Compositions and methods for identifying compounds that protect against lipofuscin cytotoxicity

Abstract

The present disclosure provides compositions and methods for treating ocular diseases (eg, retinopathies), and in particular ocular diseases associated with cytotoxic lipofuscin-associated cytotoxicity in retinal cells.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 140,533, filed January 22, 2021, the entire contents of which are incorporated herein by reference. Statement of government support This invention was made with Government support under EY027422-04 awarded by the National Institutes of Health / National Eye Institute. The Government has certain rights in this invention. The present technology relates generally to compositions and methods for treating ocular diseases (eg, retinopathies), and in particular, ocular diseases associated with cytotoxic lipofuscin-related cytotoxicity in retinal cells. [Background technology]

[0002] The following description of the background of the present technology is provided merely as an aid in the understanding of the present technology and is not admitted to describe or constitute prior art to the present technology. Retinal pigment epithelium (RPE) cell death is the primary cause of geographic atrophy (GA) in the retina in Stargardt and dry AMD, the most prevalent and incurable inherited and age-related blinding disorders among young and old people, respectively. Lipofuscin (LF) is a fine yellow-brown pigment composed of indigestible material that is believed to be a remnant after lysosomal digestion. LF is composed mostly of dimers of retinaldehyde, known as lipid bisretinoids, and small amounts of carbohydrates, oxidized proteins, and metals. Accumulation of LF in retinal cells causes retinal toxicity, which is associated with conditions such as macular degeneration, ocular degenerative diseases, and Stargardt disease. Furthermore, the mechanism and extent to which LF contributes to degeneration is partially unclear, as all attempts to target its cytotoxic effects have failed to maintain RPE viability and halt retina's decay. Thus, novel molecular targets for treating Stargardt and dry AMD followed by GA are urgently needed. Summary of the Invention

[0003] In one aspect, the present disclosure provides a method for preventing or treating an ocular disease associated with retinal cell lipofuscin-associated cytotoxicity in a subject in need thereof, comprising administering to the subject an effective amount of at least one therapeutic agent selected from the group consisting of dabrafenib, necrosulfonamide (NSA), arimoclomol, kinase inhibitor RNase attenuator (KIRA) compounds, salubrinal, SAL003, and any pharma- ceutically acceptable salt thereof, wherein the ocular disease associated with retinal cell lipofuscin-associated cytotoxicity is autosomal recessive retinitis pigmentosa (RP), Stargardt's disease (STGD), Best's disease (BD), cone-rod dystrophy, or ABCA4 mutation age-related macular degeneration (AMD). Examples of KIRA compounds include, but are not limited to, KIRA3, KIRA6, KIRA7, or KIRA8. In some embodiments, the subject comprises a mutation in ABCA4 and / or RDH12. The ABCA4 and / or RDH12 mutations can be homozygous or heterozygous. Additionally, or alternatively, in some embodiments of the methods disclosed herein, administration of an effective amount of at least one therapeutic agent prevents exacerbation of lipofuscin-associated cytotoxicity in retinal cells of the subject. In another aspect, the disclosure provides a method for preventing or treating an ABCA4 mutant eye disease associated with retinal cell lipofuscin-associated cytotoxicity in a subject in need thereof, comprising administering an effective amount of Necrostatin 7 (Nec7) or a pharma- ceutically acceptable salt thereof to the subject, wherein the ABCA4 mutant eye disease associated with retinal cell lipofuscin-associated cytotoxicity is autosomal recessive retinitis pigmentosa (RP), cone-rod dystrophy, or age-related macular degeneration (AMD). In some embodiments, administering an effective amount of Nec7 or a pharma-ceutically acceptable salt thereof prevents exacerbation of lipofuscin-associated cytotoxicity in retinal cells in the subject.

[0004] In any and all embodiments of the methods disclosed herein, the eye disease is hereditary, non-hereditary, or age-related. In some embodiments of the methods disclosed herein, the AMD is dry AMD. In other aspects of the methods disclosed herein, the cone-rod dystrophy is autosomal recessive cone-rod dystrophy. Additionally, or alternatively, in some embodiments of the methods disclosed herein, the subject is a patient with any of the following AML phenotypes: ABCA4 D2177N, ABCA4 G1961E, ABCA4 G863A, ABCA4 1847delA, ABCA4 L541P, ABCA4 T2028I, ABCA4 N247I, ABCA4 E1122K, ABCA4 W499 * , ABCA4 A1773V, ABCA4 H55R, ABCA4 A1038V, ABCA4 IVS30+1G→T, ABCA4 IVS40+5G→A, ABCA4 IVS14+1G→C, and ABCA4 F1440del1cT. In any and all embodiments of the methods disclosed herein, the control has at least one ABCA4 mutation selected from the group consisting of RDH12 G127 * , RDH12 Q189 * , RDH12 Y226C, RDH12 A269Gfs * , RDH12-L274P, RDH12-R65 * , RDH12 H151D, RDH12 T155I, RDH12 V41L, RDH12 R314W, and RDH12 V146D. Additionally or alternatively, in some embodiments of the methods disclosed herein, at least one therapeutic agent of the present technology (e.g., dabrafenib, NSA, arimoclomol, KIRA compounds, salubrinal, SAL003, Nec7, or a pharmaceutically acceptable salt thereof) reduces or eliminates lipofuscin bisretinoid (LB) lipid-induced phosphorylation and / or polymerization of MLKL. In any and all embodiments of the methods disclosed herein, at least one therapeutic agent of the present technology (e.g., dabrafenib, NSA, arimoclomol, KIRA compounds, salubrinal, SAL003, Nec7, or a pharmaceutically acceptable salt thereof) reverses the LB lipid-induced translocation of phosphorylated MLKL (pMLKL) to the plasma membrane of retinal pigment epithelial cells. In certain embodiments, the LB lipid is selected from the group consisting of N-retinylidene-N-retinylethanolamine (A2E), A2E isomers, oxidized derivatives of A2E, and all-trans retinal dimer (ATRD).

[0005] In any of the preceding embodiments of the methods disclosed herein, at least one therapeutic agent of the present technology (e.g., dabrafenib, NSA, arimoclomol, KIRA compound, salubrinal, SAL003, Nec7, or a pharma- ceutically acceptable salt thereof) reduces the mRNA or protein levels of one or more genes associated with retinal degeneration, inflammation / angiogenesis, ER stress, and / or necroptosis. Examples of one or more genes associated with retinal degeneration, inflammation / angiogenesis, ER stress, and / or necroptosis include, but are not limited to, EDN2, FGF2, GFAP, SERP, VEGF, CXCL15, XBP1s, SCAND1, CEBPA, and HMGA. Additionally, or alternatively, in some embodiments of the methods disclosed herein, at least one therapeutic agent of the present technology (e.g., dabrafenib, NSA, arimoclomol, KIRA compound, salubrinal, SAL003, Nec7, or a pharma- ceutically acceptable salt thereof) inhibits or reduces lipofuscin-induced necroptosis and / or reduces infiltration of activated microglia / macrophages in retinal pigment epithelial cells.

[0006] In any and all embodiments of the methods disclosed herein, at least one therapeutic agent of the present technology (e.g., dabrafenib, NSA, arimoclomol, KIRA compounds, salvulinal, SAL003, Nec7, or a pharmaceutically acceptable salt thereof) is administered via topical, intravenous, intraocular, subretinal, or subscleral administration. Additionally or alternatively, in some embodiments of the methods disclosed herein, at least one therapeutic agent of the present technology (e.g., dabrafenib, NSA, arimoclomol, KIRA compounds, salvulinal, SAL003, Nec7, or a pharmaceutically acceptable salt thereof) is conjugated to a drug that targets retinal pigment epithelial cells. Examples of drugs that target retinal pigment epithelial cells include, but are not limited to, tamoxifen, chloroquine (CQ) / hydroxychloroquine (HCQ), ethambutol (EMB), or sodium iodate (NaIO3). Other examples of RPE targeting agents are described in Crisostomo S, Vieira L, Cardigos J (2019) Retina: 23-28; Michaelides M (2011) Arch Ophthalmol 129(1): 30; Tsai RK, He MS, Chen ZY, Wu WC, Wu WS (2011) Mol Vis 17(June): 1564-1576; MacHalinska A et al. (2010) Neurochem Res 35(11): 1819-1827; Tsang SH, Sharma T (2018) Drug-Induced Retinal Toxicity. Atlas of Inherited Retinal Diseases, eds Tsang SH, Sharma T (Springer International Publishing, Cham), pp 227-232. [Brief description of the drawings]

