Constructs 20 As used herein, the term " vector" may refer to a vector that directs expression of an RNA or polypeptide from sequences linked to transcriptional regulatory sequences on the vector. The sequences expressed will often, but not necessarily, be heterologous to the cell. An expression vector may comprise additional elements, for example, the expression vector may have two replication systems, thus allowing it to be maintained in two 25 organisms, for example, in human cells for expression and in a prokaryotic host for cloning and amplification. The term "expression" refers to the cellular processes involved in producing RNA and proteins and as appropriate, secreting proteins, including where applicable, but not limited to, for example, transcription, transcript processing, translation and protein folding, modification and processing. "Expression products" include RNA 30 transcribed from a gene, and polypeptides obtained by translation of mRNA transcribed from a gene. As used herein, the term "viral vector" may refer to a nucleic acid vector construct that includes at least one element of viral origin and has the capacity to be packaged into a viral vector particle. The viral vector can contain a nucleic acid encoding a polypeptide as

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[0053] described herein in place of non-essential viral genes. The vector and / or particle may be utilized for the purpose of transferring nucleic acids into cells either in vitro or in vivo. Numerous forms of viral vectors are known in the art. In some embodiments, the viral vector is an adeno-associated viral, adenoviral, lentiviral, or a retroviral vector. In some 5 embodiments, the viral vector is a lentiviral vector. In some embodiments, the lentiviral vector is a second-generation lentiviral vector. In some embodiments, an adeno-associated virus (AAV)-based system is used for delivery of a nucleic acid of the technology (e.g., a polynucleotide encoding a TNFAIP-3) described herein. Non-limiting examples of AAVs include AAV1, AAV2, AAV3, AAV4, 10 AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV1, AAV12, AAV13, AAVrh.2, AAVrh.8, AAVrh.10, AAVrh.13, AAVhu.14, AAV3a / 3b, AAVrh32.33, AAVrh.39, AAVrh.43, AAvrh.46, AAVrh.64, AAV-HSC15, AAV-HSC17, AAVhu.13, AAVhu.26, AAvhu.29, AAVhu.37, AAVhu.53, AAVrh.8, CHt-P6, AAV2.5, AAV6.2, AAV2i8, AAV-HSC15 / 17, AAVM41, AAV9.45, AAV2.5T, AAV-HAE1 / 2, AAV clone 32 / 83, 15 AAVShH10, AAV2.15, AAV2.4, AAVM41, Anc80, AAV2.7m8, AAVr3.45, and pseudotyped AAV capsids. In some embodiments, the AAV capsid protein is selected from an AAV1, AAV2, AAV4, AAV5, AAV7, AAV8, AAV9, AAV10 and AAV5 capsid protein. As used herein, the term “non-viral vector” may refer to any non-viral based gene 20 delivery method that can be used to introduce nucleic acids in cells (e.g., mammalian cells) or target tissues. Such methods can be used to administer nucleic acids encoding a nucleic acid of the technology (e.g., a polynucleotide encoding a TNFAIP-3 and an inverted terminal repeat (ITR)) to a host organism (e.g., a subject). In some embodiments, a non-viral delivery-based system is used for delivery of a nucleic acid (e.g., a 25 polynucleotide encoding a TNFAIP-3) and an inverted terminal repeat (ITR)) described herein. Non-limiting examples of non-viral vector delivery systems include ribonucleoprotein (RNP) complexes, DNA plasmids, polymeric nanoparticles, or inorganic nanoparticles. As used herein, the term "recombinant vector" or “a recombinant viral genome” 30 may be a vector that includes a heterologous nucleic acid sequence or "transgene" that is capable of expression in vivo. It should be understood that the vectors described herein can, in some embodiments, be combined with other suitable compositions and therapies. In some embodiments, the vector is episomal. The use of a suitable episomal vector provides a means of maintaining the nucleotide of interest in the subject in high copy

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[0055] number extra-chromosomal DNA thereby eliminating potential effects of chromosomal integration. In some embodiments, a nucleic acid encoding a polypeptide as described herein (e.g., a TNFAIP-3) is comprised by a plasmid. The term “plasmid” may refer to a circular 5 piece of DNA the comprises an origin of replication. In some embodiments, the plasmid comprises a prokaryotic origin of replication. In some embodiments, the plasmid comprises a bacterial origin of replication. In some embodiments, the plasmid comprises a eukaryotic origin of replication. In some embodiments, the plasmid comprises a mammalian origin of replication. In some embodiments, the plasmid comprises a 10 prokaryotic and eukaryotic origin of replication. In some embodiments, the plasmid comprises an origin of replication that is active in a cell which the plasmid is located. In some embodiments, the plasmid is a lentiviral plasmid (e.g., a second-generation lentiviral plasmid). In some embodiments, a vector is selected from the group consisting of a plasmid 15 and a recombinant viral genome. In some embodiments, the recombinant viral genome is selected from the group consisting of a recombinant AAV genome and / a recombinant adenoviral genome. Methods of administration 20 In some embodiments, methods described herein comprise treating a subject having a disease or pathology associated with a retinopathy. As used herein, a “subject” means a human or animal. Usually, the animal is a vertebrate such as a primate, rodent, domestic animal or game animal. Primates include, for example, chimpanzees, cynomolgus monkeys, spider monkeys, and macaques, e.g., 25 rhesus. Rodents include, for example, mice, rats, woodchucks, ferrets, rabbits and hamsters. Domestic and game animals include, for example, cows, horses, pigs, deer, bison, buffalo, feline species, e.g., domestic cat, canine species, e.g., dog, fox, wolf, avian species, e.g., chicken, emu, ostrich, and fish, e.g., trout, catfish and salmon. In some embodiments, the subject is a mammal, e.g., a primate, e.g., a human. The terms, 30 “individual,” “patient,” and “subject” are used interchangeably herein. Preferably, the subject is a mammal. The mammal can be a human, non-human primate, mouse, rat, dog, cat, horse, or cow, but is not limited to these examples. Mammals other than humans can be advantageously used as subjects that represent animal models of disease, e.g., cancer. A subject can be male or female.