[0007] [Fig. 1A-1E]Figure 1A: Autofluorescence images of flat-mounted RPE eyecups from young and old WT and DKO mice. Individual fields at 10X were stitched together and presented in the following orientations: dorsal-superior; ventral-inferior; nasal-right; temporal-left, bar = 1 mm. Inset shows a high-magnification field of the RPE in the central-equatorial area, where yellow corresponds to LF granules, red corresponds to ZO1 borders, and blue corresponds to Hoechst-stained nuclei, bar = 10 μm. Figure 1B: LF fluorescence per RPE quantified by 63X microscopy. Borders were visualized with phalloidin and individual cells were manually selected in ImageJ. Each dot is the integrated autofluorescence / cell of RPE in central retina of 8 and 33 month old WT (n=5); 3 month old DKO (n=4); 8 month old DKO (n=6); 13 month old DKO (n=4); 26 month old DKO (n=5). LF content of 3 month old DKO was higher than 33 month old WT (p<0.01). LF / cell was also significantly different among all DKO age groups (WT 8 months vs. WT 33 months, p<0.05; DKO 3 months vs. WT 33 months, p<0.01; p<0.001; DKO 8 months / DKO 13 months / DKO 26 months vs. WT 33 months, p<0.0001). Figure 1C: 63X image of lipofuscin in RPE layer, lipofuscin did not drop at 600 and 700 days. FIG1D: 63X images of phalloidin cytoskeleton (red) and LF granules (yellow) to show structural abnormalities associated with LF accumulation, bar=10 μm. FIG1E: HPLC quantification of A2E content in RPE and DKO mice at different ages. Each dot is the content in one eye. Bars represent median values ​​per age group. [Figure 2A-2H]Degenerative changes in the retina associated with LF accumulation. Figure 2A: RPE size (μm2) in the central retina. Each dot is the area / cell in 8-month (n=5) and 27-month (n=5) WT and 8-month (n=6) and 23-month (n=5) DKO animals. The oldest DKO group showed significantly enlarged RPE compared to all other groups (*p<0.01 by unpaired t-test in Prism7). Figure 2B: Number of RPE nuclei counted at every 0.1 mm interval and plotted as a function of distance from the ONH in 23-month-old DKO (n=10) and 27-month-old WT retinas (n=8). Means (±SEM) were significantly different for each point (DKO vs. WT, p<0.05 by multiple t-test in Prism7). Figure 2C: ONL thickness measured at every 0.1 mm interval from the ONH along the vertical axis in DKO (n=5) and WT control eyes (n=7) at 27 months of age. Means (±SEM) were significantly different for each point (DKO vs. WT, p<0.05 by multiple t-test in Prism7). Figure 2D: Representative fluorescence microscopy of frozen sections of 30-month-old WT (n=3) and DKO (n=3) mice. DKO neural retinal cells expressed a massive infiltration of LF particles of about 1-3 μm, 20 μm scale. Figure 2E: The image on the left is a melanin-bleached paraffin-embedded transverse section of a DKO 20-month-old mouse showing a LF infiltration of about 1-3 μm strongly positive for Iba1. The image on the right is a brightfield / fluorescence overlay of a 10-month-old DKO showing ~1-3 μm particles positive for rhodopsin (red) and negative for melanin (scale bar = 20 μm). Figure 2F: Cross section of an 800-day DKO showing sloughed RPE (arrows) migrating into the neural retina. Figure 2G: Maximum projection of neural retinal flat mount and Z-stack cut sections obtained by confocal microscopy showing migratory RPE inserted at different depths into the photoreceptor layer. Scale bar = 20 μm. Figure 2H: H&E of paraffin-embedded cross section showing migration and lamellar stacking in DKO RPE. Migrating RPE contain melanin and are ~5-10 μm. DKO showed damage to the ONL overlying the RPE with migratory / proliferative behavior. [Fig. 2I-2J]Degenerative changes in the retina associated with LF accumulation. (Figure 2I) Representative photomicrographs show that microglial cells appearing in the outer segments of DKO mouse eyes are positive for CD11b (red) and IBA1 (green) and also accumulate LB (white). (Figure 2J) RPE cells from DKO mice of different ages show increased accumulated A2E up to 200 days, followed by less A2E accumulation thereafter, as measured by HPLC. [Figure 3A-3B] Light-independent LF cytotoxicity is the primary contributor to pigmented retinal degeneration. Figure 3A: Loss of photoreceptors and RPE between 2 and 12 months of age in animals (n=6 per light, age and genotype condition) housed in complete darkness vs. 12-h light-dark cycles. Red and blue correspond to ONL thickness, while orange and grey circles are the number of RPE nuclei, in 2- and 12-month-old mice, respectively. Only DKO showed ONL thinning and RPE loss during that time period by two-way ANOVA (p<0.01), which was similar between light-period and dark-housed mice. Figure 3B: Autofluorescence of the eyecups of 12-month-old DKO and WT housed in dark or cyclic lighting. Images from individual fields taken with a 5X objective (λexc=430 nm, λemm=610 nm) were stitched and oriented dorsal-superior; ventral-inferior; nasal-right; temporal-left. Scale bar = 1 mm. [Figure 4A-4I]Light-independent LF cytotoxicity induces atypical necroptosis. Figure 4A: Cell death assay to study LF dark toxicity. 90% confluent ARPE-19 or hfRPE were incubated overnight in serum-free medium supplemented with the indicated lipid bisretinoid (LB) concentrations. Combined autofluorescence was localized within lamp2 lysosomes. Viability was assessed at 24 hours by microscopy with AlamarBlue® or DRAQ7 / NUC405. Figure 4B: Real-time monitoring of necrotic (DRAQ7 in red = plasma membrane leakage) and apoptotic (NUC405 in blue = caspase 3 activation) cell death by automated fluorescence microscopy. Figure 4C: Neutralization of detergent activity does not protect against A2E. Compared to untreated cells, 10 μM methyl-βCD counteracted 500 μM TRITON-X100 in A2E, TRITON, and A2E with MBCD (*p<0.001). p-values ​​were determined by t-test in Prism 7.0 and did not affect the cytotoxicity of 20 μM A2E. Figure 4D: Inhibition of the effector cascade of programmed necrosis. Dose-dependent protection with NSA (p<0.01) without pan-caspase, gasdermin D, or RIPK3 inhibitors (n=4). Figure 4E: IP with anti-MLKL showed significantly increased kinase activity (p<0.01) only in pulldowns from cells with accumulated A2E, suggesting the formation of the necrosome. Figure 4F: Western blot with anti-Ser358 phospho-MLKL showing dose-dependent phosphorylation and polymerization of human MLKL. Figure 4G: Protection by necrostatins. Anti-RIPK1 necrostatins: Necrostatin 7 (Nec7) (p<0.01), but not 1, 1s, and 5, protected against A2E cytotoxicity (n=3). Figure 4H: Western blot showing that Nec7 prevents phosphorylation and polymerization of MLKL by A2E. Figure 4I: Fluorescence images of ARPE-19 monolayers showing that A2E and ATRD induced translocation of pMLKL to the plasma membrane was blocked by Nec7. [Fig. 4J-4T]Figure 4J: Viability assay used to test the toxicity of A2E 20μM and ATRD 80μM in ARPE19 cultures at different cell numbers. Fully confluent ARPE19 cells were more resistant to LB cell death. Figure 4K: Increased cell confluency affects the amount of A2E taken up by cells. Figure 4L: 20μM A2E was loaded into ARPE19 cells at different confluencies for 24 hours. The amount of A2E / cell in cultured cells was comparable to the amount of lipofuscin in RPE cells in 800-day-old DKO mice. Figure 4M: Comparison of ARPE19 and hfRPE cell viability with A2E treatment. hfRPE cells were more resistant to A2E than ARPE19 cells at 20μM, 30μM and 45μM (p<0.05, t-test). FIG. 4N: Light-independent LF cytotoxicity in hfRPE compared to ARPE19. FIG. 4O: Necroptosis cascade induced by virus, toll-like agonist, and TNF shows that the pathway is significantly dependent on the nature of the necroptotic stimulus. FIG. 4P: Dose-dependent induction of MLKL phosphorylation and polymerization in ATRD. FIG. 4Q: MLKL phosphorylation and polymerization was not prevented by either Nec1 or GSL'872 and was suppressed by Nec7 but not by Nec1 (FIG. 4R). FIG. 4S: Analysis by RNAseq of the expression of different isoforms of RIPK1, 2, 3, and 4 in ARPE19 cells. FIG. 4T: WB analyzing the activation of pMLKL, RIPK1, RIPK3 in HT29 undergoing cell death by treatment with A2E, ATRD, or STZ. [Figure 4U] FIG. 1 shows a Western blot demonstrating that Nec7, but not Nec1, prevents MLKL phosphorylation / polymerization induced by lipofuscin substances. [Figure 4V] Melanin does not affect fluorescence quantification. Lysis buffer or RPE lysates from WT C57BL6 obtained as described in M&M were spiked with 300 pmol A2E per ml and 430 nm / 600 nm fluorescence was used for quantification. [Figure 4W]FIG. 1 shows dose-dependent cell death in ARPE19 cells exposed to A2E, ATRD, and ATR. [Figure 4X] FIG. 1 shows that pretreatment with 33 μM Nec1, Nec1s, or Nec7 did not block ATR-induced cell death. [Figure 4Y] FIG. 1 shows that Western blots with anti-phospho-MLKL and GAPDH show that ATR does not alter the phosphorylation status of MLKL. [Figure 5A-E] Figure 5: LF necroptosis does not involve oxidative stress but does involve ER stress. Figure 5A: Antioxidants such as Trolox, NAC, L-Cys, Vit-C, BHA and TMB did not rescue ARPE-19 from different doses of A2E. Figure 5B: RNAseq / IPA analysis revealed that the protective effect of Nec7 mainly involved the reduction of the unfolded protein response (UPR) pathway. Figure 5C: View of the canonical UPR cascade showing multiple mRNAs in the IRE1α and PERK pathways that are downregulated by Nec7. Figure 5D: IPA predicted survival effect of downregulating UPR with Nec7. [Figure 5E] FIG. 1 shows that Nec7 neutralizes the effect of A2E as evidenced by the close clustering of Ctr-Nec7 and A2E-Nec7 in both heat-diagrams and PCA analysis. [Figures 6A-6N]LF triggers ER stress and inhibitors of IRE1α block necroptosis. Figure 6A: Western blot showing p-eIF2a, ATF4 and BiP / GRP78 were downregulated by LF without illumination. Figure 6B: ATF4 mRNA induction by A2E 25 μM and 80 μM ATRD in the dark detected by qPCR (n=2). Dose (Figure 6C) and kinetics (Figure 6D) of XBP1s induction by A2E and ATRD detected by qPCR (n=3). Figure 6E: Agarose gel confirmation of XBP1 splicing in both ARPE-19 and primary hfRPE cells (n=2). Figure 6F: Western blot showing cleavage of ATF6 in A2E-accumulated cells. Figure 6G: qPCR showing Nec7 blocks XBP1s (IRE1α branch). Figure 6H: Western blot showing that Nec7 also blocked CHOP (downstream of PERK) and that neither Nec1 nor GSK'872 affected the UPR, consistent with the lack of protection from necroptosis. Figure 6I: Viability of ARPE-19 cells against LF after individual knockdown of ER stress sensors / effectors with shRNA. Only IRE1α knockdown conferred protection from A2E and ATRD (p<0.05, n=3). Figure 6J: Selective inhibition of IRE1α kinase and / or RNase activation had no protective effect from LF, whereas a drug that blocks IRE1α dimerization (KIRA) increased viability. Figure 6K: Western blot showing that IRE1α inhibitor KIRA6 prevents LF-induced phosphorylation and polymerization of MLKL. Figure 6L: Immunostaining of ARPE19 cells accumulating LF and treated with KIRA6 shows inhibition of pMLKL plasma membrane translocation. Figure 6M: Efficacy of KIRA6 and Nec7 to promote survival against LF in hfRPE. Figure 6N: qPCR confirming induction of XBP1 splicing by LF and its prevention by Nec7 in hfRPE. [Figure 6O]Figure 2: Antioxidants fail to prevent LF-induced UPR.WB showing LF-induced light-independent phosphorylation of IRE1α remains unaffected in the presence of NAC. [Figure 7A-7F] ER stress and necroptosis in DKO cells. Fig. 7A: Flat-mounted RPE optic cup immunostained with anti-XBP1s (red) and nuclear DAPI (blue) in central equatorial RPE from aged DKO as indicated. XBP1s was negligible in aged WT but detectable in 2-month-old DKO and became stronger with age. Bar = 20 μm (n = 3 per group). Fig. 7B: ER stress monitored with anti-phospho-Ser345-MLKL (red) and nuclear DAPI (blue) showed an age-related increase in label localized to the plasma membrane in the oldest group (n = 3), scale bar = 20 μm. Fig. 7C: Demelanized paraffin cross sections showing XBP1s (green) and pMLKL (red) co-expression on the RPE layer and in small Iba1+ cells ~1-3 μm apart. FIG. 7D: Iba1+ cells in the subretinal space co-expressing Iba1 (red) / XBP1s (green) or Iba1 (red) / MLKL (green), scale bar = 20 μm. FIG. 7E: Panoramic view of the neural retina with a large area of ​​large "spots". Neural retinal flat mounts were immunostained with anti-pMLKL (red). A striking feature was a halo of pMLKL in the layer of ONL surrounding the RPE infiltrate. FIG. 7F: Enlarged view of the squared area shown in FIG. 7E, with the maximum shown on the z-axis showing that the necroptosis signal spreads in all directions around the invading RPE fragment. [Fig. 7G-7I]Figure 7G: Exemplary immunofluorescence confocal images showing co-localization of p-MLKL (red), XBP1s (green) and LB (white) on RPE flat mount samples from postnatal day 700 mice (n=3). Bar=20 μm. Figure 7H: A single 1 μl intraocular injection of Nec7 reduces pMLKL levels in the retina as shown in RPE flat mounts from treated DKOs. Figure 7I: Mean fluorescence intensity (MFI) plots of pMLKL measured at 0.1 mm intervals and plotted as a function of distance from the ONH in the upper half of the retina for vehicle-treated and Nec7-treated eyes of postnatal day 700 DKOs; (quantified by Image J, **p<0.01 for mock vs. KIRA6, n=3; determined by two-way ANOVA in GraphPad Prism). [Figure 7J] FIG. 1 shows microglia / macrophages attached to RPE flat-mounted eyecups from 20-month-old DKO retinas stained positive for phospho-MLKL. [Figure 7K] Phospho-MLKL staining (red) in areas of RPE rich in lipofuscin (yellow) and with strong migratory activity. [Figure 7L] FIG. 1 shows that a single intravitreal injection of Nec7, but not Nec1, eliminated phospho-MLKL staining in 20-month-old DKO retinas (n=4). [Figure 7M] FIG. 1 shows a representative neural retinal flat mount showing the reduction of phospho-MLKL in the photoreceptor layer after receiving intraocular Nec7 one week previously. [Figure 7N] Subretinal infiltration of CD11b cells on RPE flat mounts from 18-20 month old DKOs that received an intravitreal injection of 2 μl of vehicle (control) or Nec7 one week previously. Bar = 20 μm. [Figure 8A-8E]IRE1α inhibitors reduce inflammation and necroptosis in LF-bearing retinas. FIG. 8A: Intravitreal injection of KIRA6 reduces ER stress in DKO retinas. Center-to-periphery images along the vertical axis of RPE flat mounts from right (OD) and left (OS) eyes of 17-month-old DKOs treated with 1 μl of vehicle (mock) or KIRA6, respectively. Immunostaining for XBP1s is represented in green. Bar=20 μm. Inset is a magnified view of the RPE in the central retina in these eyes. Bar=20 μm. Plot of mean fluorescence intensity (MFI) of XBP1s (green) immunostaining measured at 0.1 mm intervals and plotted as a function of distance from the ONH in the upper half of the retina for vehicle- and KIRA6-treated eyes of postnatal day 600 DKO; (quantified by Image J, **p<0.01 for mock vs. KIRA6, n=3; determined by two-way ANOVA in GraphPad Prism). Figure 8B: Center-to-periphery images along the vertical axis of RPE flat mounts from right (control) and left (treated) eyes of postnatal day 512 DKO. Mice received a single 1 μl intravitreal injection of vehicle or KIRA6 per eye. pMLKL marker of necroptosis (red) was dramatically reduced (n=3). Bar=20 μm. Inset is a magnified view of the RPE in the central retina in these eyes, scale 20 μm. Plot of mean fluorescence intensity (MFI) of pMLKL (red) immunostaining measured at 0.1 mm intervals and plotted as a function of distance from the ONH in the upper half of the retina in vehicle- and KIRA6-treated eyes of postnatal day 512 DKO; (quantified by Image J, **p<0.01 for mock vs. KIRA6, n=3; determined by two-way ANOVA in GraphPad Prism). Figure 8C: Dot plots representing the percentage of XBP1s / pMLKL double positive cells in cryosections of 600 day DKO. Top panel is OD-vehicle and bottom panel is OS-KIRA6 treated. Figure 8D: KIRA6 reduces the number of Iba-1+ microglia / macrophages infiltrating the outer retina of 800 day DKO (n=3).(E) qPCR showing normalization of downregulated transcripts during photoreceptor degeneration to KIRA6. (F) Comparison of multiple markers of ongoing retinal degeneration in WT and DKO mice as detected by qPCR. [Figure 9A] FIG. 1 shows an exemplary model illustrating protection by Salubrinal (and SAL003) against retinal lipofuscin. [Figure 9B] 1 shows an exemplary mechanism of action of the composition of the present technology to protect against light-independent lipofuscin cytotoxicity. Lipofuscin forms solid crystals that, in large quantities, puncture the lysosomal membrane, causing LMP. Release of lysosomal enzymes triggers the formation of atypical necrosomes that phosphorylate MLKL, promoting its oligomerization and membrane translocation. Phospho-MLKL destabilizes the lysosomal membrane, promoting more LMP. The level of phospho-MLKL in the plasma membrane becomes unacceptable, and cells undergo necroptosis. [Figure 10] FIG. 1 shows antibodies used in the examples described herein. [Figure 11] FIG. 1 shows primer sequences used in the examples described herein. [Figure 12] FIG. 1 shows chemical inhibitors used in the examples described herein. [Figure 13] FIG. 1 shows antibodies and fluorescent probes used in the examples described herein. [Figure 14A] FIG. 1 shows that without illumination, cellular ROS (red) were detected in ARPE19 mitochondria but not in lipofuscin granules (green). Only after blue light exposure did ROS colocalize with lipofuscin. [Figure 14B] A2E (MW=592) cannot pass through a 0.45 μm filter, suggesting that it forms aggregates. [Figure 14C] FIG. 1 shows the correlation between MW and membrane cutoff of molecules that do not form aggregates. [Figure 14D]DIC and autofluorescence (green) show distinct A2E granules in cells co-stained with lysotracker (red). Yellow is due to green-red overlap. [Figure 14E] FIG. 1 shows A2E crystals after evaporation of the solvent on a cover-sleep. [Figure 14F] Galectin-3 puncta assay to assess lysosomal membrane damage. Both positive controls LLO and A2E induced lysosomal membrane permeabilization (LMP). [Figure 14G] FIG. 1 shows inactivation of cathepsin D in cells exposed to different doses of LLO. [Figure 14H] FIG. 1 shows cathepsin D in cells with different amounts of A2E. [Figure 14I] FIG. 1 shows that loss of cathepsin D activity can be prevented with arimoclomol or necrostatin 7. [Figure 14J] FIG. 1 shows that arimoclomol or Nec7 promote survival against A2E accumulation. [Figure 14K] FIG. 1 shows that the LMP inducer LLO promotes Nec7, but not Nec1, and arimoclomol-preventable atypical necroptosis. [Figure 15A] 1 is a heatmap showing protein levels detected by mass spectrometry in cells containing lipofuscin (15 μM-24 hr A2E, loaded overnight) versus healthy controls. Up- and down-regulated levels are represented on an orange to blue scale, respectively. [Figure 15B]Causal network association and hierarchical clustering analysis using Ingenuity Pathway Analysis (IPA) identified cellular processes induced by lipofuscin. The dendrogram constructed based on Pearson correlation metric and average clustering method shows that sublethal doses of lipofuscin predominantly induce anti-necroptosis response. The statistical significance expressed as the negative common logarithm of the p-value obtained by the right-sided Fisher exact test of IPA is the probability that the cellular process or signaling pathway identified by IPA is not due to chance. -log(p-value) is indicated by the diameter of the circle. IPA also assigned a z-score that predicted the overall activation / inhibition status of the cell / signaling pathway, shown here as the color of the circle (blue-orange scale). [Figure 15C] Figure 1 shows Ingenuity software identification of the main signaling cascades responsible for the inhibition of necroptotic cell death and survival of lipofuscin occupied cells: eIF2a, eIF4, mTOR, ubiquitin proteasome system (UPS), integrin signaling (IS), and remodeling of epithelial adherence junctions (REAJ). Circle size and color indicate statistical significance (-log(p-value)) and direction of regulation, respectively. [Figure 15D] FIG. 1 shows IPA analysis of cellular processes individually regulated by eIF2a, eIF4, mTOR, the ubiquitin proteasome system (UPS), integrin signaling (IS), and remodeling of epithelial adherens junctions (REAJ). [Figure 15E] FIG. 1 shows IPA identification of the molecular processes by which eIF2a, eIF4, mTOR, and the UPS counteract lipofuscin necro-toxicity. [Figure 16A]Figure 1 shows the identity of proteins regulated by lipofuscin and their association with the anti-necroptotic signaling pathways, eIF2a, eIF4, mTOR, and UPS. The data indicate profound reshaping of the cellular proteomics through alterations in the initiation of protein translation and ubiquitination. [Figure 16B] FIG. 1 shows the symbols of the increased degradative machinery responsible for the degradation of ER-synthesized proteins induced by sublethal doses of lipofuscin. [Figure 17] Figure 1 shows IPA analysis of signaling pathways induced by lipofuscin in vivo. Comparison of differences in mRNA levels detected by bulk RNAseq between RPE / choroid from 100-day and 800-day ABCA4- / -RDH8- / - double knockout (DKO) mice using Ingenuity pathway analysis. [Figure 18A] ARPE19 cells were pretreated for 1 h with agonists of eIF2a (salubrinal (SAL), SAL003, or guanabenz); eIF4 (briciclib or eFT508); mTOR (rapamycin, Torin-1); or a nonspecific protein translation inhibitor (cyclohexamide CHX) and then incubated with a lethal dose of A2E (25 μM) for an additional 24 h in the presence of these drugs. Viability was assessed with AlamarBlue®. Only SAL and SAL003, which target both eIF2α phosphatases in cells that are composed of PP1 bound to either catalytic subunit GADD34 or CreP, protected against lipofuscin. In contrast, guanabenz, which only disrupts PP1-GADD34 association or eIF4 and mTOR activators, did not confer significant protection. [Figure 18B] 1 shows protection by SAL against increasing doses of A2E or all-trans retinal dimer (ATRD), two of the most abundant bis-retinoids in retinal lipofuscin. Viability was assessed with AlamarBlue®. [Figure 18C]14. SAL does not protect against phototoxic degradation of lipid bisretinoids. ARPE-19 cells were incubated overnight with a non-toxic dose of A2E (5 μM) to allow its uptake into lysosomes, the medium was changed to PBS, and cells were irradiated with blue light for 10 min, after which the cells were maintained in Optimem for a further 24 h before viability was assessed with AlamarBlue®. [Figure 18D] Fluorescence microscopy of cells incubated for 24 hours with a lethal dose of A2E (25 μM) in Optimem. Green fluorescence corresponds to A2E deposits. Nuclei were stained with the DNA dye Hoechst in viable cells (blue) and with Hoechst and DRAQ7 (a DNA dye that only enters cells with disrupted membranes) in dead cells (purple). [Figure 19A] Since SAL is a known activator of eIF2a through preservation of its phosphorylation state and PERK is a kinase of eIF2a; ARPE19 cells were pretreated with SAL in the presence (or absence) of a potent and specific PERK inhibitor (GSK2606414) for 1 h and then incubated with a lethal dose of A2E (25 μM) in the presence of the drugs for an additional 24 h. Viability was assessed with AlamarBlue®. [Figure 19B] Figure 1 shows that knockdown of ATF4 did not prevent SAL from protecting against lipofuscin. Because ATF4 is the main downstream effector of PERK, ARPE-19 cells were transduced with lentivirus expressing scrambled shRNA or ATF4-shRNA for 48 hours and then incubated with 25 μM A2E in the presence or absence of SAL for an additional 24 hours. Viability was assessed with AlamarBlue®. [Figure 20A] The level of spliced ​​XBP1 (XBP1s), measured by quantitative real-time PCR as a readout of IRE1a activity, increases in a dose-dependent manner with the amount of lipofuscin accumulated in cells. IRE1a activity can be abrogated by treatment with SAL. IRE3 is a potent inhibitor of IRE1a and is used here as a control. [Figure 20B] Immunofluorescence staining of phospho-MLKL (green), indicating that cells undergoing necroptosis by A2E or ATRD, shows phospho-MLKL membrane localization that can be abrogated by treatment with SAL. IRE3 was used as a positive control for IRE1a inhibition. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0008] It will be appreciated that certain aspects, modes, embodiments, variations and features of the present methods are described below at various levels of detail in order to provide a substantial understanding of the present technology. In carrying out the present methods, many conventional techniques in molecular biology, protein biochemistry, cell biology, immunology, microbiology and recombinant DNA are used. For example, Sambrook and Russell eds. (2001) Molecular Cloning: A Laboratory Manual, 3rd edition; the series Ausubel et al. eds. (2007) Current Protocols in Molecular Biology; the series Methods in Enzymology (Academic Press, Inc., NY); MacPherson et al. (1991) PCR 1: A Practical Approach (IRL Press at Oxford University Press); MacPherson et al. (1995) PCR 2: A Practical Approach; Harlow and Lane eds. (1999) Antibodies, A Laboratory Manual;Freshney (2005) Culture of Animal Cells: A Manual of Basic Technique, 5th edition;Gait ed. (1984) Oligonucleotide Synthesis;US Patent No. 4,683,195;Hames and Higgins eds. (1984) Nucleic Acid Hybridization;Anderson (1999) Nucleic Acid Hybridization;Hames and Higgins eds. (1984) Transcription and Translation;Immobilized Cells and Enzymes (IRL Press (1986));Perbal (1984) A Practical Guide to Molecular Cloning;Miller and Calos eds.(1987) Gene Transfer Vectors for Mammalian Cells (Cold Spring Harbor Laboratory); Makrides ed. (2003) Gene Transfer and Expression in Mammalian Cells; Mayer and Walker eds. (1987) Immunochemical Methods in Cell and Molecular Biology (Academic Press, London); and Herzenberg et al. eds (1996) Weir's Handbook of Experimental Immunology. Methods for detecting and measuring levels of polypeptide gene expression products (i.e., gene translation levels) are well known in the art and include the use of polypeptide detection methods such as antibody detection and quantification techniques. (See also Strachan & Read, Human Molecular Genetics, Second Edition. (John Wiley and Sons, Inc., NY, 1999)).