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[0057] A subject can be one who has been previously diagnosed with or identified as suffering from or having a condition in need of treatment (e.g., diagnosed with a disease or pathology associated with retinopathy). In some embodiments, a subject is diagnosed with a retinopathy that is ischemic, inflammatory, or proliferative. In some embodiments, a 5 subject is diagnosed with, for example, diabetic retinopathy, macular edema, age-related macular degeneration, or retinopathy of prematurity. Alternatively, a subject can also be one who has not been previously diagnosed as having such condition or related complications. For example, a subject can be one who exhibits one or more risk factors for the condition or one or more complications related to 10 the condition or a subject who does not exhibit risk factors. A “subject in need” of treatment for a particular condition (e.g., a disease or pathology associated with a retinopathy described herein) can be a subject having that condition, diagnosed as having that condition, or at risk of developing that condition. The term "effective amount" or “amount that is effective” in treating a retinopathy 15 disclosed herein, includes the amount of a nucleic acid of the technology, a vector of the technology, a viral particle of the technology, or a composition of the technology administered to a subject. The term “therapeutically effective amount” refers to an amount of a nucleic acid of the technology, a vector of the technology, a viral particle of the technology, or a composition of the technology described herein that is sufficient to 20 treat a retinopathy described herein when administered to a typical subject. An effective amount as used herein, in various contexts, would also include an amount sufficient to delay the progression of vascular pathology in the retina. Thus, it is not generally practicable to specify an exact “effective amount.” However, for any given case, an appropriate “effective amount” can be determined by one of ordinary skill in the art using 25 only routine experimentation. In some embodiments, the methods of treating a subject having a disease or pathology associated with retinopathy described herein comprise administering a nucleic acid of the technology, a vector of the technology, a viral particle of the technology, or a composition of the technology described herein via local administration. In some 30 embodiments, the local administration is by intraocular, intravitreal, or subretinal injection. In some embodiments, a composition of the disclosure comprises a nucleic acid of the technology, a vector of the technology, or a viral particle of the technology. In some embodiments, the composition is a further comprises a pharmaceutically acceptable

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[0059] excipient. Thus, in some embodiments, a composition is a pharmaceutical composition. Pharmaceutical compositions described herein can be prepared by any method known in the art of pharmaceutics. In general, such preparatory methods include bringing a compound, agent, particle, or composition described herein (e.g., the “active ingredient”) 5 into association with a carrier or excipient, and / or one or more other accessory ingredients, and then, if necessary and / or desirable, shaping, and / or packaging the product into a desired single- or multi-dose unit. Pharmaceutically acceptable excipients may include, for example, inert diluents, dispersing and / or granulating agents, surface active agents and / or emulsifiers, disintegrating agents, binding agents, preservatives, buffering agents, 10 lubricating agents, and / or oils. All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and / or ordinary 15 meanings of the defined terms. The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.” The phrase “and / or,” as used herein in the specification and in the claims, should 20 be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and / or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and / or” clause, whether related or unrelated to 25 those elements specifically identified. Thus, as a non-limiting example, a reference to “A and / or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc. 30 As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements.

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[0061] This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non- limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, 5 equivalently “at least one of A and / or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least 10 one, optionally including more than one, B (and optionally including other elements); etc. In the claims, as well as in the specification above, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” “composed of,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases “consisting of” and “consisting essentially 15 of” shall be closed or semi-closed transitional phrases, respectively. EXAMPLES Example 1: In vitro gain- and loss-of-function studies identify A20 as a novel regulator of angiogenic responses. 20 The effect of A20 on angiogenic responses is still unresolved. To address this gap, the impact of A20 overexpression in HREC on angiogenic responses using tube formation (TF) and EC migration assays was investigated. HREC transduced with rAd.A20 for 30h, then plated into reduced growth factor Matrigel (rGF) for 48 hours, developed significantly fewer tube-like structures than control rAd.βgal-transduced cells, as 25 quantified by Angiogenesis Analyzer measuring several TF vectorial elements (36). Total mesh area (MA), measuring areas delineated by segment-forming tubes, and total master segment length (MS) were significantly lower in rAd.A20 vs. rAd.βgal-transduced HREC, totaling 1.42±0.34 x106vs.3.39±0.22 x106pixels / high power field (HPF) and 1.9±0.14 x104vs.2.94±1.15 x104pixels / HPF, respectively (FIG.1A; n=3-4, p<0.01). Notably, A20 30 overexpression in HREC did not affect number of nodes or total branch length, suggesting that A20 disrupts late rather than early stages of TF (FIG.1A). This A20 effect was not reversed in full GF Matrigel (fGF) supplemented with optimal concentrations of VEGF. Both total MA (1.73±0.49 x106vs.3.05±0.13 x106pixels / HPF) and total MS (1.96±0.25 x104vs.2.87±0.18 x104pixels / HPF) remained significantly lower in rAd.A20 vs.

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[0063] rAd.βgal-transduced HREC (FIG.1A, n=4-5, p<0.05). Overexpression of A20 in HREC also hindered cell migration. The residual wound area 9h post-scratch was significantly larger in rAd.A20 vs. non-transduced (NT) and rAd.βgal-transduced HREC (90.6±2.5% vs.42.8±5.9% and 47.8±2.5%; p<0.05 and p<0.01, respectively, n=3, FIG.1B). These 5 findings provide the first direct evidence that overexpression of A20 in HREC impedes their angiogenic responses. This novel function of A20 did not stem from decreased VEGF bioavailability / expression level or expression level / membrane localization of its VEGF- R2 / Flk1 receptor. Indeed, A20 overexpression in HREC did not affect basal or hypoxia- 10 induced upregulation of VEGF and VEGF-R2 mRNA levels in HREC (FIGs.13A-13B). Basal VEGF-R2 protein levels were even higher in rAd.A20-transduced compared to rAd.βgal-transduced or non-transduced (NT) HREC, both at baseline (FIG.1C, n=3, p<0.01) and after hypoxia (FIG.13C). Downstream phosphorylation of VEGF-R2 Tyr- 1175 was also comparable in all groups, confirming VEGF-R2 functionality and cell 15 surface expression (FIG.13D). Next, the effect of A20 knockdown in HREC on TF and EC migration was evaluated. A significant 65-75% A20 protein knockdown was achieved in HREC transduced with rAd.shA20 or transfected with A20siRNA (FIG.14A). The TF results show that rAd.shA20-transduced HREC exhibit significantly higher total branch length 20 compared to rAd.shScr-transduced controls, under both rGF and fGF conditions (1.82±0.13 x104vs.0.98±0.31 x104, and 2.14±0.08 vs.1.53±0.22 pixels / HPF respectively, p<0.05, n=4). They also demonstrate a trend towards a lower number of nodes / HPF in rAd.shA20 vs. rAd.shScr-transduced HREC cultured in rGF (1371.2±131.5 vs. 886.7±163.5; p=0.06, n=4-5). However, A20 knockdown had no impact on total MA or 25 total MS, indicating that increased branching and node numbers in these cells does not convert into a full-fledged pro-angiogenic effect (FIG.2A). Notably, A20 knockdown did not affect wound closure in the scratch assay (FIG.2B, n=5). To address concerns that increased susceptibility of A20-deficient EC to apoptosis may affect the reliability of these assays (28), the impact of A20 knockdown on angiogenesis in the mouse aortic ring assay 30 was evaluated, a physiologically more relevant assay that maintains EC in their tissue environment. Ex vivo aortic rings from wild-type (WT) and A20-null (KO) or heterozygous (Het) mice were isolated, and the endothelium was allowed to sprout in Matrigel. The vascular sprouting area was significantly larger in KO vs. WT aortic rings (53.5±4.9 vs.9±1.5 mm2 / ring, p<0.001, n=4 done in 6 replicates, FIG.2C). Aortic rings