[0009] Methods and compositions are provided for maintaining viability of the RPE layer in subjects with Stargardt's disease (STGR1 and STGR3); vitelliform macular degeneration (Best's macular dystrophy or Best's disease); autosomal recessive cone-rod dystrophy (ar-CRD) and autosomal recessive retinitis pigmentosa (ar-RP) secondary to mutations in either the ABCA4 or RDH12 genes; and ultimately individuals with dry age-related macular degeneration (dry AMD); choroidal melanoma; or severe ocular damage associated with increased fundus autofluorescence (FAF) (CJ Kennedy et al., Eye (Lond). 9 (Pt 6) (1995) 763-71; SK Verbakel et al., Prog. Retin. Eye Res. 66 (2018) 157-186; M. a van Driel et al., Ophthalmic Genet. 19 (1998) 117-122;A. Maugeri et al., Am. J. Hum. Genet. 67 (2000) 960-966;AV Cideciyan et al., Hum. Mol. Genet. 13 (2004) 525-534).

[0010] Excessive ER stress in photoreceptors has only been associated with autosomal dominant retinitis pigmentosa (adRP) but never with autosomal recessive retinitis pigmentosa (arRP). Comitato et al., Human Molecular Genetics, Vol. 25, No. 13 2801-2812 (2016). Chemical chaperones, drugs that increase the folding capacity in cells and decrease the activity of all (IRE1α, PERK and ATF) UPR sensors, were beneficial for adRP (SX Zhang et al., Exp. Eye Res. 125 (2014) 30-40; MS Gorbatyuk et al., Prog. Retin. Eye Res. (2020) 100860), but had no effect on ER stress caused by lipofuscin. Moreover, while treatment with salubrinal did indeed increase IRE1α activity in adRP retinas (Comitato et al., Human Molecular Genetics, Vol. 25, No. 13 2801-2812 (2016)), the Examples herein show that salubrinal reduced the same activity in cells with lipofuscin-induced ER stress.

[0011] In another model of retinal degeneration due to ER stress induced by administration of drugs that cause oxidative stress, inhibition of IRE1α had deleterious consequences (SX Zhang et al., Exp. Eye Res. 125 (2014) 30-40;T. McLaughlin et al., Mol. Neurodegener. 13 (2018) 1-15). KIRA inhibitors of IRE1α have been shown to be useful in protecting photoreceptors with large amounts of misfolded proteins from apoptosis (most cases of adRP) (R. Ghosh et al., Cell. (2014) 1-15;HC Feldman et al., ACS Chem. Biol. 11 (2016) 2195-2205), but were never used to protect PREs from lipid cytotoxicity.

[0012] The disclosed method is based on the following unexpected discoveries that change current thinking in the field of lipofuscin pathogenesis: 1) lipid bisretinoids render the lysosomes in which they are trapped leaky (increased lysosomal membrane permeabilization (LMP)); 2) cytoplasmic lipofuscin triggers the unfolded protein response (UPR); 3) lipofuscin-induced UPR induces the formation of atypical necrosomes that phosphorylate MLKL via the ER stress sensor IRE1α. Phospho-MLKL then self-assembles into pores that damage the ER, lysosomal membranes, and plasma membranes, creating an amplification loop "ER stress ←→ phospho-MLKL" that culminates in necrosis of lipofuscin-occupied cells. The lipofuscin-induced cell death pathway is fundamentally different from previously reported cell death mechanisms as it does not involve oxidative stress, apoptosis, or the typical necrosome, involving RIPK1 and RIPK3 kinases.

[0013] The disclosed method preserves visual function in subjects suffering from lipofuscin pathologies such as Stargardt's disease (STGD), autosomal recessive retinitis pigmentosa (RP), age-related macular degeneration (AMD), Best's disease (BD), or autosomal recessive cone-rod dystrophy by: i) administering an effective amount of a KIRA compound (e.g., KIRA3, KIRA6, KIRA7, KIRA8); ii) administering an effective amount of a salvulinal derivative; iii) administering an effective amount of necrostatin 7, necrosulfonamide (NSA), dabrafenib, or arimoclomol, which inhibits the formation of phospho-MLKL, thus interrupting the "phospho-MLKL→ER stress→IRE1α→phospho-MLKL" loop. These strategies may be applied individually or in combination to halt the degenerative process. The above-described approach is completely different from all previous attempts used to date to protect the retina from lipofuscin cytotoxicity, including inhibition of lipid bisretinoid formation, anti-apoptotic agents, antioxidants, or the use of light-blocking lenses.

[0014] definition Unless otherwise defined, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this technology belongs. As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, a reference to a "cell" includes a combination of two or more cells, etc. In general, the terminology used herein and the laboratory methods in cell culture, molecular genetics, organic chemistry, analytical chemistry, and nucleic acid chemistry and hybridization described below are well known and commonly employed in the art.

[0015] As used herein, the term "about" in reference to a number is generally understood to include numbers within 1%, 5%, or 10% in either direction (greater or lesser) of the number, unless otherwise stated or apparent from the context (except where such number is less than 0% or greater than 100% of a possible value). As used herein, "administration" of an agent or drug to a subject includes any route by which a compound is introduced or delivered to a subject to perform its intended function. Administration may be by any suitable route, including, but not limited to, oral, intranasal, parenteral (intravenous, intramuscular, intraperitoneal, or subcutaneous), rectal, intrathecal, or topical. Administration includes self-administration and administration by another.

[0016] As used herein, the term "biological sample" refers to sample material derived from living cells. Biological samples can include tissues, cells, protein or membrane extracts of cells, and biological fluids (e.g., ascites fluid or cerebrospinal fluid (CSF)) isolated from a subject, as well as tissues, cells, and fluids present within a subject. Biological samples of the present technology include, but are not limited to, samples taken from eye, breast tissue, kidney tissue, cervix, endometrium, head and neck, gallbladder, parotid tissue, prostate, brain, pituitary gland, kidney tissue, muscle, esophagus, stomach, small intestine, colon, liver, spleen, pancreas, thyroid, heart tissue, lung tissue, bladder, adipose tissue, lymph node tissue, uterus, ovarian tissue, adrenal tissue, testicular tissue, tonsil, thymus, blood, hair, buccal, skin, serum, plasma, CSF, semen, prostatic fluid, seminal plasma, urine, feces, sweat, saliva, sputum, mucus, bone marrow, lymph, and tears. Biological samples may also be obtained from biopsies of internal organs. Biological samples may be obtained from subjects for diagnosis or investigation, or from non-diseased individuals as controls or for basic research. Samples may be obtained by standard methods, including, for example, venipuncture and surgical biopsy. In certain embodiments, the biological sample is a tissue sample obtained by needle biopsy. As used herein, a "control" is an alternative sample used in an experiment for comparison purposes. A control can be "positive" or "negative". For example, if the purpose of an experiment is to determine the correlation of efficacy of a therapeutic agent for treating a particular type of disease, a positive control (a compound or composition known to exhibit the desired therapeutic effect) and a negative control (a subject or sample that does not receive therapy or receives a placebo) are typically employed.

[0017] As used herein, the term "effective amount" refers to an amount sufficient to achieve a desired therapeutic and / or prophylactic effect, such as an amount that results in the prevention or reduction of a disease or condition described herein or one or more signs or symptoms associated with a disease or condition described herein. In the context of therapeutic or prophylactic use, the amount of the composition administered to a subject will vary depending on the composition, the extent, type, and severity of the disease, as well as individual characteristics such as general health, age, sex, weight, and tolerance to drugs. Those skilled in the art will be able to determine appropriate dosages depending on these and other factors. The composition may also be administered in combination with one or more additional therapeutic compounds. In the methods described herein, the therapeutic composition may be administered to a subject having one or more signs or symptoms of a disease or condition described herein. As used herein, a "therapeutically effective amount" of a composition refers to a level of the composition at which the physiological effects of the disease or condition are ameliorated or eliminated. A therapeutically effective amount may be given in one or more administrations.

[0018] As used herein, "expression" includes one or more of the following: transcription of a gene into pre-mRNA; splicing and other processing of the pre-mRNA to yield mature mRNA; mRNA stability; translation of the mature mRNA into protein (including codon usage and tRNA availability); and glycosylation and / or other modifications of the translation product as necessary for proper expression and function.

[0019] As used herein, the terms "individual," "patient," or "subject" may be an individual organism, a vertebrate, a mammal, or a human. In some embodiments, the individual, patient, or subject is a human. As used herein, the term "pharmaceutical acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal compounds, isotonic and absorption delaying compounds, etc., that are compatible with pharmaceutical administration. Pharmaceutically acceptable carriers and their formulations are known to those skilled in the art and are described, for example, in Remington's Pharmaceutical Sciences (20th edition, ed. A. Gennaro, 2000, Lippincott, Williams & Wilkins, Philadelphia, Pa.). Examples of pharmaceutical acceptable carriers include liquid or solid fillers, diluents, excipients, manufacturing aids (e.g., lubricants, talc, magnesium, calcium or zinc stearate, or steric acid, or solvent encapsulating materials that are useful for introducing active agents into the body.

[0020] As used herein, "prevention," "prevent" or "preventing" of a disorder or condition refers to one or more compounds that, in a statistical sample, reduce the occurrence of a disorder or condition in a treated sample compared to an untreated control sample, or delay the occurrence of one or more symptoms of a disorder or condition compared to an untreated control sample. As used herein, the term "separate" therapeutic application refers to the administration of at least two active ingredients simultaneously or substantially simultaneously by different routes. As used herein, the term "sequential" therapeutic use refers to the administration of at least two active ingredients at different times, with the same or different routes of administration. In particular, sequential use refers to the whole administration of one of the active ingredients before the administration of the other or other begins. Thus, one of the active ingredients can be administered minutes, hours, or days before the administration of the other active ingredient or ingredients. In this case, there is no simultaneous treatment. As used herein, the term "simultaneous" therapeutic use refers to the administration of at least two active ingredients by the same route and at the same time or substantially the same time.

[0021] "Treating" or "treatment," as used herein, encompasses treatment of a disease or disorder described herein in a subject, such as a human, including: (i) inhibiting the disease or disorder, i.e., preventing its development; (ii) relieving the disease or disorder, i.e., causing regression of the disorder; (iii) slowing the progression of the disorder; and / or (iv) inhibiting, alleviating, or slowing the progression of one or more symptoms of the disease or disorder. In some embodiments, treating means that the symptoms associated with the disease are, for example, alleviated, reduced, cured, or placed in remission. It will also be appreciated that the various modes of treatment of the disorders described herein are intended to mean "substantially" which includes total but also less than total treatment, resulting in some biologically or medically relevant result. Treatment may be continuous, long-term treatment of chronic diseases, or may be a single or several doses for treatment of acute conditions.

[0022] Ocular diseases associated with retinal cell lipofuscin cytotoxicity Lipofuscin accumulates with age and can be increased due to genetic predisposition and certain underlying diseases. Molday RS, Zhong M, Quazi F, Biochim Biophys Acta 1791(7):573-83 (2009);Zaneveld J, et al. Genet Med 17(4):262-270 (2015);Allikmets R et al., Science 277(5333):1805-7 (1997);van Driel M a, Maugeri a, Klevering BJ, Hoyng CB, Cremers FP, Ophthalmic Genet 19(3):117-122 (1998);Fishman GA, Ophthalmic Genet 31(4):183-9 (2010);Lim LS, Mitchell P, Seddon JM, Holz FG, Wong TY, Lancet 379(9827):1728-1738 (2012);Swaroop A, See Chew EY, Rickman CB, Abecasis GR, Annu Rev Genomics Hum Genet 10:19-43 (2009);Charbel Issa P, Barnard AR, Herrmann P, Washington I, MacLaren RE (2015) Proc Natl Acad Sci 112(27):8415-20 (2017). ABCR mutations can occur in patients with age-related macular degeneration (AMD), Stargardt disease, fundus flava, cone dystrophy (COD, where only cones undergo degeneration), and cone-rod dystrophy (CRD, where both rods and cones undergo degeneration), as well as retinitis pigmentosa. Examples of such ABCR mutations include, but are not limited to, ABCA4 D2177N, ABCA4 G1961E, ABCA4 G863A, ABCA4 1847delA, ABCA4 L541P, ABCA4 T2028I, ABCA4 N247I, ABCA4 E1122K, ABCA4 W499 *, ABCA4 A1773V, ABCA4 H55R, ABCA4 A1038V, ABCA4 IVS30+1G→T, ABCA4 IVS40+5G→A, ABCA4 IVS14+1G→C, ABCA4 F1440del1cT, and those disclosed in Allikmets R et al., Science 277(5333):1805-7 (1997).

[0023] Retinitis pigmentosa (RP) is a group of diseases in which photoreceptor cells die. RP is the most common inherited retinal dystrophy (IRD) with a global prevalence of approximately 1:4000 (SK Verbakel, et al., Prog. Retin. Eye Res. 66, 157-186 (2018)). RP can be inherited in an autosomal dominant, autosomal recessive or X-linked manner. More than 40 genes have been linked to RP so far, the majority of which are expressed in either photoreceptors or the retinal pigment epithelium. The genetics of RP are complicated due to the enormous heterogeneity of the disease. Ferrari et al., Current Genomics, 12, 238-249 (2011).

[0024] Typical fundus abnormalities include spicule-like pigmentation, mainly in the peripheral and / or mid-peripheral retina, from which the disease gets its name. Typical spicules of dark pigmentation are visible with an ophthalmoscope and represent RPE cells that have detached from Bruch's membrane after photoreceptor degeneration and migrated to intraretinal perivascular sites, where they form melanin pigmentation around blood vessels. These spicules often originate from the mid-periphery, where the concentration of rod cells is highest. It is not known exactly what triggers RPE migration, but migration may be facilitated by the reduced distance between the inner retinal vessels and the RPE due to photoreceptor degeneration. Almost all forms of RP go through a stage in which no pigmentary changes are present in the retina. This stage may be present decades before the appearance of typical RP symptoms.

[0025] There are two autosomal recessive RP subtypes, due to mutations in either the ABCA4 or RDH12 genes, which are very severe forms of RP with early onset in life (RF Mullins, et al., Invest. Ophthalmol. Vis. Sci. 53, 1883-94 (2012); A. Schuster, et al., Investig. Ophthalmol. Vis. Sci. 48, 1824-1831 (2007)). Studies in Asian populations have revealed that they represent at least 3% and 2% of all RP, respectively (L. Huang, et al., Sci. Rep. 7, 1-10 (2017)). Examples of such RDH12 mutations include, but are not limited to, RDH12 p.G127X, RDH12 p.Q189X, RDH12 p.Y226C, RDH12 p.A269GfsX1, RDH12 p.L274P, RDH12 p.R65X, RDH12 p.H151D, RDH12 p.T155I, RDH12 p.V41L, RDH12 p.R314W, and RDH12 p.V146D. Unlike autosomal dominant RP, autosomal recessive RP is characterized by a high content of retinal lipofuscin in the RPE.

[0026] Treatment method of this technology The present disclosure provides compositions that protect against lipofuscin cytotoxicity in retinal cells, such as arimoclomol, dabrafenib, necrosulfonamide (NSA), necrostatin 7 (Nec7), KIRA compounds (e.g., KIRA3 / 6 / 7 / 8), salubrinal, or SAL003, or a pharmaceutically acceptable salt thereof.

[0027] In one aspect, the present disclosure provides a method for preventing or treating an ocular disease associated with retinal cell lipofuscin-associated cytotoxicity in a subject in need thereof, comprising administering to the subject an effective amount of at least one therapeutic agent selected from the group consisting of dabrafenib, necrosulfonamide (NSA), arimoclomol, kinase inhibitor RNase attenuator (KIRA) compounds, salubrinal, SAL003, and any pharma- ceutically acceptable salt thereof, wherein the ocular disease associated with retinal cell lipofuscin-associated cytotoxicity is autosomal recessive retinitis pigmentosa (RP), Stargardt's disease (STGD), Best's disease (BD), cone-rod dystrophy, or ABCA4 mutation age-related macular degeneration (AMD). Examples of KIRA compounds include, but are not limited to, KIRA3, KIRA6, KIRA7, or KIRA8. In some embodiments, the subject comprises a mutation in ABCA4 and / or RDH12. The ABCA4 and / or RDH12 mutations can be homozygous or heterozygous. Additionally, or alternatively, in some embodiments of the methods disclosed herein, administration of an effective amount of at least one therapeutic agent prevents exacerbation of lipofuscin-associated cytotoxicity in retinal cells of the subject.