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[0065] from Het mice showed an intermediate phenotype with sprouting areas significantly smaller than KO (16.4 ± 2.6 mm2 / ring, p<0.001, n=3, 6 replicates) and larger, albeit not quite significantly, than WT. These findings, which demonstrate that full or partial A20 knockout promotes angiogenic responses, highlight A20’s role as a physiologic regulator 5 of angiogenesis. Example 2: Eye-directed A20 gene therapy protects from OIR while A20 knockdown aggravates disease severity. Having identified A20 as a regulator of angiogenesis in HREC, the relevance of 10 this finding in the OIR mouse model of ROP was further investigated. In gain-of-function studies, the left eye of C57BL / 6 P12 mouse pups, which do not carry any gene for retinal degeneration, received intravitreal injections of saline, rAd.A20, or rAd.βgal upon exit from the hyperoxic chamber. At P17, mouse retinae were analyzed via real-time 4- quadrant retinal fluorescence angiography, or animals were perfused with a FITC-dextran 15 solution, and their eyes were recovered for retinal mount processing and analysis by fluorescence microscopy. The results show that the percentage of central avascular area per retina, quantified on nasal and temporal retinal field images of the fluorescence angiography, significantly increased in saline and rAd.βgal-treated eyes compared to control age-matched retinae of pups kept in normoxia (p<0.01, n=3, FIG.3A). 20 Remarkably, the percent of avascular retinal area in rAd.A20-treated eyes was comparable to normoxia controls, and significantly smaller than in saline or rAd.βgal-treated eyes, reaching 19.2±2.3% vs.50.0±6.5% and 44.0±4.0%, respectively (p<0.01 and p<0.05; n=3- 4, FIG.3A). Similar benefits were evident in retinal whole mounts with rAd.A20-treated eyes exhibiting significantly smaller areas of central retinal VO (expressed as percentage 25 of whole retinal area) than saline and rAd.βgal-treated eyes, reaching 2.9±0.7% vs. 17.2±0.9% and 20.0±1.2%, respectively (p<0.0001; n=3-4; FIG.3B). Peripheral retinal areas occupied by abnormal NV were also significantly smaller in rAd.A20 vs. saline and rAd.βgal-treated eyes, reaching 4.4±0.3% vs.8.9±0.5% and 11.4±0.6%, respectively (p<0.001; n=3-4, FIG.3B). VO and NV areas in untreated contralateral retinae were 30 comparable in all groups. These findings demonstrate, for the first time, that delivery of A20 gene therapy to the ischemic retina prevents lesions of ROP. In loss-of-function studies, A20 Het and KO mice along their WT littermates (34) were used. Using IHC, the absence of A20 expression in retinae of P17 KO mice was confirmed, while A20 was still detected in retinae of WT and Het mice, albeit staining

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[0067] intensity was stronger in WT (FIG.15). Due to the vulnerability of KO pups to anesthesia during live angiography, OIR lesions were only assessed on retinal whole mounts following FITC-Dextran perfusion. P17 retinae of KO pups exposed to hyperoxia exhibited significantly larger retinal VO areas compared to WT (24.1±0.7%. vs. 5 13.7±1.9%, p<0.001, n=3), with Het pups fairing in between (18.9±0.5%, p<0.05 vs. WT and KO, n=3; FIG.3C). Retinae of KO pups also showed a markedly higher percentage of NV areas than WT (24.9±0.8% vs.8.2±1.0%, p<0.0001, n=3), with Het pups in between (13.1±1.5%, p<0.05 vs. WT and p<0.001 vs. WT, n=3). In normoxia, retinal NV area remained below 3%. These findings confirm A20 as a physiologic regulator of ischemia- 10 driven pathologic angiogenesis. Example 3: Eye-directed A20 gene therapy prevents pathologic retinal neovascularization by interrupting proliferative signals downstream of VEGF-R2, while A20 knockdown promotes pathologic retinal neovascularization. 15 To elucidate the mechanisms of A20's protective effect in OIR, immunohistochemistry (IHC), immunofluorescence (IF), Western blot (WB) analysis, and qPCR were conducted on P17 mouse pup retinae to assess markers of angiogenesis and EC proliferation. A20 overexpression significantly decreased the number of CD31+vascular structures (32.8±9.7 / retinal section, n=5) in the nerve fiber and ganglion cell 20 layers (NFL / GCL), primary sites of pathologic neovascularization in ROP, compared to saline (53.6±6.9 / section; n=5, p<0.01) and rAd.βgal-treated (50.7±6.7 / section n=4, p<0.05) retinae (FIG.4A). Additionally, rAd.A20-treated retinae showed normal vascular plexus morphology, while saline and rAd.βgal-treated ones displayed typical OIR lesions, including enlarged vessel lumen and intravitreal invasion (FIG.4A). These findings 25 underscore A20’s ability to prevent NV formation while facilitating normal revascularization of the hypoxic retina. Using IHC, it was verified that intravitreal injection of 1x108MOI / eye (multiplicity of infection) of rAd.A20 or rAd.βgal, expressed under the control of the universal CMV promoter, resulted in transgene expression throughout all retinal layers and cell types, consistent with previous gene therapy reports. 30 Indeed, A20 or βgal expression was robust in the GCL, which encompasses the retinal superficial vascular plexus and astrocytes and remained substantial in the inner nuclear layer (INL), which harbors the inner retinal vascular plexus as well as amacrine, bipolar, and horizontal cells, in addition to Müller cell bodies. It was also detected in

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[0069] photoreceptors (rods and cones) rich outer nuclear layer (ONL) (FIG.4B, n=4-5). Expression of A20 and βgal in treated retinae was also confirmed by qPCR (n=2, Fig.4C). By IHC, there was a significant yet comparable increase in retinal VEGF and VEGF-R2 expression in rAd.A20 and rAd.βgal-treated P17 retinae compared to age- 5 matched pups kept in normoxia (FIG.5A). VEGF immunostaining localized to the GCL and the outer and inner nuclear membranes likely originating from astrocytes and Müller cells, primary VEGF sources in hypoxic retinae. VEGF-R2 immunostaining was observed in the NFL / GCL and the INL, following a vascular pattern. These findings suggest that A20 likely reduces pathologic retinal neovascularization by modulating angiogenic signals 10 downstream of VEGF-R2. Next, cell proliferation using Ki67 immunostaining was assessed. P17 mouse retinae treated with rAd.A20 had very few Ki67+cells compared to a significant increase in Ki67+cells mostly lining the GCL vascular structures of rAd.βgal-treated retinae (2.1±1 vs.82.7±7.5 / HPF; p<0.001, n=4-6, FIG.5B). Next, activating phosphorylation levels of 15 PKCβII (P-PKCβII) and ERK1 / 2 (P-ERK1 / 2), key drivers of VEGF and FGF proliferative and migratory signals in EC, was assessed. Immunostaining for P-PKCβII Ser-660, predominating in the GCL and INL, was significantly lower in rAd.A20 vs. rAd. βgal- treated retinae, as measured by integrated density of staining and expressed in arbitrary unit (AU) (0.12±0.05 x106vs.5.77±1.49 x106, p<0.01, n=5, FIG.5B). WB analysis of P- 20 ERK1 / 2 expression also showed significantly lower levels in P17 rAd.A20 vs. saline and rAd.βgal-treated retinae (p<0.05, n=3 / group, FIG.5C). In loss-of-function studies, it was observed that greater, albeit not quite significant, CD31+staining in the GCL and INL of P17 Het and KO mouse retinae compared to WT, indicating exacerbated neovascularization (FIG.6A). Retinal vessels in P17 Het and KO 25 mice displayed larger lumens and greater tortuosity than in WT retinae, which corresponded with significantly higher number of Ki67+cells lining abnormal vascular structures in KO and Het compared to WT retinae (30.3±3.0 and 31.5±3.8 vs.15.3±1.3 Ki67+cells / HPF, p<0.01 and p<0.001, respectively, n=4-8; FIG.6B). Heightened vascular proliferative index in KO and Het retinae associated with a trend, albeit not significant, for 30 higher VEGF-R2 expression levels compared to WT (FIG.6A). However, they matched with significantly higher P-PKCβII immunostaining in GCL and INL of KO and Het retinae compared to WT (FIG.6B). Integrated density of P-PKCβII staining reached 2.77±0.94x106and 1.9±0.5x106AU / section in KO and Het, respectively, compared to 1.15±0.34x106in WT (n=3-7; p<0.05 KO vs. WT). It also corresponded with a 7-fold

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[0071] increase in P-ERK1 / 2 protein levels in KO and Het vs. WT retinae, as determined by WB (p<0.01, n=2-4, FIG.6C). These findings further ascertain the key physiologic role of A20 in modulating angiogenic responses in the ischemic retina. 5 Example 4: Intravitreal A20 gene therapy reduces hypoxia-induced microglial and Muller cell activation and degeneration, while A20 knockdown exacerbates these features. Reactive retinal gliosis, involving activation of Müller cells, astrocytes, and microglia, and the consequential degeneration of these cells that are vital for endothelial 10 and neuronal cell survival and function, significantly contribute to OIR pathogenesis and severity. Reactive gliosis was assessed by immunostaining with the microglial activation marker, ionized calcium-binding adapter molecule-1 (Iba-1). The results showed a significant increase in the number of Iba-1+cells, primarily in the GCL, inner (IPL), and outer plexiform (OPL) layers, in P17 mouse retinae treated with saline or rAd.βgal 15 compared to normoxia controls (405±49 and 350±63 vs.153±33 cells / mm2, p<0.01 and p<0.05; n=3-5; FIG.7A). Iba-1+cells were hypertrophic with distorted morphology, indicative of activation (43). Conversely, the number and morphology of Iba1+cells in rAd.A20-treated retinae was comparable to normoxia controls and significantly lower (196±38 cells / mm2, n=6) than in saline and rAd.βgal-treated retinae (p<0.01 and p<0.05; 20 FIG.7A). Reactive retinal gliosis, also indicated by increased expression of GFAP (glial fibrillary acidic protein) in Müller cells (44), was markedly increased in the IPL of saline and rAd.βgal-treated P17 retinae compared to normoxia controls, and co-stained with fragmented vimentin, indicating structural damage (FIG.7B). Remarkably, rAd.A20- treated retinae displayed a normal GFAP and vimentin co-IF staining pattern. Conversely, 25 A20 deficiency exacerbated reactive retinal gliosis. P17 KO mice retinae exhibited significantly higher Iba-1+cells / mm2than WT (759±170 vs.383±35 cells / mm2, n=3 and 6 respectively, p<0.05), with Het mice showing an intermediate number (489±121 cells / mm2, n=5; FIG.7C). 30 Example 5: Eye-directed A20 gene therapy protects the neuronal retina by activating the AKT survival pathway, while A20 knockdown aggravates neuronal demise. The severity of OIR, which can result in vision loss, correlates with the extent of retinal damage, particularly the neuronal retina. A20-mediated protection from OIR associated with significantly lower numbers of TUNEL+apoptotic nuclei in rAd.A20

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[0073] compared to rAd.βgal-treated P17 pups’ retinae (7.8±2.0 vs.48.5±13.8 TUNEL+nuclei / HPF, p<0.01, n=6-7; FIG.8A). Fluoro-Jade C (FJC) staining, specifically detecting neuronal apoptosis (45), showed significantly lower FJC fluorescence intensity in rAd.A20 vs. rAd.βgal-treated retinae (13.4±1.76 vs.21.4±2.26x106AU / retinal section, 5 p<0.05, n=6), including at the level of the optic nerve (OpN) (FIG.8A). FJC fluorescence in rAd.A20-treated retinae was comparable to normoxia controls. P17 retinal sections were also stained with an antibody against phosphorylated AKT Ser-473, a surrogate marker for the activation of the PI3K / AKT survival pathway crucial for neuronal, Müller / glial cells, and EC health (11, 14, 15, 46, 47). Retinae of rAd.A20-treated eyes exhibited significantly 10 higher P-AKT immunostaining compared to rAd.βgal-treated eyes (9.0±2.3 vs.2.0±0.6 x106AU / retinal section, p<0.01, n=6-7, FIG.8B). P-AKT expression in rAd.A20 treated retinae was also notably higher than in normoxia controls (3.5±0.8 x106AU / retinal section, p<0.05, n=5), highlighting that A20 advantageously amplifies the AKT survival pathway while interrupting proliferative pathways downstream of VEGF. 15 Loss-of-function studies showed a significant, yet comparable, increase in the number of TUNEL+cells in retinae of KO, Het, and WT P17 mouse pups. However, FJC fluorescence was significantly higher in P17 KO compared to Het and WT retinae (20.9±2.1 vs.15.1±2.2 and 13.9±0.7 AU / retinal section, p<0.05 and p<0.01, respectively, n=3-9), including at the level of the OpN (FIG.9B). Heightened neuronal apoptosis in 20 A20-deficient mice retinae correlated with a significant decrease in P-AKT immunostaining in KO vs. WT retinae (4.4±1.7 x106vs.10.2±1.8 x106AU / retinal section, n= 3 and 10 respectively, p<0.05; FIG.9B. Het pups displayed intermediate P-AKT staining (6.5±0.7 x106, n=10). These results underscore the neuroprotective function of A20 in the retina. 25 The impact of the potent NF-kB inhibitory protein, A20, on PR was investigated as described herein. The choice of A20 was informed by its comprehensive anti- inflammatory and anti-apoptotic effects in EC, neuroprotective functions, and ability to contain reactive gliosis. However, controversy regarding A20’s impact on angiogenesis 30 needed to be addressed first. Previous in vitro loss-of-function studies assigned contradictory pro- and anti-angiogenic effects to A20, with A20 silencing preventing TF in human umbilical vein EC but increasing hypoxia-induced angiogenesis in pulmonary artery EC. To resolve this, the impact of A20 overexpression and knockdown on EC migration and TF was revisited, using HREC as a relevant cell type to PR. Using the