[0028] In another aspect, the disclosure provides a method for preventing or treating an ABCA4 mutant eye disease associated with retinal cell lipofuscin-associated cytotoxicity in a subject in need thereof, comprising administering an effective amount of Necrostatin 7 (Nec7) or a pharma- ceutically acceptable salt thereof to the subject, wherein the ABCA4 mutant eye disease associated with retinal cell lipofuscin-associated cytotoxicity is autosomal recessive retinitis pigmentosa (RP), cone-rod dystrophy, or age-related macular degeneration (AMD). In some embodiments, administering an effective amount of Nec7 or a pharma-ceutically acceptable salt thereof prevents exacerbation of lipofuscin-associated cytotoxicity in retinal cells in the subject. In any and all embodiments of the methods disclosed herein, the eye disease is hereditary, non-hereditary, or age-related. In some embodiments of the methods disclosed herein, the AMD is dry AMD. In other aspects of the methods disclosed herein, the cone-rod dystrophy is autosomal recessive cone-rod dystrophy.

[0029] Additionally, or alternatively, in some embodiments of the methods disclosed herein, the subject is a patient with any of the following AML phenotypes: ABCA4 D2177N, ABCA4 G1961E, ABCA4 G863A, ABCA4 1847delA, ABCA4 L541P, ABCA4 T2028I, ABCA4 N247I, ABCA4 E1122K, ABCA4 W499 * , ABCA4 A1773V, ABCA4 H55R, ABCA4 A1038V, ABCA4 IVS30+1G→T, ABCA4 IVS40+5G→A, ABCA4 IVS14+1G→C, and ABCA4 F1440del1cT. In any and all embodiments of the methods disclosed herein, the control has at least one ABCA4 mutation selected from the group consisting of RDH12 G127 * , RDH12 Q189 * , RDH12 Y226C, RDH12 A269Gfs * , RDH12-L274P, RDH12-R65 * , RDH12 H151D, RDH12 T155I, RDH12 V41L, RDH12 R314W, and RDH12 V146D.

[0030] Additionally or alternatively, in some embodiments of the methods disclosed herein, at least one therapeutic agent of the present technology (e.g., dabrafenib, NSA, arimoclomol, KIRA compounds, salubrinal, SAL003, Nec7, or a pharmaceutically acceptable salt thereof) reduces or eliminates lipofuscin bisretinoid (LB) lipid-induced phosphorylation and / or polymerization of MLKL. In any and all embodiments of the methods disclosed herein, at least one therapeutic agent of the present technology (e.g., dabrafenib, NSA, arimoclomol, KIRA compounds, salubrinal, SAL003, Nec7, or a pharmaceutically acceptable salt thereof) reverses the LB lipid-induced translocation of phosphorylated MLKL (pMLKL) to the plasma membrane of retinal pigment epithelial cells. In certain embodiments, the LB lipid is selected from the group consisting of N-retinylidene-N-retinylethanolamine (A2E), A2E isomers, oxidized derivatives of A2E, and all-trans retinal dimer (ATRD).

[0031] In any of the preceding embodiments of the methods disclosed herein, at least one therapeutic agent of the present technology (e.g., dabrafenib, NSA, arimoclomol, KIRA compound, salubrinal, SAL003, Nec7, or a pharma- ceutically acceptable salt thereof) reduces the mRNA or protein levels of one or more genes associated with retinal degeneration, inflammation / angiogenesis, ER stress, and / or necroptosis. Examples of one or more genes associated with retinal degeneration, inflammation / angiogenesis, ER stress, and / or necroptosis include, but are not limited to, EDN2, FGF2, GFAP, SERP, VEGF, CXCL15, XBP1s, SCAND1, CEBPA, and HMGA. Additionally, or alternatively, in some embodiments of the methods disclosed herein, at least one therapeutic agent of the present technology (e.g., dabrafenib, NSA, arimoclomol, KIRA compound, salubrinal, SAL003, Nec7, or a pharma- ceutically acceptable salt thereof) inhibits or reduces lipofuscin-induced necroptosis and / or reduces infiltration of activated microglia / macrophages in retinal pigment epithelial cells.

[0032] In any and all embodiments of the methods disclosed herein, at least one therapeutic agent of the present technology (e.g., dabrafenib, NSA, arimoclomol, KIRA compounds, salvulinal, SAL003, Nec7, or a pharmaceutically acceptable salt thereof) is administered via topical, intravenous, intraocular, subretinal, or subscleral administration. Additionally or alternatively, in some embodiments of the methods disclosed herein, at least one therapeutic agent of the present technology (e.g., dabrafenib, NSA, arimoclomol, KIRA compounds, salvulinal, SAL003, Nec7, or a pharmaceutically acceptable salt thereof) is conjugated to a drug that targets retinal pigment epithelial cells. Examples of drugs that target retinal pigment epithelial cells include, but are not limited to, tamoxifen, chloroquine (CQ) / hydroxychloroquine (HCQ), ethambutol (EMB), or sodium iodate (NaIO3). Other examples of RPE-targeted drugs are described in Crisostomo S, Vieira L, Cardigos J (2019) Retina: 23-28; Michaelides M (2011) Arch Ophthalmol 129(1): 30;Tsai RK, He MS, Chen ZY, Wu WC, Wu WS (2011) Mol Vis 17(June): 1564-1576;MacHalinska A et al. (2010) Neurochem Res 35(11): 1819-1827;Tsang SH, Sharma T (2018) Drug-Induced Retinal Toxicity. Atlas of Inherited Retinal Diseases, eds Tsang SH, Sharma T (Springer International Publishing, Cham), pp 227-232.

[0033] The term "pharmaceutically acceptable salt" refers to a salt prepared from a base or acid acceptable for administration to a patient, such as a mammal (e.g., a salt with acceptable mammalian safety for a given dosing regimen). However, it should be understood that the salt need not be a pharmaceutically acceptable salt, such as a salt of an intermediate compound not intended for administration to a patient. A pharmaceutically acceptable salt may be derived from a pharmaceutically acceptable inorganic or organic base, or from a pharmaceutically acceptable inorganic or organic acid. Also, one or more compositions of the present technology (e.g., arimoclomol, dabrafenib, necrosulfonamide (NSA), necrostatin 7 (Nec7), KIRA compounds (e.g., KIRA3 / 6 / 7 / 8), salubrinal, or SAL003) contain both a basic moiety, such as an amine, pyridine, or imidazole, and an acidic moiety, such as a carboxylic acid or tetrazole, and zwitterions may be formed and are included within the scope of the term "salt" as used herein.

[0034] In one embodiment, one or more compositions of the present technology (e.g., arimoclomol, dabrafenib, necrosulfonamide (NSA), necrostatin 7 (Nec7), KIRA compounds (e.g., KIRA3 / 6 / 7 / 8), salubrinal, or SAL003) may contain one or more basic functional groups, such as amino or alkylamino, and thereby form pharma- ceutically acceptable salts by reaction with a pharma-ceutically acceptable acid. These salts may be prepared in situ during the administration vehicle or dosage form manufacturing process, or by separately reacting a purified compound of the present technology in its free base form with a suitable organic or inorganic acid and isolating the formed salt during subsequent purification. In another embodiment, one or more compositions of the present technology (e.g., arimoclomol, dabrafenib, necrosulfonamide (NSA), necrostatin 7 (Nec7), KIRA compounds (e.g., KIRA3 / 6 / 7 / 8), salubrinal, or SAL003) may contain one or more acidic functional groups, thereby forming pharma- ceutically acceptable salts by reaction with a pharma- ceutically acceptable base. These salts may similarly be prepared in situ during the administration vehicle or dosage form manufacturing process, or by separately reacting the purified compound in its free base form (e.g., hydroxyl or carboxyl) with a suitable base and then isolating the formed salt during purification.

[0035] Salts derived from pharmaceutically acceptable inorganic bases include ammonium, aluminum, calcium, copper, ferric, ferrous, lithium, magnesium, manganic, manganous, potassium, sodium, and zinc salts, etc. Salts derived from pharmaceutically acceptable organic bases include salts of primary, secondary, and tertiary amines, including substituted amines, cyclic amines, naturally occurring amines, etc., such as arginine, betaine, caffeine, choline, N,N'-dibenzylethylenediamine, ethylamine, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, diethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperadine, polyamine resins, procaine, purines, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine, etc. Pharmaceutically acceptable salts derived from inorganic acids include salts of boric acid, carbonic acid, hydrohalic acids (hydrobromic acid, hydrochloric acid, hydrofluoric acid or hydroiodic acid), nitric acid, phosphoric acid, sulfamic acid and sulfuric acid.Pharmaceutically acceptable salts derived from organic acids include salts of aliphatic hydroxyl acids (e.g., citric acid, gluconic acid, glycolic acid, lactic acid, lactobionic acid, malic acid, and tartaric acid), aliphatic monocarboxylic acids (e.g., acetic acid, butyric acid, formic acid, propionic acid, and trifluoroacetic acid), amino acids (e.g., aspartic acid and glutamic acid), aromatic carboxylic acids (e.g., benzoic acid, 2-acetoxybenzoic acid, p-chlorobenzoic acid, diphenylacetic acid, gentisic acid, hippuric acid, and triphenylacetic acid), aromatic hydroxyl acids (e.g., o-hydroxybenzoic acid, p-hydroxybenzoic acid, 1-hydroxynaphthalene-2-carboxylic acid, and 3-hydroxynaphthalene-2-carboxylic acid), ascorbic acid, dicarboxylic acids (e.g., fumaric acid, maleic acid, oxalic acid, and succinic acid), glucuronic acid, mandelic acid, and the like. Examples of suitable salts include salts of cyclic acid, mucic acid, nicotinic acid, orotic acid, pamoic acid, pantothenic acid, sulfonic acids (e.g., benzenesulfonic acid, camphorsulfonic acid, edisylic acid, ethanesulfonic acid, isethionic acid, methanesulfonic acid, naphthalenesulfonic acid, naphthalene-1,5-disulfonic acid, naphthalene-2,6-disulfonic acid, and p-toluenesulfonic acid), xinafoic acid, valeric acid, oleic acid, palmitic acid, stearic acid, lauric acid, toluenesulfonic acid, methanesulfonic acid, ethanedisulfonic acid, citric acid, ascorbic acid, maleic acid, oxalic acid, fumaric acid, phenylacetic acid, isothionic acid, succinic acid, tartaric acid, glutamic acid, salicylic acid, sulfanilic acid, napthylic acid, lactobionic acid, gluconic acid, and laurylsulfonic acid.

[0036] Additionally, or alternatively, in some embodiments, administration of an effective amount of one or more compositions of the present technology (e.g., arimoclomol, dabrafenib, necrosulfonamide (NSA), necrostatin 7 (Nec7), KIRA compounds (e.g., KIRA3 / 6 / 7 / 8), salubrinal, or SAL003) or a pharma- ceutically acceptable salt thereof prevents the exacerbation of lipofuscin-associated retinal cytotoxicity in a subject. In any and all embodiments of the methods disclosed herein, administration of an effective amount of one or more compositions of the present technology (e.g., arimoclomol, dabrafenib, necrosulfonamide (NSA), necrostatin 7 (Nec7), KIRA compounds (e.g., KIRA3 / 6 / 7 / 8), salubrinal, or SAL003) or a pharma- ceutically acceptable salt thereof blocks, reduces, or reverses lipofuscin-associated retinal cytotoxicity in retinal pigment epithelial cells.

[0037] In some embodiments of the methods disclosed herein, administration of an effective amount of one or more compositions of the present technology (e.g., arimoclomol, dabrafenib, necrosulfonamide (NSA), necrostatin 7 (Nec7), KIRA compounds (e.g., KIRA3 / 6 / 7 / 8), salubrinal, or SAL003) or a pharma- ceutically acceptable salt thereof prevents, delays the onset of, or reduces the severity of lipofuscin-associated damage or a disease or condition directly or indirectly associated with lipofuscin-associated damage in RPE cells of a subject. The subject may be of any gender (e.g., male or female) and / or of any age, such as elderly (generally at least 60, 70, or 80 years of age or older), subjects in the transition age between elderly and adulthood, adults, subjects in the transition age between adulthood and minors, and minors, including adolescents (e.g., 13 to 16, 17, 18, or 19 years of age), children (generally under 13 years of age, prior to the onset of puberty), and infants. The subject may also be of any ethnic group or genotype. Some examples of human ethnic groups include Caucasian, Asian, Latino, African, African American, Native American, Semitic, and Pacific Islander. Additionally, or alternatively, in some embodiments, one or more compositions of the present technology (e.g., arimoclomol, dabrafenib, necrosulfonamide (NSA), necrostatin 7 (Nec7), KIRA compounds (e.g., KIRA3 / 6 / 7 / 8), salubrinal, or SAL003) or a pharma- ceutical acceptable salt thereof are configured to localize to RPE cells. Additionally, or alternatively, in certain embodiments, one or more compositions of the present technology (e.g., arimoclomol, dabrafenib, necrosulfonamide (NSA), necrostatin 7 (Nec7), KIRA compounds (e.g., KIRA3 / 6 / 7 / 8), salubrinal, or SAL003) or a pharma- ceutically acceptable salt thereof are localized to the RPE cells by being administered directly at, to, or in close proximity to the RPE cells, such as by injection or implantation.

[0038] In other embodiments, one or more compositions of the present technology (e.g., arimoclomol, dabrafenib, necrosulfonamide (NSA), necrostatin 7 (Nec7), KIRA compounds (e.g., KIRA3 / 6 / 7 / 8), salubrinal, or SAL003) or a pharma- ceutically acceptable salt thereof can be administered in combination with one or more compositions of the present technology (e.g., arimoclomol, dabrafenib, necrosulfonamide (NSA), necrostatin 7 (Nec7), KIRA compounds (e.g., KIRA3 / 6 / 7 / 8), salubrinal, or SAL003) or a pharma- ceutically acceptable salt thereof, By coupling with a targeting agent that selectively targets RPE cells, one or more compositions of the present technology (e.g., arimoclomol, dabrafenib, necrosulfonamide (NSA), necrostatin 7 (Nec7), KIRA compounds (e.g., KIRA3 / 6 / 7 / 8), salubrinal, or SAL003) or a pharma- ceutically acceptable salt thereof can be administered at, to, or in close proximity to RPE cells, or remotely from RPE cells (e.g., by systemic administration). A cell-targeting agent (i.e., a "targeting agent") is any chemical moiety that has the ability to bind to (i.e., "target") RPE cells. A cell-targeting agent can target any part of an RPE cell, such as the cell membrane, organelles (e.g., lysosomes or endosomes), or the cytoplasm. In one embodiment, the cell-targeting agent selectively targets a component of an RPE cell. By selectively targeting a component of an RPE cell, the cell targeting agent can, for example, selectively target a particular component of a cell over other types of cellular components. In other embodiments, the targeting agent non-selectively targets a cellular component, for example, by targeting a cellular component found in most or all cells.

[0039] In various embodiments, the targeting agent may be, for example, a peptide, dipeptide, tripeptide (e.g., glutathione), tetrapeptide, pentapeptide, hexapeptide, higher oligopeptide, protein, monosaccharide, disaccharide, trisaccharide, tetrasaccharide, higher oligosaccharide, polysaccharide (e.g., carbohydrate), nucleobase, nucleoside (e.g., adenosine, cytidine, uridine, guanosine, thymidine, inosine, and S-adenosylmethionine), nucleotide (i.e., mono-, di-, or triphosphate form), dinucleotide, trinucleotide, tetranucleotide, higher oligonucleotide, nucleic acid, cofactor (e.g., TPP, FAD, NAD, coenzyme A, biotin, lipoamide, metal ion (e.g., Mg 2+ ), metal-containing clusters (e.g., iron-sulfur clusters), or non-biological (i.e., synthetic) targeting groups. Some specific types of proteins include enzymes, hormones, antibodies (e.g., monoclonal antibodies), lectins, and steroids.

[0040] Antibodies for use as targeting agents are generally specific for one or more cell surface antigens. In certain embodiments, the antigen is a receptor. The antibody may be a complete antibody, or alternatively, may be a fragment of an antibody that retains the recognition portion of the antibody (i.e., the hypervariable region). Some examples of antibody fragments include Fab, Fc, and F(ab')2. In certain embodiments, the antibody or antibody fragment may be chemically reduced to derivatize the antibody or antibody fragment with sulfhydryl groups, particularly to facilitate crosslinking of the antibody to one or more compositions of the present technology described herein (e.g., arimoclomol, dabrafenib, necrosulfonamide (NSA), necrostatin 7 (Nec7), KIRA compounds (e.g., KIRA3 / 6 / 7 / 8), salubrinal, or SAL003) or pharma-ceutically acceptable salts thereof. In certain embodiments, the targeting agent is a ligand of an internalized receptor of the target cell. For example, the targeting agent may be a targeting signal of acid hydrolase precursor protein, which transports various substances to lysosomes. One such targeting agent of particular interest is mannose 6-phosphate (M6P), which is recognized by the mannose 6-phosphate receptor (MPR) protein in the trans-Golgi endosomes known to be involved in the transport of M6P-labeled material to lysosomes.