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[0075] improved Angiogenesis Analyzer software for precise analysis of TF stages, it was shown that A20 overexpression in HREC cultures significantly decreased MA and MS, while the number of nodes remained unaffected, which suggested that A20 overexpression disrupted later stages of TF. A20 overexpression also hindered EC migration. These findings are the 5 first to support a direct link between A20 overexpression and decreased angiogenic responses in vitro. A20’s effect on TF was refractory to growth factors, including VEGF, despite heightened protein expression of VEGF-R2 in these cells. Increased VEGF-R2 expression in A20-transduced HREC did not correspond with higher mRNA levels, suggesting it resulted from post-transcriptional regulation. Since VEGF-R2 expression and 10 activity are regulated by endosomal / lysosomal trafficking and ubiquitin-dependent proteasomal degradation, one can infer that a lysosomal deubiquitinating enzyme, such as A20, might either interfere with VEGF-R2 delivery to and / or degradation in the lysosome or limit its ubiquitination and subsequent proteasomal degradation. Notably, A20 overexpression did not impact the activating Tyr-1175 phosphorylation of VEGF-R215 following VEGF treatment, confirming receptor functionality and placing A20’s anti- angiogenic target downstream of it. Additional experiments are planned to identify A20’s targets within this pathway. Loss-of-function experiments presented more complexities. A20 knockdown significantly enhanced the early steps of TF, specifically node count and total branch 20 length. However, this early positive effect did not translate into higher total MA or MS. Further experiments are needed to elucidate the underlying basis for these results, which could potentially be attributed to EC dysfunction in A20-deficient cells due to decreased eNOS expression / activity and / or increased susceptibility to apoptosis, especially when cultured in isolation. To circumvent these limitations, the aortic ring assay was used, 25 which increases resilience by maintaining A20-deficient EC in their natural cell support environment. The results showing increased vascular sprouting in aortic rings from A20 KO and Het vs. WT mice support a net pro-angiogenic effect of A20 knockdown and emphasize A20’s role as a physiologic regulator of angiogenesis. The conclusion differs from a previous report which attributed an anti-angiogenic effect to A20 on the sole basis 30 of decreased TF in A20-silenced EC without considering the impact of A20 knockdown on EC homeostasis. Having resolved A20 as a negative regulator of angiogenesis in HREC, the impact of A20 gain or loss in the OIR mouse model of PR was investigated. Intravitreal administration of rAd.A20 significantly reduced abnormal neovascularization and central

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[0077] avascular area compared to rAd.βgal- treated and contralateral untreated control eyes, restoring a quasi-normal retinal vasculature by P17. Akin in HREC cultures, A20 overexpression in the mouse retina did not affect hypoxia-driven upregulation of VEGF and VEGF-R2, validating that these mice experienced a similar hypoxic insult than 5 controls. Despite NF-kB’s documented ability to activate HIF-1α upstream of VEGF transcription, VEGF upregulation in A20-treated ischemic retinae was unaffected by A20’s NF-kB inhibitory function. This underscores the complexity of VEGF transcriptional regulation, involving multiple transcription factors such as STAT3, which has been shown to be positively regulated by A20 in hepatocytes. 10 Despite similar levels of VEGF and VEGF-R2, A20-treated retinae exhibited a lower proliferative index. This was associated with significantly reduced expression of P- PKCβII and P-ERK1 / 2, placing A20's targets upstream of these two mediators of VEGF / VEGF-R2-driven proliferative and migratory signaling in EC. The discovery that A20 also inhibits PKCβII activation adds to its potential as a PR therapy, even if inhibition 15 of PKCβ (Ruboxistaurin) was insufficient on its own. Conversely, A20 KO and Het retinae showed increased abnormal retinal vessels with significantly higher proliferative indices. This was associated with higher expression of P-PKCbII and P-ERK1 / 2 despite similar VEGF-R2 levels, confirming A20’s target(s) downstream of VEGF-R2 signaling. Without wishing to be bound by theory, A20’s ability to inhibit pathologic angiogenesis 20 while preserving VEGF and VEGF-R2 expression in the retina suggests it may offer a safer alternative than classic anti-VEGF therapies, which interrupt all VEGF signaling, including VEGF-driven survival pathways in endothelial, neuronal and glial cells. The rationale for exploring A20 in PR was primarily informed by its potent anti- inflammatory properties. Indeed, increasing evidence implicates inflammation, mainly 25 driven by glial cell activation, as a prime driver of abnormal retinal neovascularization and damage. In conditions Like DR and ROP, cytokine-producing Iba-1+microglia with shortened ramifications and increased soma size accumulate in the retina and contribute to pathologic neovascularization and neuronal demise. These studies uncovered A20 as a novel regulator of microglial activation in the retina. Whilst A20 overexpression 30 significantly reduced the number of Iba-1+microglia and normalized their morphology, A20 deficiency significantly increased the number of Iba-1+cells in mouse retinae. A20’s ability to temper gliosis without eliminating these cells is advantageous compared to strategies aimed at depleting them, which may exacerbate rather than reduce NV formation.

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[0079] A20 also mitigated Müller cell activation. Activation of these cells, predominant glial cells in the retina, significantly contributes to NV severity and delays in reparative revascularization in ROP. The acquisition of GFAP expression by Müller cells, along with stiffening and fragmentation of their filaments as observed by Vimentin co- 5 immunolabeling, in response to retinal hypoxia / injury marks their activation. These findings, demonstrating that eye-directed A20 gene therapy significantly reduces GFAP expression and prevents process fragmentation in Müller cells, is the first evidence documenting A20’s ability to safeguard Müller cell integrity and prevent its activation. By doing so, A20 not only mitigates inflammation and its detrimental effects on EC and 10 photoreceptors but also preserves Müller cell’s homeostasis. The latter is crucial for sustaining these cells’ ability to provide nutrients to photoreceptors and produce necessary transporters for neurotransmitter uptake and recycling. By inhibiting gliopathy and preserving microglial and Müller cell integrity and function, A20’s potential applications could extend beyond PR to other retinopathies that share gliosis as a pathogenic culprit, 15 including DME and retinitis pigmentosa. Apoptosis, induced by hypoxia and inflammation, is also a critical determinant of outcomes in PR. Early EC loss exacerbates hypoxia, amplifying microglial cell demise. In turn, loss of microglia deprives EC and neuronal cells from survival cues, further aggravating neuronal cell death and worsening blindness. Therefore, without wishing to be 20 bound by theory, a therapy that prevents apoptosis in all these cells could be beneficial for PR. A20’s documented anti-apoptotic effects in EC, glial, and neuronal cells provided an additional rationale for evaluating it as a therapeutic candidate in PR. Analysis of P17 rAd.A20 treated retinae showed a significant reduction in TUNEL+apoptotic nuclei, particularly evident in the neuron and glia-rich INL and ONL. 25 Fewer apoptotic cells were detected in the GCL where the superficial vascular plexus is located, indicating a probable earlier occurrence of EC apoptosis. Neuron-specific FJC apoptosis staining confirmed a significant reduction in neuronal cell death in rAd.A20- treated retinae. Conversely, P17 retinae of A20-deficient mice displayed significantly higher neuronal death compared to their WT littermates. Differences in tissue distribution 30 between TUNEL and FJC likely reflect TUNEL’s limited ability to capture neuronal death, inferring that TUNEL+cells detected in the INL / ONL are likely glial cells. A20’s ability to support neuronal survival in the ischemic retina is multifactorial. A20 reduces gliosis, thereby mitigating inflammation-induced neuronal damage. It preserves neurotrophic and EC-protective capabilities of glial cells, bolstering neuronal resilience

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[0081] against ischemia while accelerating reparative vascularization to alleviate hypoxia. Additionally, it directly exerts cytoprotective effects in neurons. Unlike ischemic neurons, healthy ones do not produce vaso-repellents that impede reparative revascularization. Decreased apoptosis in A20-treated retinae correlated with significantly higher 5 immunostaining for Ser-473 P-AKT, marking activation of the AKT pro-survival pathway downstream of VEGF. P-AKT expression was observed across retinal layers and cell types, including GCL vascular structures, Müller cell filaments in the IPL, and glial and photoreceptor-rich ONL. A20 overexpression increased P-AKT levels while inhibiting VEGF-induced phosphorylation of pro-angiogenic P-ERK1 / 2 and P-PKCβII. This 10 suggests that A20 selectively blocks VEGF proliferative signals while maintaining AKT activation or, alternatively, induces AKT phosphorylation directly. Conversely, A20 KO and Het mice showed reduced P-AKT and increased P-ERK1 / 2 expression in their retinae. By providing survival advantages to EC, neuronal cells, and glial cells, A20- mediated activation of the AKT pro-survival pathway is bound to benefit outcomes of PR. 15 Indeed, blockade of apoptosis solely in retinal EC is sufficient to accelerate retinal revascularization, as shown in the EC-specific bax and bak KO mouse model of OIR (75). Moreover, AKT activation can dampen angiogenesis through inhibitory phosphorylation of c-Raf (Ser-259) upstream of ERK1 / 2 activation (77). The data showing that A20 overexpression increases P-AKT and decreases P-ERK1 / 2 expression, while its 20 knockdown yields the opposite, suggest that this crosstalk might be implicated in A20’s ability to limit pathologic NV in vivo. Additional mechanistic experiments are planned to address this question, especially since the role of AKT in angiogenesis remains controversial, with conflicting reports of both loss and gain of AKT causing pathologic angiogenesis. While these reports fail to take into consideration the complex temporal, 25 contextual, and threshold-dependent fine-tuning of angiogenesis by AKT, the experimental system in which A20-mediated increase in P-AKT remains forcibly contained within physiologic total AKT levels, provides a better-suited context to gauge the impact of AKT signaling on retinal EC angiogenic responses. This disclosure provides the first evidence that ocular A20 gene therapy prevents 30 lesions of OIR and promotes reparative neovascularization in the ischemic retina. Mechanistically, A20’s beneficial effects are manifold, encompassing its ability to comprehensively suppress hypoxia-driven glial cell inflammation while preserving these cells’ neurotrophic and vasculoprotective functions. A20 also promotes EC and neuronal cell resistance to hypoxic cell death and safeguards their cross-talks to accelerate

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[0083] revascularization while simultaneously preventing pathologic neo-angiogenesis. This novel function of A20 as a modulator of angiogenesis was validated in HREC cultures, highlighting its relevance to human physiology. Notably, the in vitro and in vivo loss-of- function studies, which emphasized the crucial role of endogenous A20 in determining PR 5 severity, strongly support the promise of an A20-based therapy to treat PR. Developing an A20 gene therapy to treat PR may be particularly beneficial for patients with defective A20 expression due to genetic polymorphisms or increased degradation, as seen in diabetes. The data demonstrating a 30% reduction in TNF-induced upregulation of A20 protein in HREC cultured under high D-glucose (15 and 30 mM / L), mimicking poorly 10 controlled diabetes, versus physiologic D-glucose concentrations (5mM / L) (FIG.16) agrees with other reports of lower A20 expression in mouse microglial cells cultured in high glucose (84). The feasibility of a novel eye-directed A20 gene therapy described herein to treat ischemic PR, including ROP and PDR, is bolstered by the availability of safe gene therapy 15 platforms. Notably, recombinant adeno-associated viruses (AAV) have demonstrated effectiveness in various eye disease models, including the pioneering FDA-approved AAV therapy for treating Leber congenital amorausis. An AAV-based A20 therapy would advantageously exert low toxicity and minimal immunogenicity, provide sustained expression, and be easily delivered through intravitreal injection. Furthermore, using a 20 universal promoter, as in this study, would ensure A20 expression across multiple cell types involved in PR pathogenesis, boosting therapeutic potential. Although intravitreal AAV delivery may not directly target the retinal pigment epithelium (RPE), A20 expression in RPEC was still verified to be safe. The data show that A20 overexpression in a human RPEC cell line is not only safe but, in fact, protects against inflammation and 25 oxidative stress (FIGs.17A-17D), hint that the benefits of an A20 gene therapy may extend to RPEC-centric ocular diseases, including dry age-related macular degeneration, and auto-immune uveitis. Methods corresponding to the Examples 30 Cell culture and reagents Primary human retinal endothelial cells (HREC) from 6 different donors were purchased from Cell Systems (Kirkland, WA), cultured according to the manufacturer’s instructions, and used between passages 4 and 8. In select experiments, full medium was removed and replaced by basal serum-free and growth factor-free medium for 2 hours (h)