[0041] In other embodiments, the targeting agent is a peptide that contains an RGD sequence, or a variant thereof, which binds to the RGD receptor on the surface of many types of cells.Other targeting agents include, for example, transferrin, insulin, amylin, etc.Receptor internalization can be used to facilitate intracellular delivery of one or more compositions of the technology described herein (e.g., arimoclomol, dabrafenib, necrosulfonamide (NSA), necrostatin 7 (Nec7), KIRA compounds (e.g., KIRA3 / 6 / 7 / 8), salubrinal, or SAL003) or pharma- ceutical acceptable salts thereof. In certain embodiments, a cell targeting molecule or group, or several (e.g., 2, 3 or more) of the same type of cell targeting molecule or group is attached directly or via a linker to one or more compositions of the present technology (e.g., arimoclomol, dabrafenib, necrosulfonamide (NSA), necrostatin 7 (Nec7), KIRA compounds (e.g., KIRA3 / 6 / 7 / 8), salubrinal, or SAL003) or a pharmaceutically acceptable salt thereof. In other embodiments, two or more different types of targeting molecules are attached directly or via a linker to one or more compositions of the present technology (e.g., arimoclomol, dabrafenib, necrosulfonamide (NSA), necrostatin 7 (Nec7), KIRA compounds (e.g., KIRA3 / 6 / 7 / 8), salubrinal, or SAL003) or a pharmaceutically acceptable salt thereof.

[0042] Additionally or alternatively, in some embodiments, a fluorophore may be attached to one or more compositions of the present technology (e.g., arimoclomol, dabrafenib, necrosulfonamide (NSA), necrostatin 7 (Nec7), KIRA compounds (e.g., KIRA3 / 6 / 7 / 8), salubrinal, or SAL003) or a pharmaceutically acceptable salt thereof. The incorporation of one or more fluorophores may have several purposes. In some embodiments, one or more fluorophores are included to quantify the cellular uptake and cellular retention of one or more compositions of the present technology (e.g., arimoclomol, dabrafenib, necrosulfonamide (NSA), necrostatin 7 (Nec7), KIRA compounds (e.g., KIRA3 / 6 / 7 / 8), salubrinal, or SAL003) or a pharmaceutically acceptable salt thereof (e.g., fluorescence spectroscopy).

[0043] As used herein, "fluorophore" refers to any species capable of emitting fluorescence (i.e., fluorescent). For example, in one embodiment, the fluorophore is an organic fluorophore. The organic fluorophore can be, for example, a charged (i.e., ionic) molecule (e.g., sulfonic acid or ammonium group), an uncharged (i.e., neutral) molecule, a saturated molecule, an unsaturated molecule, a cyclic molecule, a bicyclic molecule, a tricyclic molecule, a polycyclic molecule, an acyclic molecule, an aromatic molecule, and / or a heterocyclic molecule (i.e., by being ring-substituted by one or more heteroatoms selected from, for example, nitrogen, oxygen, and sulfur). In the particular case of an unsaturated fluorophore, the fluorophore comprises one, two, three, or more carbon-carbon and / or carbon-nitrogen double and / or triple bonds. In certain embodiments, the fluorophore comprises at least two (e.g., two, three, four, five, or more) conjugated double bonds, apart from any aromatic groups that may be in the fluorophore. In other embodiments, the fluorophore is a fused polycyclic aromatic hydrocarbon (PAH) containing at least 2, 3, 4, 5, or 6 rings (e.g., naphthalene, pyrene, anthracene, chrysene, triphenylene, tetracene, azulene, and phenanthrene), where the PAH may be ring substituted or derivatized with one, two, three, or more heteroatoms or heteroatom-containing groups.

[0044] In other embodiments, the organic fluorophore is a xanthene derivative (e.g., fluorescein, rhodamine, Oregon Green, eosin, and Texas Red), a cyanine or a derivative or subclass thereof (e.g., streptocyanins, hemicyanins, closed cyanins, phycocyanins, allophycocyanins, indocarbocyanins, oxacarbocyanines, thiacarbocyanines, merocyanins, and phthalocyanines), a naphthalene derivative (e.g., dansyl and prodan derivatives), a coumarin and its derivatives, an oxadiazo and its derivatives (e.g., pyridyloxazole, nitrobenzoxadiazole, and benzoxadiazole), pyrene and its derivatives, oxazine and its derivatives (e.g., Nile red, Nile blue, and cresyl violet), acridine derivatives (e.g., proflavine, acridine orange, and acridine yellow), arylmethine derivatives (e.g., auramine, crystal violet, and malachite green), and tetrapyrrole derivatives (e.g., porphyrins and bilirubin). Some specific families of dyes contemplated herein are the Cy® family of dyes, the Alexa® family of dyes, the ATTO® family of dyes, and the Dy® family of dyes. The ATTO® dyes may have several structural motifs, including coumarin-based, rhodamine-based, carbopyronine-based, and oxazine-based structural motifs, among others.

[0045] Fluorophores may be attached to one or more compositions of the present technology (e.g., arimoclomol, dabrafenib, necrosulfonamide (NSA), necrostatin 7 (Nec7), KIRA compounds (e.g., KIRA3 / 6 / 7 / 8), salubrinal, or SAL003) or pharma- ceutically acceptable salts thereof by any of the linking methods known in the art. For example, fluorophores may be linked to one or more molecules that contain an appropriate reactive group (e.g., amino, thiol, hydroxy, aldehyde, or ketone group) using commercially available single-reactive fluorophores (e.g., NHS-Cy5) or dual-reactive fluorophores (e.g., bis-NHS-Cy5 or bis-maleimide-Cy5). Alternatively, one or more compositions of the present technology (e.g., arimoclomol, dabrafenib, necrosulfonamide (NSA), necrostatin 7 (Nec7), KIRA compounds (e.g., KIRA3 / 6 / 7 / 8), salubrinal, or SAL003) or a pharma- ceutically acceptable salt thereof can be derivatized with one, two or more such reactive groups, and these reactive moieties reacted with a fluorophore (e.g., an amino-containing fluorophore) containing a suitable reactive group.

[0046] One or more compositions of the present technology (e.g., arimoclomol, dabrafenib, necrosulfonamide (NSA), necrostatin 7 (Nec7), KIRA compounds (e.g., KIRA3 / 6 / 7 / 8), salubrinal, or SAL003) or a pharma- ceutically acceptable salt thereof may be administered by any route that allows contact with RPE cells. Administration may be, for example, ocular, parenteral (e.g., subcutaneous, intramuscular, or intravenous), topical, transdermal, intravitreal, retro-orbital, subretinal, subscleral, oral, sublingual, or buccal modes of administration. Some of the aforementioned exemplary modes of administration may be achieved by injection. However, in some embodiments, injection is avoided by the use of a slow-release implant in the vicinity of the retina (e.g., subscleral route) or by administering drops to the conjunctiva. One or more compositions of the present technology (e.g., arimoclomol, dabrafenib, necrosulfonamide (NSA), necrostatin 7 (Nec7), KIRA compounds (e.g., KIRA3 / 6 / 7 / 8), salubrinal, or SAL003) or a pharma- ceutically acceptable salt thereof may be administered locally to the eye of patients suffering from lipofuscin cytotoxicity or at risk of developing retinal degeneration due to lipofuscin cytotoxicity, including Stargardt, carriers of the defective ABCA4 gene, dry AMD. Topical administration includes intravitreal, topical ocular, transdermal patch, subcutaneous, parenteral, ocular, subconjunctival, or retrobulbar or subtenon injection, transscleral (including iontophoretic), posterior juxtascleral delivery, or slow release biodegradable polymers or liposomes. One or more compositions of the present technology (e.g., arimoclomol, dabrafenib, necrosulfonamide (NSA), necrostatin 7 (Nec7), KIRA compounds (e.g., KIRA3 / 6 / 7 / 8), salubrinal, or SAL003) or a pharma- ceutically acceptable salt thereof may also be delivered in an ocular irrigation solution. The concentration may range from about 0.001 μM to about 100 μM, preferably from about 0.01 μM to about 5 μM.

[0047] In some embodiments, one or more compositions of the present technology (e.g., arimoclomol, dabrafenib, necrosulfonamide (NSA), necrostatin 7 (Nec7), KIRA compounds (e.g., KIRA3 / 6 / 7 / 8), salubrinal, or SAL003) or a pharma- ceutically acceptable salt thereof are administered, at least initially, at a level lower than that required to achieve the desired therapeutic effect, and the dosage is gradually or suddenly increased until the desired effect is achieved. In other embodiments, one or more compositions of the present technology (e.g., arimoclomol, dabrafenib, necrosulfonamide (NSA), necrostatin 7 (Nec7), KIRA compounds (e.g., KIRA3 / 6 / 7 / 8), salubrinal, or SAL003) or a pharma- ceutically acceptable salt thereof are administered, at least initially, at a level higher than that required to promote the desired therapeutic effect, and the dosage is gradually or suddenly decreased until the desired effect is achieved.

[0048] The selected dosage level will depend on several factors, as determined by the physician, including the type of disease or condition being treated, the stage or severity of the condition or disease, the potency of the therapeutic compound being used and its bioavailability profile, and the details (e.g., genotype and phenotype) of the subject being treated, such as age, sex, weight, and general condition.

[0049] In particular, for systemic modes of administration, dosages may range, for example, from about 0.01, 0.1, 0.5, 1, 5, or 10 mg per kg of body weight per day to about 20, 50, 100, 500, or 1000 mg per kg of body weight per day, or every other day, or 2, 3, 4 or more times per day. In particular, one or more compositions of the present technology (e.g., arimoclomol, dabrafenib, necrosulfonamide (NSA), necrostatin 7 (Nec7), KIRA compounds (e.g., KIRA3 / 6 / 7 / 8), salubrinal, or SAL003) or pharma- ceutically acceptable salts thereof are administered non-systemically directly at the retina, and dosages may be independent of body weight and may be smaller (e.g., 1 to 1000 μg per dose). In some embodiments, the daily dose of one or more compositions of the present technology (e.g., arimoclomol, dabrafenib, necrosulfonamide (NSA), necrostatin 7 (Nec7), KIRA compounds (e.g., KIRA3 / 6 / 7 / 8), salubrinal, or SAL003) or a pharmaceutically acceptable salt thereof is the lowest dose effective to produce a therapeutic effect. In some embodiments, one or more compositions of the present technology (e.g., arimoclomol, dabrafenib, necrosulfonamide (NSA), necrostatin 7 (Nec7), KIRA compounds (e.g., KIRA3 / 6 / 7 / 8), salubrinal, or SAL003) or a pharmaceutically acceptable salt thereof are not administered in separate doses, but are administered continuously, such as provided by a slow release implant or intravenous line. In one aspect, the disclosure provides pharmaceutical compositions comprising arimoclomol, dabrafenib, necrosulfonamide (NSA), necrostatin 7 (Nec7), KIRA compounds (e.g., KIRA3 / 6 / 7 / 8), salubrinal, SAL003, and pharmaceutically acceptable salts thereof.

[0050] One or more compositions of the present technology (e.g., arimoclomol, dabrafenib, necrosulfonamide (NSA), necrostatin 7 (Nec7), KIRA compounds (e.g., KIRA3 / 6 / 7 / 8), salubrinal, or SAL003) or a pharma- ceutically acceptable salt thereof may be formulated with one or more pharma- ceutically acceptable carriers (additives) and / or diluents known in the art. The pharmaceutical compositions of the present technology may be specially formulated for administration in solid or liquid form, including those adapted for: (1) oral administration, e.g., drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., buccal, sublingual, and those targeted for systemic absorption, boluses, powders, granules, pastes for application to the tongue; (2) parenteral administration, e.g., by subcutaneous, intramuscular, intravenous, or epidural injection, e.g., as a sterile solution or suspension, or sustained release formulation; (3) topical application, e.g., as a cream, ointment, or sustained release patch or spray applied to the skin; (4) sublingual; (5) ophthalmic; (6) transdermal; or (7) nasal.

[0051] In some embodiments, the pharmaceutical composition of the present technology may mainly comprise one or more "pharmaceutical acceptable carriers", which as used herein generally refers to pharmaceutical acceptable compositions such as liquid or solid fillers, diluents, excipients, manufacturing aids (e.g., lubricants, magnesium talc, calcium or zinc stearate, or stearic acid), or solvent encapsulating materials, which are useful for introducing active agents into the body. Each carrier should be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not harmful to the patient. Examples of suitable aqueous and non-aqueous carriers that may be employed in the pharmaceutical composition of the present technology include, for example, water, ethanol, polyols (such as glycerin, propylene glycol, polyethylene glycol, etc.), vegetable oils (such as olive oil), and injectable organic esters (such as ethyl oleate), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

[0052] In some embodiments, the formulation may include one or more of the following: sugars, such as lactose, glucose, and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethylcellulose, ethylcellulose, and acetylcellulose; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository wax; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol, and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; alginic acid; buffers, such as magnesium hydroxide and aluminum hydroxide; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; pH buffer solutions; polyesters, polycarbonates, and / or polyanhydrides; preservatives; lubricants; fillers; and other non-toxic compatible substances employed in pharmaceutical formulations.

[0053] Various auxiliary agents such as wetting agents, emulsifiers, lubricants (e.g., sodium lauryl sulfate and magnesium stearate), coloring agents, release agents, coating agents, sweeteners, flavoring agents, preservatives, and antioxidants may also be included in the pharmaceutical formulation. Some examples of pharma-ceutically acceptable antioxidants include: (1) water-soluble antioxidants such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium disulfite, sodium sulfite, etc.; (2) oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, α-tocopherol, etc.; and (3) metal chelating agents such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc. In some embodiments, the pharmaceutical formulation comprises an excipient selected from, for example, cellulose, liposomes, micelle-forming agents (e.g., bile acids), and polymeric carriers such as polyesters and polyanhydrides. The suspension may contain, in addition to the active compound, suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar and tragacanth, and mixtures thereof. Prevention of the action of microorganisms on the active compounds can be ensured by the inclusion of various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and the like. It is also desirable to include isotonic agents in the composition, such as sugars, sodium chloride, and the like. Prolonged absorption of the injectable pharmaceutical form can also be brought about by the inclusion of agents which delay absorption, such as aluminum monostearate and gelatin.

[0054] The pharmaceutical formulations of the present technology may be prepared by any of the methods known in the pharmaceutical art. The amount of active ingredient that may be combined with a carrier material to produce a single dosage form will vary depending on the host treated and the particular mode of administration. The amount of active ingredient that may be combined with a carrier material to produce a single dosage form will generally be that amount of the compound that produces a therapeutic effect. In general, the amount of active compound will range from about 0.1-99%, more typically from about 5-70%, and more typically from about 10-30%.

[0055] The compositions of the present technology may be administered locally to the eye of patients suffering from lipofuscin cytotoxicity, including Stargardt, carriers of the defective ABCA4 gene, dry AMD, or patients at risk of developing retinal degeneration due to lipofuscin cytotoxicity. One or more compositions of the present technology (e.g., arimoclomol, dabrafenib, necrosulfonamide (NSA), necrostatin 7 (Nec7), KIRA compounds (e.g., KIRA3 / 6 / 7 / 8), salubrinal, or SAL003) or pharma- ceutically acceptable salts thereof may be incorporated into various types of ophthalmic formulations for delivery to the eye (e.g., topically, intracamerally, juxtasclerally, or via an implant). One or more compositions of the present technology (e.g., arimoclomol, dabrafenib, necrosulfonamide (NSA), necrostatin 7 (Nec7), KIRA compounds (e.g., KIRA3 / 6 / 7 / 8), salubrinal, or SAL003) or a pharma- ceutically acceptable salt thereof may be combined with ophthalmically acceptable preservatives, surfactants, thickening agents, gelling agents, permeation enhancers, buffers, sodium chloride, and water to form aqueous, sterile ophthalmic suspensions or solutions, or preformed or in situ formed gels.

[0056] In some embodiments, the compositions of the present technology (e.g., arimoclomol, dabrafenib, necrosulfonamide (NSA), necrostatin 7 (Nec7), KIRA compounds (e.g., KIRA3 / 6 / 7 / 8), salubrinal, SAL003, and pharmaceutically acceptable salts thereof) are administered 1-10 times per day, once per day, twice, three times, four or more times per day, 1-3 times per day, 2-4 times per day, 3-6 times per day, 4-8 times per day, or 5-10 times per day. In some embodiments, the compositions of the present technology (e.g., arimoclomol, dabrafenib, necrosulfonamide (NSA), necrostatin 7 (Nec7), KIRA compounds (e.g., KIRA3 / 6 / 7 / 8), salubrinal, SAL003, and pharmaceutically acceptable salts thereof) are administered daily, every other day, 2-3 times per week, or 3-6 times per week. In some embodiments, the dose of the compositions of the present technology (e.g., arimoclomol, dabrafenib, necrosulfonamide (NSA), necrostatin 7 (Nec7), KIRA compounds (e.g., KIRA3 / 6 / 7 / 8), salubrinal, SAL003, and pharma- ceutically acceptable salts thereof) may range, for example, from about 0.01, 0.1, 0.5, 1, 5, 10, or 100 mg per kg of body weight per day to about 20, 50, 100, 500, or 1000 mg per kilogram of body weight per day. In particular, in embodiments in which the active agent is administered directly at the retina, the dosage administered may be independent of body weight and may be smaller (e.g., 1 to 1000 μg per dose).