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[0085] prior to stimulation with VEGF. Recombinant human VEGF-165 / VEGF-A was purchased from R&D SYSTEMS® (Minneapolis, MN). Recombinant adenoviruses and silencing RNA Recombinant A20 adenovirus (rAd.A20) expressing human A20 / TNFAIP3, and 5 control β-galactosidase adenovirus (rAd.βgal) were generated and titrated in the laboratory. In vitro, tube formation loss-of-function experiments were conducted using a rAd. expressing GFP-tagged short hairpin (sh) strand-specific human A20 siRNA (Ad- GFP-U6-h-TNFAIP3-shRNA), referred to as rAd.shA20, and control scrambled shRNA Ad-GFP-U6-shRNA (rAd.shScr) purchased from Vector Biolabs (Malvern, PA). HREC 10 were transduced at a multiplicity of infection (MOI) of 100, resulting in 95-100% of cells expressing the transgene 48 hours post-transduction with negligible toxicity. For scratch assays, A20 knockdown was achieved by transfecting HREC with human A20 siRNA (Hs_TNFAIP3_1 FlexiTube) at a final concentration of 40nM, using the Hiperfect transfection reagent, both from Qiagen (Germantown, MD). AllStars fluorescent siRNA 15 served as control. Transduction / transfection efficiency and transgene expression (A20 and βgal) or knockdown (A20) were confirmed by Western blot (WB) analysis, immunohistochemistry (IHC, A20), Xgal staining (βgal), and fluorescence microscopy (GFP, Alexa Fluor), as described. Endothelial Cell Angiogenesis and Migration Assays 20 HREC angiogenesis was assessed by the endothelial tube formation assay (TF), as described (94). CORNINGTMfull (fGF, CB-40234A) and reduced growth factor (rGF, CB-40234A) MATRIGELTMmembrane matrices were used in this assay (FISHER SCIENTIFIC®, Waltham, MA). HREC (2×105cells / well) were plated in basal medium (Cell Applications Inc. San Diego, CA) in a 96-well plate, cultured for 48 hours, then 25 imaged at 40X magnification with a Nikon Eclipse Ts2 inverted microscope. Quantitative analysis of TF parameters, including total mesh area (MA), total segment length (MS), number of nodes, and total branch length, was performed using the Fiji software and a plugin extension of the “Angiogenesis Analyzer” written in the ImageJ macro language. EC migration was evaluated by the T-scratch assay. At confluence, HREC transduced with 30 rAd.A20 or rAd.βgal, or transfected with A20siRNA or AllStars siRNA underwent wounding by clearing a cross-shaped area within the cell monolayer using a pipette tip. Bright field micrographs centered on the intersection of the vertical and horizontal lines were captured at 40X magnification immediately after wounding and 9h later. The

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[0087] percentage of residual wounded area 9h post-scratching was calculated using the TScratch software. Ex vivo aortic ring assay A20-null (KO), A20 heterozygous (Het), and wild type (WT) littermates (kind gift 5 of Dr. Averil Ma, UCSF) were bred in-house (34). Thoracic aortae from 3-4 weeks old mice were transversely sliced into 0.5 mm-thick rings and cultured in Matrigel supplemented with VEGF (50 ng / mL) and 1.5% horse serum. After 5 days, phase-contrast micrographs were captured, and capillary sprouting was quantified using Image J-based calculation of the area covered by branching microvessels. 10 Western Blot Analysis Total protein cell lysates were extracted from HREC or mouse eyes and subjected to Western blot (WB) analysis to assess target protein expression. Primary antibodies used included rabbit anti-VEGF-R2, total-ERK1 / 2 (t-ERK1 / 2), phospho-VEGF-R2 Tyr-1175 (P-VEGFR2), phospho-ERK1 / 2 Thr-202 / Tyr-204 (ERK1) and Thr-185 / Tyr-187 (ERK2),15 and phospho-AKT Ser-473 (P-AKT) from Cell Signaling (Beverley, MA); rabbit anti- human IκBα and mouse anti-human β-actin from SANTA CRUZ BIOTECHNOLOGY® Inc. (Santa Cruz, CA); rabbit anti-t-AKT and chicken anti-human A20 from Abcam (Waltham, MA); and mouse anti-GAPDH from EMD Chemicals (Bedford, MA). Secondary donkey anti-rabbit and goat anti-mouse HRP-conjugated antibodies were 20 purchased from Thermo Scientific (Rockford, IL). Protein bands were visualized using the enhanced chemiluminescence kit (ECL) from PerkinElmer Life Science (Waltham, MA) and quantified via ImageJ-based densitometry. Alternatively, membranes were probed with IRDYE® infrared secondary antibodies, compatible with the LI-COR ODYSSEY® CLx imaging System (LI-COR Inc, Lincoln, NE), and bands were quantified using 25 IMAGE STUDIOTMSoftware (LI-COR). Quantitative RT-PCR RNA from HREC and mouse retinae was extracted using the RNeasy Mini Kit (Qiagen, Germantown, MD), followed by reverse transcription with iScript cDNA Synthesis Kit (BIO-RAD®, Hercules, CA). Quantitative PCR (qPCR) was conducted to 30 analyze gene expression of human VEGF, VEGF-R2, TNFAIP3, Intercellular adhesion molecule-1 / ICAM1 primers and iTaq Universal SYBR Green Supermix (BIO-RAD®), or TaqMan Mm00627280_m1 for A20 (Applied Biosystems, Foster City, CA) on a 7500 Fast Real-Time PCR System (Applied Biosystems). Gene expression levels were

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[0089] determined using the Pfaffl method (97), and normalized to human and mouse 18S or 28S housekeeping genes (HKG). Primers used are listed in Supplementary Table I. MTT and LDH Assays Viability of RPEC after oxidative stress, modeled by treatment with 250 µM of 5 hydrogen peroxide (H2O2) for 16 and 24 hours, was evaluated by the MTT (Sigma- Aldrich) and LDH release (G-Biosciences, St Louis, MO) assays, as per manufacturer’s instructions. Oxygen-Induced Retinopathy (OIR) Mouse litters from C57BL / 6 (WT x WT, gain-of-function) and A20 Heterozygous 10 (Het x Het, loss-of-function) breeding pairs were housed with their nursing mothers in sealed hyperoxia chambers under 75% O2from postnatal day 7 (P7) to P12. This induces vaso-obliteration (VO) of developing retinal vessels within 2 days of hyperoxia exposure. At P12, mice were returned to room air, triggering the central hypoxic retina to produce pro-angiogenic VEGF, mainly by glial cells and EC. Elevated intra-retinal VEGF levels 15 cause pathological neovascular tufts (NV) at the junction of the avascular and vascularized retina between P17 and P21, with NV tufts being most severe at P17. In gain-of-function experiments, mice exiting the hyperoxia chamber were anesthetized, and their left eye was intravitreally injected with 1 µL of saline, rAd.A20, or rAd.βgal at 1x108MOI / eye. Non- treated contralateral right eyes, along with eyes of mouse pups raised in normoxia, served 20 as controls. Transgene delivery to the retina was confirmed by immunostaining using rabbit polyclonal anti-βgal (Novus Biologicals, Littleton, CO) and rabbit monoclonal anti- TNFAIP3 (Abcam) antibodies, as well as by RT-PCR. Lesions of OIR were assessed at P17, and all animals were sacrificed at this time. In loss-of-function experiments, a piece of tail was obtained at the time of sacrifice for DNA extraction and genotyping, as 25 described. All animal procedures were conducted in compliance with the U.S. Department of Health and Human Services Guide for Use and Care of Laboratory Animals and were approved by the Beth Israel Deaconess Medical Center Institutional Committee for Use and Care of Laboratory Animals. Retinal angiograms 30 Retinal angiograms were conducted following established procedures known in the art. At P17, pups’ eyes were dilated and a contact lens was applied onto the rAd-injected left eye. Sodium fluorescein solution (5 mg / kg) was perfused through the jugular vein, and a laser ophthalmoscope was used to acquire 4-quadrant fluorescence real-time angiograms.

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[0091] Pertinent digitized images from each quadrant were used to calculate the percentage of avascular areas. Retinal whole mounts High-molecular-mass FITC Dextran (mol wt 2x106) from Sigma-Aldrich was 5 injected into the inferior vena cava of anesthetized mouse pups and allowed to circulate for 2 minutes prior to sacrificing the animals and collecting their eyes. Retinae were isolated, mounted on slides, and images were captured using an inverted fluorescence microscope. The percentage of VO and NV areas per retina was quantified using the Image J-based SWIFT NV module software. 10 Immunohistochemistry, immunofluorescence, and TUNEL Staining Six-mm zinc-fixed, paraffin-embedded eye sections were stained with various antibodies, including the proliferation marker Ki67 (Agilent / DAKO, Santa Clara, CA), P- AKT (Cell Signaling), the endothelial cell-specific marker CD31 (BD Pharmingen), VEGF (Santa Cruz), VEGF-R2 (Abcam), Iba-1(Abcam), human TNFAIP3 (Abcam), P-15 PKCβII Serine-660 (Santa Cruz), and βgalactosidase (Novus Biologicals). HRP- conjugated avidin-biotin secondary antibodies and DAB ImmPact substrate were purchased from Vector Labs (Burlingame, CA). Counterstaining was done with Hematoxylin / Eosin (H&E) (Newcomer Supply Inc. Middleton, WI) or methyl green (Vector Labs). Immunofluorescence (IF) staining for glial fibrillary acid protein (GFAP) 20 and vimentin was done using a rabbit polyclonal anti-GFAP antibody (Agilent / DAKO, red) and a rabbit anti-vimentin monoclonal antibody (Cell Signaling, green), followed by VectaFluor dye-conjugated secondary antibodies (Vector Labs). Nuclear counterstaining was performed, using 4’, 6-diamidino-2-phenylindole (DAPI, 1 µg / mL, Sigma-Aldrich). Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL, 25 Millipore, Billerica, MA) and Fluoro-Jade C (FJC) labeling (Biosensis, Thebarton, South Australia) were used to detect apoptosis and degenerating neurons, respectively, according to manufacturer’s instructions. Positive FJC staining was visualized by fluorescence microscopy (475-650 nm). Unless indicated, for IHC and IF staining, four-to-six 200X magnification images were captured / retinal section using the Olympus DP72 microscope 30 camera and CellSens software (Tokyo, Japan). Quantification included the number of Ki67+and TUNEL+nuclei per high-power field (HPF), Iba-1+cells per square millimeter, and CD31+vessels in the superficial vascular plexus per whole retinal section in gain-of- function studies. In loss-of-function studies, where more extensive pathologic angiogenesis was observed, the sum of vascular CD31+pixels per HPF was quantified.

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[0093] Additionally, integrated staining density / retinal section for VEGF, VEGF-R2, P-PKCβII, and P-AKT was determined using the ImageJ Fiji 2.9.0 software (NIH, Bethesda, MD). The integrated density of grayscale-transformed images was calculated based on the average pixel product value (0-255) multiplied by retinal area and reported as arbitrary 5 units (AU). Statistical Analysis Quantitative data were expressed as mean ± standard error of mean (SEM). Statistical analysis was conducted using Prism 9 software (GraphPad, La Jolla CA). Analysis of variance (ANOVA) followed by Tukey’s post hoc test was primarily utilized 10 in most experiments. Unpaired t-tests were used in selected experiments as appropriate. A probability (p) value of <0.05 was considered statistically significant. Table 1: List of human and mouse primers 15 References 1. Sapieha P, Joyal JS, Rivera JC, Kermorvant-Duchemin E, Sennlaub F, Hardy P, et al. Retinopathy of prematurity: understanding ischemic retinal vasculopathies at an extreme of life. J Clin Invest.2010;120(9):3022-32.

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Claims

CLAIMS What is claimed is:

1. A nucleic acid comprising a polynucleotide encoding a Tumor Necrosis Factor Alpha Inducible Protein-3 (TNFAIP-3) and an inverted terminal repeat (ITR) as described in FIG.

10.

2. The nucleic acid of claim 1, wherein the TNFAIP-3 is human TNFAIP-3.

3. The nucleic acid of claim 1 or 2, wherein the ITR is an adeno-associated virus 2 (AAV2) ITR.

4. The nucleic acid of any one of claims 1-3, further comprising a promoter.

5. The nucleic acid of claim 4, wherein the promoter comprises a CMV enhancer and a chicken β-actin promoter sequence.

6. The nucleic acid of any one of claims 1-5, further comprising a polyadenylation (poly A) sequence.

7. The nucleic acid of claim 6, wherein the poly A is a human growth hormone poly A.

8. A vector comprising a nucleic acid of any one of claims 1-7.

9. The vector of claim 8, wherein the vector is selected from the group consisting of a plasmid and a recombinant viral genome.

10. The vector of claim 9, wherein the recombinant viral genome is selected from the group consisting of a recombinant AAV genome and / a recombinant adenoviral genome.

11. A viral particle comprising a nucleic acid of any one of claims 1-7 or a vector of any one of claims 8-10.55 / 5712. The viral particle of claim 11, further comprising a viral capsid protein.

13. The viral particle of claim 12, wherein the viral capsid protein is selected from an AAV capsid protein and an adenoviral capsid protein.

14. The viral particle of 13, wherein the AAV capsid protein is selected from an AAV1, AAV2, AAV4, AAV5, AAV7, AAV8, AAV9, AAV10 and AAV5 capsid protein.

15. A composition comprising a nucleic acid of any one of claims 1-7, a vector of any one of claim 8-10, or a viral particle of any one of claims 11-14.

16. The composition of claim 15, further comprising a pharmaceutically acceptable excipient.

17. A method of treating a retinopathy in a subject in need thereof, the method comprising administering to the subject a nucleic acid of any one of claims 1-7, a vector of any one of claims 8-10, a viral particle of any one of claims 11-14, or a composition of claim 15 or 16 in an amount that is effective in treating the retinopathy, wherein the treating comprises prophylactic treatment and therapeutic treatment.

18. The method of claim 17, wherein the retinopathy is ischemic, inflammatory, or proliferative retinopathy.

19. The method of claim 17 or 18, wherein the retinopathy is diabetic retinopathy, macular edema, age-related macular degeneration, or retinopathy of prematurity.

20. The method of any one of claims 17-19, wherein the administering is by local administration.

21. The method of claim 20, wherein the local administration is by intraocular, intravitreal, or subretinal injection.56 / 57

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