[0057] When administered topically, one or more compositions of the present technology (e.g., arimoclomol, dabrafenib, necrosulfonamide (NSA), necrostatin 7 (Nec7), KIRA compounds (e.g., KIRA3 / 6 / 7 / 8), salubrinal, or SAL003) or a pharma- ceutically acceptable salt thereof may be formulated as a topical ophthalmic suspension or solution having a pH of about 4 to 8. One or more compositions of the present technology (e.g., arimoclomol, dabrafenib, necrosulfonamide (NSA), necrostatin 7 (Nec7), KIRA compounds (e.g., KIRA3 / 6 / 7 / 8), salubrinal, or SAL003) or a pharma- ceutically acceptable salt thereof are typically included in these formulations in an amount of 0.001% to 5% by weight, or in an amount of 0.01% to 2% by weight. Thus, for topical application, 1-2 drops of these formulations are delivered to the ocular surface 1-4 times per day, as determined by the skilled clinician. In some embodiments, a therapeutically effective amount of at least one composition of the present technology (e.g., arimoclomol, dabrafenib, necrosulfonamide (NSA), necrostatin 7 (Nec7), KIRA compounds (e.g., KIRA3 / 6 / 7 / 8), salubrinal, or SAL003, or a pharma- ceutical acceptable salt thereof) is delivered intravitreally, either by injection (possibly microspheres), through an intravitreal device, or by injection, gel, or implant, or by other methods described above, placed in the subtenon's space. When delivered as a solution, the therapeutically effective amount of one or more compositions of the present technology (e.g., arimoclomol, dabrafenib, necrosulfonamide (NSA), necrostatin 7 (Nec7), KIRA compounds (e.g., KIRA3 / 6 / 7 / 8), salubrinal, or SAL003) or a pharma- ceutically acceptable salt thereof in the composition can be about 18-44 μM, with a concentration of about 20-50%. When formulated as a suspension, the therapeutically effective amount of one or more compositions of the present technology (e.g., arimoclomol, dabrafenib, necrosulfonamide (NSA), necrostatin 7 (Nec7), KIRA compounds (e.g., KIRA3 / 6 / 7 / 8), salubrinal, or SAL003) or a pharma- ceutically acceptable salt thereof is about 20-80%.

[0058] In another embodiment, a therapeutically effective amount of one or more compositions of the present technology (e.g., arimoclomol, dabrafenib, necrosulfonamide (NSA), necrostatin 7 (Nec7), KIRA compounds (e.g., KIRA3 / 6 / 7 / 8), salubrinal, or SAL003) or a pharma- ceutically acceptable salt thereof is administered in the form of minitablets, each weighing from about 1 mg to about 40 mg, or about 5 mg. One to twenty such minitablets may be injected (dry) through a trocar into the subtenon's space such that a total single dose of 50-100 mg [44-88 μM] is injected. In some embodiments, the compositions of the present technology (e.g., arimoclomol, dabrafenib, necrosulfonamide (NSA), necrostatin 7 (Nec7), KIRA compounds (e.g., KIRA3 / 6 / 7 / 8), salubrinal, SAL003, and pharmaceutically acceptable salts thereof) are administered 1-10 times per day, once per day, twice per day, three times per day, four or more times per day, 1-3 times per day, 2-4 times per day, 3-6 times per day, 4-8 times per day, or 5-10 times per day. In some embodiments, the compositions of the present technology (e.g., arimoclomol, dabrafenib, necrosulfonamide (NSA), necrostatin 7 (Nec7), KIRA compounds (e.g., KIRA3 / 6 / 7 / 8), salubrinal, SAL003, and pharmaceutically acceptable salts thereof) are administered daily, every other day, 2-3 times per week, or 3-6 times per week.

[0059] In some embodiments, the dose of the compositions of the present technology (e.g., arimoclomol, dabrafenib, necrosulfonamide (NSA), necrostatin 7 (Nec7), KIRA compounds (e.g., KIRA3 / 6 / 7 / 8), salubrinal, SAL003, and pharma- ceutically acceptable salts thereof) may range, for example, from about 0.01, 0.1, 0.5, 1, 5, 10, or 100 mg per kg of body weight per day to about 20, 50, 100, 500, or 1000 mg per kg of body weight per day. In particular, in embodiments in which the active agent is administered directly at the retina, the dosage administered may be independent of body weight and may be smaller (e.g., 1 to 1000 μg per dose). Formulations of the present technology suitable for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges (using a flavored base, usually sucrose and acacia or tragacanth), powders, granules, or as a solution or suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia), and / or as mouthwashes and the like, each containing a predetermined amount of a compound of the present technology as an active ingredient. The active compound may also be administered as a bolus, electuary, or paste.

[0060] The method of preparing these formulations generally includes mixing the composition of the present technology or a pharma- ceutically acceptable salt thereof with a carrier, and may also include mixing with one or more auxiliary agents. In the case of solid dosage forms (e.g., capsules, tablets, pills, powders, granules, lozenges, etc.), the active compound can be mixed with a finely divided solid carrier, and typically formed by pelleting, tableting, granulation, powdering, coating, etc. Generally, solid carriers include, for example, sodium citrate or dicalcium phosphate, and / or the following: (1) fillers or extenders, such as starch, lactose, sucrose, glucose, mannitol, and / or silicic acid; (2) binders, such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and / or acacia; (3) humectants, such as glycerin; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarding agents, such as paraffin. (6) absorption enhancers such as quaternary ammonium compounds and surfactants, such as poloxamers and sodium lauryl sulfate; (7) wetting agents, such as cetyl alcohol, glycerol monostearate, and non-ionic surfactants; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, zinc stearate, sodium stearate, stearic acid, and mixtures thereof; (10) coloring agents; and / or (11) sustained-release agents, such as crospovidone or ethylcellulose. In the case of capsules, tablets, and pills, the pharmaceutical compositions may also include buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard-shelled gelatin capsules, using excipients such as lactose or milk sugar, and high molecular weight polyethylene glycols, and the like.

[0061] Tablets may be produced by compression or molding, and may be produced with one or more accessory ingredients. Compressed tablets may be prepared using binders (e.g., gelatin or hydroxypropylmethylcellulose), lubricants, inert diluents, preservatives, disintegrants (e.g., sodium starch glycolate or cross-linked sodium carboxymethylcellulose), surfactants or dispersants. Tablets, as well as other solid dosage forms of active agents, such as capsules, pills and granules, may be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical formulation art. Dosage forms may also be formulated to provide slow or sustained release of the active ingredient, for example, using various percentages of hydroxymethylcellulose, other polymer matrices, liposomes and / or microspheres to provide the desired release profile. Alternatively, dosage forms may be formulated, for example, lyophilized, for immediate release.

[0062] In general, the dosage form must be sterile. For this purpose, the dosage form can be sterilized, for example, by filtration through a bacteria-retaining filter, or by incorporating a sterilizing agent in the form of a sterile solid composition that can be dissolved in sterile water or some other sterile injectable solvent immediately before use. The pharmaceutical composition can also contain an opacifying agent, and can be a composition that releases the active ingredient only or preferentially in a certain part of the digestive tract, or the release can be delayed. Examples of embedding compositions that can be used include polymeric substances and waxes. The active ingredient can also be in the form of a microencapsulation, if appropriate, with one or more of the above-mentioned excipients.

[0063] Liquid dosage forms are typically pharma- ceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, or elixirs of active agents.In addition to active ingredients, liquid dosage forms can contain other solvents, solubilizers and emulsifiers that are commonly used in the art, such as water, ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (especially cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil and sesame oil), glycerin, tetrahydrofuran alcohol, polyethylene glycol and fatty acid esters of sorbitan, and other inert diluents, solubilizers and emulsifiers, and mixtures thereof.

[0064] Dosage forms intended for topical or transdermal administration in particular may be in the form of, for example, powders, sprays, ointments, pastes, creams, lotions, gels, solutions, or patches. Ophthalmic preparations such as eye ointments, powders, solutions, and the like are also contemplated herein. The active compound may be mixed under sterile conditions with a pharma- ceutically acceptable carrier, and with any preservatives, buffers, or propellants that may be required. The topical or transdermal dosage forms may contain, in addition to the active compound of the present technology, one or more excipients, such as those selected from animal and vegetable fats, oils, waxes, paraffins, starches, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonite, silicic acid, talc, and zinc oxide, and mixtures thereof. The propellant may also contain customary propellants, such as chlorofluorohydrocarbons and volatile unsaturated hydrocarbons, such as butane and propane. For the purposes of this technology, transdermal patches may offer the advantage of allowing sustained release of the compounds of this technology into the body. Such dosage forms may be prepared by dissolving or dispersing the compounds in a suitable medium. Absorption enhancers may also be included to increase the flux of the compounds through the skin. The rate of such flux may be controlled by either providing a release rate-controlling membrane or dispersing the compounds in a polymer matrix or gel.

[0065] Pharmaceutical compositions of the present technology suitable for parenteral administration generally contain one or more compounds of the present technology in combination with one or more pharma- ceutically acceptable sterile, isotonic aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, or sterile powders that can be reconstituted into a sterile injectable solution or dispersion before use, which may contain sugars, alcohols, antioxidants, buffers, bacteriostats, or solutes that render the formulation isotonic with the blood of the intended recipient. In some cases, it may be desirable to slow the absorption rate of a drug from subcutaneous or intramuscular injection in order to prolong the effect of the drug.This can be achieved by using a liquid suspension of crystalline or amorphous material with poor water solubility.The rate of absorption of the drug then depends on the dissolution rate, which in turn can depend on the crystal size and crystalline form.Alternatively, delayed absorption of a parenterally administered drug form can be achieved by dissolving or suspending the drug in an oil vehicle.

[0066] Injectable depot forms can be prepared by forming microencapsulated matrices of active compounds in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of drug to polymer and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations can also be prepared by entrapping the drug in liposomes or microemulsions that are compatible with body tissues. The pharmaceutical composition may also be in the form of a microemulsion. In the form of a microemulsion, the bioavailability of the active agent may be improved. See Dordunoo, SK, et al., Drug Development and Industrial Pharmacy, 17(12), 1685-1713, 1991, and Sheen, PC, et al., J. Pharm. Sci., 80(7), 712-714, 1991, the entire contents of which are incorporated herein by reference. The pharmaceutical composition may also include micelles formed from the compounds of the present technology and at least one amphiphilic carrier, wherein the micelles have an average diameter of less than about 100 nm. In some embodiments, the micelles have an average diameter of less than about 50 nm, or an average diameter of less than about 30 nm, or an average diameter of less than about 20 nm.

[0067] While any suitable amphiphilic carrier is contemplated herein, the amphiphilic carrier is generally one that has been given the FDA Acceptable for Use (GRAS) status, can solubilize the compounds of the present technology, and can be microemulsified at a later stage when the solution comes into contact with a complex aqueous phase (such as that found in living biological tissues). Typically, amphiphilic components that meet these requirements have an HLB (hydrophilic-lipophilic balance) value of 2 to 20, and their structures include straight-chain aliphatic radicals ranging from C-6 to C-20. Some examples of amphiphilic agents include polyethylene glycolated fatty acid glycerides and polyethylene glycols.

[0068] Some amphiphilic carriers are saturated and monounsaturated polyethylene glycolated fatty acid glycerides, such as those obtained from various fully or partially hydrogenated vegetable oils. Such oils may advantageously consist of tri-, di- and mono-fatty acid glycerides, such as fatty acid compositions containing 4-10 capric acid, 3-9 capric acid, 40-50 lauric acid, 14-24 myristic acid, 4-14 palmitic acid and 5-15% stearic acid, as well as di- and mono polyethylene glycol esters of the corresponding fatty acids. Another useful class of amphiphilic carriers includes partially esterified sorbitan and / or sorbitol with saturated or monounsaturated fatty acids (SPAN series) or the corresponding ethoxylated analogs (TWEEN series). Commercially available amphiphilic carriers, including the Gelucire® series, Labrafil®, Labrasol®, or Lauroglycol®, PEG-monooleate, PEG-dioleate, PEG-monolaurate and dilaurate, lecithin, polysorbate 80 are specifically contemplated.

[0069] Hydrophilic polymers suitable for use in pharmaceutical compositions are generally those that are readily soluble in water, can be covalently attached to vesicle-forming lipids, and are tolerated in vivo without substantial toxic effects (i.e., biocompatible). Suitable polymers include, for example, polyethylene glycol (PEG), polylactic acid (also called polylactide), polyglycolic acid (also called polyglycolide), polylactic acid-polyglycolic acid copolymers, and polyvinyl alcohol. Exemplary polymers are those having a molecular weight of about 100 or 120 daltons to about 5,000 or 10,000 daltons, more preferably about 300 daltons to about 5,000 daltons. In certain embodiments, the polymer is a polyethylene glycol having a molecular weight of about 100 to about 5,000 daltons, or a molecular weight of about 300 to about 5,000 daltons, or a polyethylene glycol having a molecular weight of 750 daltons, i.e., PEG(750). Polymers may also be defined by the number of monomers therein. In some embodiments, the pharmaceutical compositions of the present technology utilize polymers of at least about three monomers, such PEG polymers being composed of at least three monomers, or approximately 150 Daltons.Other hydrophilic polymers that may be suitable for use with the present technology include polyvinylpyrrolidone, polymethoxazoline, polyethyloxazoline, polyhydroxypropylmethacrylamide, polymethacrylamide, polydimethylacrylamide, and derivatized celluloses such as hydroxymethylcellulose or hydroxyethylcellulose.

[0070] In certain embodiments, the pharmaceutical composition comprises a biocompatible polymer selected from polyamides, polycarbonates, polyalkylenes, polymers of acrylic methacrylic acid, polyvinyl polymers, polyglycolides, polysiloxanes, polyurethanes and copolymers thereof, cellulose, polypropylene, polyethylene, polystyrene, polymers of lactic and glycolic acid, polyanhydrides, poly(ortho)esters, poly(butyric acid), poly(valeric acid), poly(lactide-co-caprolactone), polysaccharides, proteins, polyhyaluronic acid, polycyanoacrylates, and blends, mixtures and copolymers thereof.

[0071] The pharmaceutical composition may also be in the form of liposomes. Liposomes contain at least one lipid bilayer membrane encapsulating an aqueous interior compartment. Liposomes may be characterized by membrane type and size. Small unilamellar vesicles (SUVs) have a single membrane and are typically in the range of 0.02-0.05 μm in diameter; large unilamellar vesicles (LUVs) are typically 0.05 μm in diameter, while oligolamellar large vesicles and multilamellar vesicles have multiple, usually concentric, membrane layers and are typically larger than 0.1 μm. Liposomes may also contain several smaller vesicles contained within a larger vesicle, i.e., multivesicular vesicles.

[0072] In some embodiments, the pharmaceutical composition comprises a liposome comprising one or more compositions of the present technology (e.g., arimoclomol, dabrafenib, necrosulfonamide (NSA), necrostatin 7 (Nec7), KIRA compounds (e.g., KIRA3 / 6 / 7 / 8), salubrinal, or SAL003) or a pharmaceutically acceptable salt thereof, wherein the liposomal membrane is formulated to provide increased carrying capacity. Alternatively, or in addition, one or more compositions of the present technology (e.g., arimoclomol, dabrafenib, necrosulfonamide (NSA), necrostatin 7 (Nec7), KIRA compounds (e.g., KIRA3 / 6 / 7 / 8), salubrinal, or SAL003) or a pharmaceutically acceptable salt thereof may be contained within or adsorbed to the liposomal bilayer of the liposome. In some embodiments, the active agent may be aggregated with a lipid surfactant and carried within the interior space of the liposome. In such cases, the liposomal membrane is formulated to resist the disruptive effects of active agent-surfactant aggregates. In certain embodiments, the lipid bilayer of the liposome comprises lipids derivatized with polyethylene glycol (PEG), such that the PEG chains extend from the inner surface of the lipid bilayer into the interior space enclosed by the liposome, and from the outside of the lipid bilayer into the surrounding environment.

[0073] The active agent contained within the liposome is preferably in solubilized form. Aggregates of surfactant and active agent (such as emulsions or micelles containing the active agent of interest) may be entrapped within the interior space of the liposome. The surfactant typically acts to disperse and solubilize the active agent. The surfactant may be selected from any suitable aliphatic, alicyclic or aromatic surfactant, including but not limited to biocompatible lysophosphatidylcholine (LPC) with varying chain lengths, for example, from about 14 to 20 carbons. Polymer-derivatized lipids, such as PEG lipids, may also be utilized for micelle formation, as they act to inhibit micelle / membrane fusion and the addition of polymers to the surfactant molecule reduces the critical micelle concentration (CMC) of the surfactant, aiding in micelle formation. Surfactants with CMCs in the micromolar range are preferred; surfactants with higher CMCs may be utilized to prepare micelles entrapped within the liposomes of the present technology, but the micelle surfactant monomers may affect liposome bilayer stability and are a factor in designing liposomes of desired stability.

[0074] The liposomes of the present technology may be prepared by any of a variety of techniques known in the art, such as those described in U.S. Pat. No. 4,235,871 and WO 96 / 14057, the entire contents of which are incorporated herein by reference. For example, liposomes may be prepared by diffusing a lipid derivatized with a hydrophilic polymer into a preformed liposome, such as by exposing the preformed liposome to micelles composed of lipid-grafted polymers at a lipid concentration corresponding to the final mole percent of derivatized lipid desired in the liposome. Liposomes containing hydrophilic polymers may also be formed by homogenization, lipid-field hydration, or extrusion techniques known in the art. By another method, the active agent is first dispersed by sonication in phosphatidylcholine or other low critical micelle concentration (CMC) surfactants (including polymer-grafted lipids) that readily solubilize hydrophobic molecules. The resulting micellar suspension of active agent is then used to rehydrate a dried lipid sample containing the appropriate mole percent of polymer-grafted lipid or cholesterol. The lipid and active agent suspension is then forced into liposome form using extrusion techniques well known in the art, and the resulting liposomes are separated from the non-encapsulated solution by standard column separation.

[0075] In some embodiments, liposomes can be prepared to have a substantially uniform size in a selected size range. One effective sizing method involves extruding an aqueous suspension of liposomes through a series of polycarbonate membranes with a selected uniform pore size. The membrane pore size corresponds roughly to the maximum size of liposomes that will be produced by extrusion through the membrane (U.S. Pat. No. 4,737,323, the entire contents of which are incorporated herein by reference).

[0076] The release characteristics of the formulations of the present technology depend on several factors, including, for example, the type and thickness of the encapsulating material, the concentration of the encapsulated drug, and the presence of release modifiers. If desired, the release can be engineered to be pH-dependent, such as by the use of a pH-sensitive coating that releases only at low pH, such as in the stomach, or at higher pH, such as in the intestine. An enteric coating can be used to prevent release from occurring until after passage through the stomach. Multiple coatings or mixtures of cyanamide encapsulated in different materials can be used to obtain initial release in the stomach and subsequent release in the intestine. Release can also be engineered by including salts or pore formers that can increase the release of the drug by water uptake or diffusion out of the capsule. Excipients that modulate the solubility of the drug can also be used to control the release rate. Agents that promote degradation of the matrix or release from the matrix may also be incorporated. The agent may be added to the drug, added as a separate phase (i.e., granules), or co-dissolved in the polymer phase depending on the compound. In all cases, the amount is preferably between 0.1-30% (w / w polymer). Some types of degradation accelerators include inorganic salts such as ammonium sulfate and ammonium chloride; organic acids such as citric acid, benzoic acid, and ascorbic acid; inorganic bases such as sodium carbonate, potassium carbonate, calcium carbonate, zinc carbonate, and zinc hydroxide; organic bases such as protamine sulfate, spermine, choline, ethanolamine, diethanolamine, and triethanolamine; and surfactants such as the commercially available surfactants Tween™ or Pluronic™. Pore formers (i.e., water-soluble compounds such as organic salts and sugars) that add microstructure to the matrix are generally included as particulates. Uptake can also be manipulated by changing the residence time of the particle in the body. This can be achieved, for example, by coating the particle with a mucoadhesive polymer or by selecting a mucoadhesive polymer as the encapsulating material. Examples include most polymers with free carboxyl groups, such as chitosan, cellulose, and especially polyacrylates (as used herein, polyacrylates refers to polymers containing acrylate groups and modified acrylate groups such as cyanoacrylates and methacrylates). EXAMPLES

[0077] The present technology is further illustrated by the following examples, which should not be construed as limiting in any way.

[0078] Example 1 Materials and Methods Cell culture. Human retinal pigment epithelium (ARPE-19) cells were obtained from ATCC and cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS and 1% penicillin / streptomycin. Cells were maintained in an incubator at 37°C with 5% CO2 and 95% humidified air. Human fetal RPE (hfRPE) cells from donors aged 16–18 weeks were cultured in RPE medium at 37°C and 5% CO2. To grow polarized hfRPE cells, cells were seeded in 12-well transwell plates in 1% RPE medium with Rock kinase inhibitor for the first week. After the first week, cells were cultured in regular 1% RPE medium for 3 weeks. Cells were used at passage 1.

[0079] Blue light treatment. ARPE19 cells were grown at 70% confluence and treated with or without 20 μM A2E in culture medium for 24 hours. The cell culture medium was then replaced with HBSS prior to blue light treatment. ARPE19 cells with or without A2E uptake were irradiated with 460+20 nm wavelength light for 20 minutes.

[0080] Cell viability assay. ARPE19 cells (80% confluent) pretreated with or without inhibitors (FIG. 12) were subjected to A2E / ATRD or vehicle (control) and incubated for 24 hours at 37° C. in serum-free OptiMEM medium. At 24 hours, viability was assessed with AlamarBlue® (Sigma, Cat. No. 199303) or by microscopic observation with NUC405 / DRAQ7. After 1 hour, medium supernatant was replaced using 1× AlamarBlue solution prepared in OptiMEM and plate fluorescence was measured with SpectraMax M (Molecular Devices, CA, USA) at 555 nm excitation / 585 nm emission. For real-time monitoring of cell death by automated fluorescence microscopy, 2 μM final concentration of NUCView Caspase 3 Substrate 405 (Biotium, Cat. No. 10407) and 0.6 μM of DRAQ7 (Invitrogen, Cat. No. D15106) were added per well and the fluorescent signal was monitored every 30 min according to the manufacturer's instructions. The precise timing of the appearance of far-red (DRAQ7 = plasma membrane leakage) and blue (NUC405 = caspase 3 activation) fluorescent signals was important to distinguish apoptosis and necrosis from secondary events such as membrane damage and general proteolytic activation during the later stages of apoptosis and necrosis, respectively. For blue light treatment, the medium of A2E / ATRD or medium-loaded cells was replaced with HBSS and the cells were exposed to a 90 watt high-power LED lamp (Cat. No. 2506BU) with 430±20 nm wavelength illumination for 15 min, and HBSS was replaced with OptiMEM medium at 0 h of treatment.

[0081] Real-time monitoring of necrotic and apoptotic cell death. Cell death assays were performed to study the dark toxicity of lipofuscin. 90% confluent ARPE-19 or hfRPE were incubated overnight in serum-free medium supplemented with the indicated LB concentrations. Viability was assessed at 24 hours by microscopy with AlamarBlue® or NUC405 / DRAQ7. Real-time monitoring of necrotic (red) and apoptotic (blue) cell death was performed by automated fluorescence microscopy. The precise timing of the appearance of far-red (DRAQ7=plasma membrane leakage) and blue (NUC405=caspase 3 activation) fluorescent signals was important to distinguish apoptosis and necrosis from secondary events such as membrane damage and general proteolytic activation during the later stages of apoptosis and necrosis, respectively.

[0082] Chemical inhibitors. The chemical inhibitors used in this study are listed in Figure 12. ARPE19 cells were treated with A2E / ATRD for 24 hours in 48-well plates with or without the inhibitors being tested.

[0083] Kinase activity assay. ARPE19 cells were treated with A2E for 6 hours, then whole cell lysates were prepared by syringe (20 times) using lysis buffer (20 mM TrisHCl, pH 7.5, 150 mM NaCl, 1% Triton, protease-phosphatase inhibitor 1X). After centrifugation at 15000×g for 10 minutes, the supernatant was collected. 200 μl (3 mg / ml) of protein was taken and incubated with primary RIP3 (13526, CS) or MLKL antibody overnight at 4°C with rotation. Then, 20 μl of agarose beads A (9863, CS) was added and kept at 4°C with rotation for 3 hours, followed by centrifugation at 15000×g for 30 seconds. The pellet was washed and dissolved in 20 μl of kinase buffer (40 mM TrisHCl pH 7.4, 20 mM MgCl2, 0.1 mg / ml BSA) with substrate and ATP (1 mM) and incubated at 30° C. for 1 h (700 speed) for kinase assay. At the end of the incubation, 10 μl of sample was taken in a 384-well plate and 5 μl of ADP-Glo ​​(ADP glo) solution was added (V6930, Promega) and kept in the dark for 40 min at room temperature, after which 10 μl of kinase detection reagent was added. After 20 min at room temperature, a luminometer reading was taken.

[0084] Animals. Pigmentation without rd8 mutation ABCA4 - / - RDH8 - / -Double knockout mice (DKO) were purchased from the Jackson laboratory and backcrossed internally to a C57BL6J control strain every 10 generations to prevent genetic drift. Genotyping was performed at Transnetyx (Memphis, TN). Only mice with ABCA4, RDH8, RPE65-Leu450 but without the crb1 mutation (retinal degeneration delayed) were maintained in the colony. Control C57BL / 6J (Rpe65-Leu450, crb1 negative) were also from the Jackson laboratory. All mice were housed in the Weill Cornell Medicine animal facility in a 12-h light (approximately 10 lux) / 12-h dark cycle environment or in complete darkness. Experimental manipulations in the dark were performed under dim red light transmitted through a Kodak No. 1 safelight filter (transmittance >560 nm). No retinal degeneration or necroptosis markers were clearly detected in C57BL6 / N (Rpe65-Met450, crb1 positive) mice aged ≥20 months obtained from the NAI / NIH. All animal procedures and experiments were approved by the Institutional Animal Care and Use Committee of Weill Cornell Medical College in accordance with guidelines established by the NIH Office of Laboratory Animal Welfare and the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic Research.

[0085] Tissue preparation. Mice were euthanized with CO2 and mouse eyes were immediately enucleated. For H&E staining, mouse eyes were directly immersed in 4% paraformaldehyde (PFA), 16.8% isopropyl alcohol, 2% trichloroacetic acid and 2% ZnCl2 in phosphate buffer and sent for paraffin embedding and sectioning. For lipofuscin images, mouse eyes were immersed in 4% PFA for 1 h before dissection. The RPE layer was dissected from mouse eyes and carefully flat-mounted on slides for lipofuscin evaluation under fluorescence microscopy. Immunofluorescence images were taken using a Zeiss spinning disk confocal microscope (Zeiss, Jena, Germany).

[0086] RPE and neural retina flat mounts. Mouse eyes were enucleated and placed in 4% PFA in PBS for 1 hour at room temperature. After fixation, a hole was made in the limbal region using a 23G needle, the periphery of the limbus was cut using iris scissors, the cornea, iris and lens were removed, and the neural retina and sclera were separated by microforceps. The neural retina and RPE layers were excised and incubated with blocking buffer (1% BSA and 0.3% Triton-X-100 in PBS) for at least 20 minutes. Primary antibodies were added to the blocking buffer and kept at 4°C overnight. The retina and RPE layers were washed and incubated with secondary antibodies for at least 30 minutes at room temperature, then washed three times with PBS. Under a dissecting microscope, the retina or RPE layer was cut into four-leaf clover shapes and mounted on slides in mounting medium (EMS glycerin mounting medium with DAPI and DABCO, catalog number 17989-61). Slides were stored at 4°C until imaging.

[0087] Cryosectioning and immunofluorescence staining. Mouse eyes were fixed with 4% PFA, infiltrated with 30% sucrose overnight at 4°C, then cryopreserved in Tissue-Tek OCT compound (Sakura Finetek, Torrance, CA, USA) and sectioned into 15 μm sections. Sections were blocked with 1% BSA and 0.1% Triton-X-100 PBS, and then immunofluorescence staining was performed using standard methods and appropriate dilutions of primary antibodies against pp-MLKL (cell signaling), XBP1s (Biogend), Iba-1 (Abcam), CD11b (Millipore), Lamp2 (hybridoma bank), CellRox and DRAQ7 (Invitrogen), rhodopsin (Abcam), phalloidin-CF660 (Biotium, Cat. No. 0052), Hoechst 33324, caspase 3 (clone 9664, Cell signaling). Slides were then incubated with Alexa-647, Alexa-594 secondary antibodies and counterstained with Hoechst 33324. Negative controls were included for each staining and slides were mounted with antifade mounting medium. Slides were stored at 4°C before analysis on an SD microscope using Zen software. For paraffin sections, mouse eyes were embedded in paraffin and cut into 5 μm sections. After deparaffinization using standard protocols, slides were incubated with blocking buffer for 2 h and then incubated with primary antibodies overnight. All antibodies used for immunofluorescence staining are listed in Figures 10 and 13.

[0088] Demelanization. Paraffin was removed from paraffin slides using standard protocols as described herein. Slides were washed 4 times in xylene at 4 min / wash, dipped 20 times in 100% ethanol, repeated 4 times, and then slides were air-dried for 10 min. For melanin bleaching, slides were immersed in PBS with 10% H2O2 at 55°C for 5 min or until melanin was bleached. Slides were blocked with blocking buffer and immunofluorescence staining was performed as described above.

[0089] H&E. H&E staining was performed using standard protocols as described herein. Paraffin was removed from slides using protocols as described above. Slides were air-dried. Slides on racks were placed in xylene for 2 minutes (repeated once); then in 100% ethanol for 2 minutes (repeated once); then in 95% ethanol for 2 minutes, once. Slides were then placed in hematoxylin for 3 minutes, then in eosin for 45 seconds, 95% ethanol for 1 minute, 100% ethanol for 1 minute, twice, then in mounting medium, and finally cover slipped.

[0090] Pharmacological treatment of mice. KIRA6 (Cayman Chemical, product number 19151) was injected intravitreally in a total volume of 1 μL. The KIRA6 concentration is 20 μg / ml. The control eye received an equal volume of mock reagent (DMSO). The mice were weighed and anesthetized with 10 mg / kg ketamine cocktail, and then the mouse eyes were dilated with tropicamide. The exact volume of mock reagent or Kira6 was measured by a 10 μl Hamilton syringe. Under an operating microscope, the mouse eye was centered in the field of view, and a 34-gauge needle was inserted into the mouse eye at the ora serrata, toward the ONH. Once the needle was inside the mouse eye, 2 μl of the contents in the Hamilton syringe was injected. The needle was slowly withdrawn to prevent backflow of the solution. Nec7 (2 μL of 33 mM stock in DMSO) was injected intravitreally into the left eye and an equal volume of vehicle (DMSO) was administered to the right eye. After intravitreal injection, mice were placed on a warm surface until fully awake. A single intravitreal injection of Nec7 reduced pMLKL staining, as was done in each fellow eye. Immunofluorescent staining with anti-p-MLKL (red) and XBP1s (green) and LB (white) of RPE flat mount samples from postnatal day 700 mice is shown (n=3). Bar=20 μm. Lipofuscin synthesis. A2E was synthesized and purified by HPLC (>97%) according to published protocols. The quality of the material was assessed by mass spectrometry and UV absorbance between 250-600 nm.

[0091] HPLC analysis of bisretinoid content. Bisretinoids were extracted from mouse eyecups under red dim illumination. Briefly, single mouse eyecups (containing RPE / choroid / sclera without neural retina) or ARPE-19 cells were washed with phosphate buffered saline (PBS) and homogenized in 1 mL PBS. Four milliliters of chloroform / methanol (2:1, vol / vol) were added, and samples were extracted by adding 4 mL chloroform and 3 mL dH2O, followed by centrifugation at 1000×g for 10 min. Extraction was repeated with the addition of 4 mL chloroform. The organic phases were pooled, filtered, dried under a stream of argon, and redissolved in 100 μL 2-propanol. Bisretinoid extracts were analyzed by normal-phase HPLC on a silica column (Zorbax-Sil 5 μm, 250×4.6 mm; Agilent Technologies, Wilmington, Del.) as previously described (Sparrow JR, et al. (2003) J Biol Chem 278(20):18207-13). The mobile phase was hexane / 2-propanol / ethanol / 25 mM potassium phosphate / glacial acetic acid (485:376:100:45:0.275 vol / vol), filtered prior to use. The flow rate was 1 mL / min. Column and solvent temperatures were maintained at 40° C. Absorbance units at 435 nm were converted to picomoles using a calibration curve of authentic A2E standards and the published molar extinction coefficient of A2E; the identity of each bisretinoid peak was confirmed by online spectroscopic analysis.

[0092] RNA isolation and quantitative PCR. Total RNA was extracted from cultured cells or mouse ocular RPE layers using RNeasy Mini kit (QIAGEN). Total RNA was digested with DNase I to prevent amplification of genomic DNA. Total RNA was then reverse transcribed using High Capacity cDNA Reverse Transcription Kit (ThermoFisher Scientific, Cat. No. 4368814) and gene expression was analyzed using SYBR Green Master Mix (ThermoFisher Scientific, Cat. No. 4472908) in an Applied Biosystems StepOne real-time PCR instrument. GAPDH was used as a reference gene. The primer sequences are shown in FIG. 11. After the genes were amplified by real-time PCR, a portion of the PCR product was separated by 2.5% agarose gel.

[0093] RNAseq. Cultured ARPE19 cells were treated with or without 15uM A2E for 24 hours, then cells were harvested and total RNA was extracted. Proteins were prepared for mass spectrometry. RNAseq profiles were further analyzed with Ingenuity Pathway Analysis (IPA, Qiagen).

[0094] Amount of lipofuscin per cell. RPE cells were seeded overnight in DMEM-10%FBS. The next day, the medium was replaced with Opti-MEM supplemented with the indicated amount of A2E (0, 10, 20, 30 μM) for 24 hours. A2E-loaded cells were trypsinized, counted, and resuspended in lysis buffer. For ocular RPE, 12-month-old WT and DKO mice were sacrificed, their eyes enucleated, and their RPE were isolated from the neural retina and underlying choroid using Qiagen's RNAprotect (RNA Protect) Cell Reagent (Cat. No. 76526) at 100 uL / eye cup for 10 min as previously described (Xin-Zhao Wang C et al., (2012) Exp Eye Res 102:1-9). Mouse RPE cells were counted and resuspended in lysis buffer. The lysis buffer used was 2% Triton X-100 1% SDS in water, as it efficiently dissolves cell membranes and lipid bisretinoids. Fluorescence of the lysates (arbitrary fluorescence units) was measured using a Spectramax M5s plate reader at 430 nm for excitation and 600 nm for detection. Detection of lipid bisretinoid aggregation. To demonstrate aggregate formation of lipid bisretinoids, the fluorescence of 500 μM A2E or ATRD solutions in PBS was measured before and after passage through 13 mm Nylon syringe disc filters 0.45 μm and 3 μm (Tisch scientific, Ohio).

[0095] Immunoblotting. After incubation of specific sample groups in 48-well plates, whole cell lysates were extracted using RIPA lysis buffer (50 mM TrisHCl, Ph8.0, 150 mM NaCl, 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS, protease-phosphatase inhibitors). Sonication or syringeing (20 times) was performed, followed by centrifugation at 12,000×g for 10 min, and the supernatants were collected and stored at −80° C. Protein concentration from homogenates was assessed by Pierce Rapid Gold BCA Protein Assay Kit (ThermoFisher Scientific, Cat. No. A53225). 30 μg of protein was then analyzed by standard SDS-PAGE gel. Samples were mixed 4:1 (volume:volume) with Nupage loading buffer with or without 8% β-mercaptoethanol and heated at 72° C. for 10 min. SDS-PAGE was performed using 4–12% Nupage gels and buffer (Life, NP0335BOX). The SDS-PAGE gel was then transferred to a nitrocellulose membrane (Whatman, PROTAN BA83) overnight at 20V. The next day, the membrane was blocked using 5% milk in TBS for 2 h, then primary antibodies were added in TBST (TBS, 0.1% Tween-20) overnight at 4°C. The next day, the membrane was washed and incubated with HRP-conjugated secondary antibody (catalog no. G21234, Invitrogen) at 1:10,000 dilution at room temperature for 2 h. After washing the membrane three more times, it was probed with enhanced chemiluminescence (ECL) reagent and detected using X-ray film for chemiluminescence images (GE healthcare, RPN2106). Scanning / imaging and quantification of images were performed using silverfast8 application software and Fiji ImageJ. Antibodies for Western blots are listed in Figure 10.

[0096] Retinal ONL thinning quantification. Whole eyes were embedded in paraffin and sectioned at a thickness of 5 μm. Sections were counterstained with hematoxylin and eosin (H&E). Digital images were taken using light microscopy and stitched by Zen software. Outer nuclear layer (ONL) thickness was measured at 0.2 mm intervals superior and inferior to the edge of the optic nerve head (ONH) along the vertical meridian. The center of the ONH was used as the start of the measurement. ONL thickness was measured by Zen software.

[0097] Retinal RPE layer nuclei quantification. RPE nuclei were quantified in H&E counterstained eyecup cross sections. Images were taken at 40x and stitched by Zen software. The number of RPE nuclei was counted at every 0.1 mm interval in 23-month-old DKO (n=10) and 27-month-old WT retinas (n=8) and plotted as a function of distance from the ONH. Means (±SEM) were significantly different at each point (p<0.05). Retinal RPE cell size quantification. Retinal RPE optic cups were carefully dissected from mouse eyes and stained with Alexa647-phalloidin for 1 h at room temperature before flat mounting on slides. A spinning disk confocal microscope with a 63x lens was used to image the RPE layer from the optic nerve head to the periphery of the layer. The outlines of individual RPE cells were visualized with phalloidin. Cell borders were manually selected and cell area was measured using ImageJ software, and area was measured using ImageJ after manually selecting cell borders. The number of RPE nuclei was counted at every 0.1 mm interval in 23-month-old DKO (n=10) and 27-month-old WT retinas (n=8) and plotted as a function of distance from the ONH. Means (±SEM) were significantly different at each point (p<0.05).

[0098] Statistics. All data were processed with Prism 7.0 software. ANOVA, Student's t-test or multiple t-test were used where appropriate. A p-value of less than 0.05 was considered statistically significant.

[0099] Example 2 LF accumulation and retinal degeneration Although HPLC, and more recently quantitative fundus autofluorescence (qFAF), have become the gold standard for measuring LB content in the retina of animal models (Sparrow JR, et al. (2013) Investig Ophthalmol Vis Sci 54(4):2812-2820), the amounts reported by each method are still not fully comparable. Mouse strains with excessive accumulation of LF, such as Abca4 - / - or Abca4- / -RDH8 - / - Experiments in (double KO or DKO mice) report an unusual dichotomy: qFAF shows that the combined content of LBs in the RPE and PR increases continuously throughout life, whereas HPLC shows a rapid decrease in LBs in the RPE after the first few months (Sparrow JR, et al. (2013) Investig Ophthalmol Vis Sci 54(4):2812-2820). This dichotomy is attributed to an early and sudden loss of RPE cells that contain LFs above a threshold level that promotes new LF formation in the PR and impairs phagocytic clearance, permanently compromising the functionality of the epithelium (Flynn E, Ueda K, Auran E, Sullivan JM, Sparrow JR (2014) Invest Ophthalmol Vis Sci 55(9):5643-52).

[0100] To clarify the cytological and pathological aspects of this process, experiments in DKO versus control mice were performed to compare the accumulation of HPLC-extractable LBs with the accumulation of fluorescent granules in the cytoplasm of RPE cells, measured by confocal microscopy, as a function of retinal age. Using confocal microscopy, the number of LF granules per RPE cell increased to completely occupy the entire cytoplasm in aged DKOs (Figure 1A). LF levels were 5-10X higher in DKOs compared to aged WT RPE cells (Figure 1B). Retinal cross-section images of DKOs revealed that the relative amount of LF between the RPE and PR did not obviously change after 8 months, with the RPE still being the main source of autofluorescence (Figure 1C). Phalloidin staining to visualize the actin cytoskeleton and cell boundaries (Figure 1D) revealed that WT RPE displayed a typical uniform hexagonal geometric shape that was maintained with age. In contrast, DKO RPE organization progressively deteriorated to a predominant phenotype consisting of diffuse multinucleated giant cells with high content of LF and intracellular stress fibers at 24 months. The continuous accumulation of granules per RPE cell throughout life is consistent with the qFAF data and supports the idea that the severity of LF burden increases progressively with age instead of reaching a threshold that damages the RPE at younger ages. Furthermore, we assessed the lipofuscin content of RPE cells by high pressure liquid chromatography (Figure 1E). The overall trend was a continuous increase in lipofuscin with age, consistent with the confocal microscopy data.

[0101] To quantify the extent of this phenomenon, flat mounts of DKO RPE and age-matched controls (8-27 months) separated from the neural retina between 8 and 23 months of age were used to measure the size (μm) of cells at random central locations. 2 The average cell size was 324–411 μm in WT. 2 , 330-1000μm for DKO 2(p<0.01), indicating a significant expansion of RPE cells in retinas with LF. This correlated with a significant reduction in the number of RPE nuclei in DKO mice over 25 months of age compared to age-matched WT mice (p<0.05) (Figure 2B). Progressive retinal degeneration was also evidenced by the observation that 27-month-old DKO mice had significantly fewer PRs from center to periphery than age-matched WT controls (Figure 2C). Furthermore, confocal microscopy of cryosections from aged DKO retinas revealed the presence of small (~1-3 μm) lipofuscin dots in the neural retina that were absent in aged WT controls (Figure 2D). These particles stained positively for Iba-1, rhodopsin (Figure 2E) and CD11b (Figure 2I) but negative for melanin (Figure 2E), indicating that they represent activated microglia carrying phagocytosed debris of degraded photoreceptor outer segments (Figure 2E).

[0102] Furthermore, larger (~5-10 μm) epithelial sized fragments filled with LF (Figures 2F-2G) and melanin (Figure 2H) were observed in the neural retina. These LF fragments could potentially represent migrating RPE cells / fragments undergoing epithelial-mesenchymal transition (EMT); this observation could explain the origin of the macula and the sloughed RPE features of Stargardt and AMD retinas, respectively. Importantly, the outer nuclear layer (ONL), in close proximity to areas with RPE migratory activity, appeared thin and disorganized (Figure 2H), reflecting damage caused to photoreceptors (RPs) by the inflammatory process. Indeed, TUNEL staining of frozen sections showed dead PRs surrounding the migratory RPE fragments (Figure 2J). Overall, these results indicate a direct link between the accumulation of LF granules in the RPE and accelerated retinal erosion, possibly due to direct LF toxicity, as well as the recruitment of activated microglia and promotion of EMT behavior in the RPE.

[0103] Lipofuscin was quantified in mouse RPE cells in DKO and WT mice of different ages by microscopy. Mouse RPE cells were photographed from the center of the mouse eyecup (ONH) to the periphery. Lipofuscin in central RPE cells was quantified by ImageJ and graphed. More than 300 RPE cells were quantified in each group, and each dot in the graph represents a single cell. Lipofuscin in 8-, 13-, and 26-month-old DKOs was significantly higher than 3-month-old DKOs (p<0.01 by unpaired t-test). 3-month-old DKOs were higher than 8- and 33-month-old WTs (p<0.01 by unpaired t-test). 33-month-old WT group was significantly higher than 8-month-old WT group (p<0.01 by unpaired t-test).

[0104] In summary, confocal images of RPE from DKO eye cups showed that the number of LF granules per cell increased seamlessly with age, accompanied by an expansion in size and a decrease in the number of RPE (Figures 1A-1D), reflecting the expansion of surviving cells to fill the gaps created by RPE death or migration into the neural retina. The observed RPE death could be due to direct toxicity or indirect damage caused by activated microglia / macrophages recruited to the LF-bearing layer (Figures 2A-2H). However, the content of LB in the RPE dropped significantly after the 8th month of life (Figures 2I-2J), although the number of granules per cell continued to increase. This dichotomy could be attributed to an age-associated increase in the chloroform insolubility of the LF granule content, rather than a loss of LF-laden RPE.

[0105] Example 3 LF photooxidation and retinal degeneration Photooxidative degradation of LBs contributes to retinal degeneration in albino animals, but whether photooxidation occurs at a significant level in the pigmented retina remains unclear. Since photooxidation can be stopped by rearing animals in complete darkness without affecting the accumulation of new LBs (Ueda K et al., (2016) Proc Natl Acad Sci US A. 113(25):6904-9; Boyer NP et al., (2012) J Biol Chem 287(26):22276-22286), studies were performed to determine whether RPE and PR degenerate in DKO retinas that are never exposed to light. Therefore, WT and DKO pigmented mice were housed from birth in either continuous darkness or a 12-hour light cycle. Changes in their ONL thickness and number of RPE nuclei were evaluated up to 1 year. As shown in Figure 3A, LF-associated thinning of the ONL and loss of RPE number progressed at a similar rate independent of whether the mice were light- or dark-housed. The WT group did not show significant loss of PR or RPE during the same time period regardless of lighting conditions. To confirm that LBs still accumulate in the absence of light, LF autofluorescence was quantified in each of the four groups in RPE flat mounts from 12-month-old animals (Figure 3B). Dark-housed mice, both WT and DKO, contained approximately 2.8-fold more autofluorescent material than their respective counterparts under light-cycle conditions (p<0.05), and DKO contained approximately 5-fold more LF than WT (Figure 3B). These results reveal that an in vivo light-independent toxic cascade contributes to the deterioration of RPE and PR in pigmented retinas with excessive LF levels. Taken together, the substantial loss of PR and RPE cells between dark- and light-reared DKO retinas (Fig. 3A) and the significant photobleaching of LF autofluorescence in light-exposed eyes (Fig. 3B) indicate that the intrinsic toxicity of granules plays a role in the degenerative process independent of their propensity to photooxidize, but suggest that the pigmented retina may be capable of handling moderate LB photooxidation.

[0106] Example 4 Light-independent cell death in vitro. To study the mechanisms involved in light-independent cell death, an existing protocol for the uptake of LB into the lysosomes of RPE cells was employed (Sparrow JR et al., (1999) Invest Ophthalmol Vis Sci 40(12):2988-95, Sparrow JR, Kim SR, Wu Y Chapter 18 Experimental Approaches to the Study of A2E, a Bisretinoid Lipofuscin Chromophore of Retinal Pigment Epithelium. 315-327, Boulton ME (2014) Exp Eye Res 126:61-7), which reproducibly induced dose-dependent RPE cell death under light-free conditions (Figure 4A).

[0107] Sensitivity to LF was highly dependent on cell confluency (Figure 4J), as denser cell cultures were more resistant to LB uptake (Figure 4K). Nevertheless, the efficiency of uptake was constant at different LB doses (Figure 4L). By default, 80% confluency was chosen for the assay, since under these conditions ARPE-19 absorbed 80% of the added LB, reaching LB levels within the concentration range seen in RPE from DKO retinas (Figure 4M). Importantly, the fluorescent readout of lysates from ARPE19 and pigmented RPE from retinas was not affected by melanin (Figure 4V). Using this in vitro configuration, LF also triggered light-independent cell death in highly differentiated hfRPE (Figure 4N). LF-induced apoptosis and necrosis at the single cell level was investigated by adding NucView® 405 (a non-fluorescent cell-permeable substrate that stains nuclear DNA blue when cleaved by caspase 3 during the executioner phase of apoptosis) and DRAQ7 (a dye that stains DNA red only if cells have compromised membrane integrity) to the culture medium. LF induced necrosis without light, but apoptosis was induced when exposed to blue light (Figure 4B). ATRD has the same aldehyde group touted to be responsible for its higher toxicity than its precursor all-trans retinal (ATR), but was a less potent inducer of necrosis and apoptosis than A2E (Maeda A et al. (2012) Nat Chem Biol 8(2):170-8). In contrast, A2E, but not ATRD, contains both a hydrophobic retinoid-derived chain and a hydrophilic pyridinium head group, conferring amphipathic properties (Soma De S, Sakmar T (2002) J Gen Physiol 120(2):147-157).

[0108] To determine whether A2E induces necrosis by inserting into membranes, methyl β-cyclodextrin (MβCD) was used. MβCD is a cyclic sugar that protects against detergent effects by forming a soluble complex with amphiphilic molecules. Remarkably, MβCD complexed with both A2E and Triton-X100, but did not protect against A2E, and completely protected against Triton-X100-induced lethality (Figure 4C). This result, together with the slow kinetics of cell death (several hours vs. immediate for Triton-X100), suggested that LF triggered programmed necrosis. Therefore, the protective effects of inhibitors of the two main effector cascades of regulated necrosis: caspase / gasdermin-D and RIPK3 / MLKL were tested (Figure 4D).

[0109] Pretreatment with the pan-caspase inhibitor z-VAD(OMe)-FMK and the Gasdermin-D inhibitor disulfiram did not provide protection, nor did GSK'872, a selective inhibitor of RIPK3 (the only known kinase that phosphorylates human MLKL at Ser358 (pMLKL)).

[0110] However, dabrafenib, an ATP-competitive inhibitor of B-Raf and RIPK3, or necrosulfonamide (NSA), a drug that prevents the spontaneous assembly of pMLKL into oligomeric pores that eventually insert into the membrane, increased RPE survival in a dose-dependent manner. For its phosphorylation, MLKL and its kinases must be recruited to a multiprotein complex known as the necrosome (Figure 4O). Necrosomes are not well conserved between species, and the nature of the stimuli that trigger their formation differs. Anti-MLKL immunoprecipitation (IP) yielded kinase-associated activity and pulldown only when prepared from lysates arising from cells containing LF, demonstrating that MLKL is part of the necrosome (Figure 4E). Given that phosphorylation of human MLKL at Ser358 (pMLKL) is a telltale triggering event and marker of necroptosis, we determined the Ser358 phosphorylation and polymerization state of MLKL in cells with LF. Using Western blots (WB) under non-reducing conditions that preserve and increase the chance of detecting phospho-oligomers, we showed that A2E (Fig. 4F) and ATRD (Fig. 4P) caused dose-dependent phosphorylation and polymerization of MLKL in the absence of light. WB analysis of anti-MLKL pull-downs (Co-IP) did not reveal bands corresponding to RIPK1 or RIPK3, the most common partners in the necrosome (data not shown). A prerequisite for the assembly of these RIP kinases into the necrosome was their phosphorylation.

[0111] To exclude that low expression of these proteins in ARPE19 would preclude their detection, experiments were performed in human intestinal HT29 cells after treatment with LB or TNFα / caspase inhibitors (Figure 4T). Although HT29 cells expressed high levels of these proteins, pRIPK1 and pRIPK3 remained undetectable in cells with LF, but were readily visible upon treatment with a TNFα inhibitor cocktail. The spectrum of protection offered by necrostatins was also investigated. Necrostatins were initially identified for their strong inhibition of TNFα-induced necroptosis in FADD-deficient Jurkat T cells (Zheng W, Degterev A, Hsu E, Yuan J, Yuan C (2008) Bioorganic Med Chem Lett 18(18):4932-4935, Teng X, et al. (2005) Bioorganic Med Chem Lett 15(22):5039-5044). Necrostatin-1 (Nec1), Nec1s, Nec2 and Nec5 all target RIPK1 (Degterev A, et al. (2008) Nat Chem Biol 4(5):313-321), whereas Nec7 targets an unknown regulatory molecule in the pathway. Thus, RIPK1-targeting necrostatins did not provide a survival benefit, whereas Nec7 was highly protective (Figure 4G). WB results show that treatment with Nec7 (Figure 4H and Figure 4U), but not Nec1 or GSK'872, reduced LF-induced phosphorylation and polymerization of MLKL (Figure 4U).

[0112] In contrast, Nec1 did not block phosphorylation and polymerization of MLKL (Figure 4R). Finally, using confocal fluorescence microscopy, we showed that Ser358 phospho-MLKL localized to the membrane in cells with abnormal levels of A2E and ATRD, but not in healthy controls (Figure 4I), and Nec7 treatment effectively reverted the pMLKL membrane localization pattern. Together, these results indicate that LF induces a novel and uncharacterized type of necrosome that does not contain RIPK1 and potentially lacks RIPK3. Chronic accumulation of polymerized pMLKL accumulates LF and may explain the progressive damage in the pigmented retina.

[0113] These results indicate that light-independent cytotoxicity of A2E- or ATRD-loaded LF granules induces necroptosis, a type of programmed cell death that results in cell membrane destruction causing areas of atrophy and release of cellular components known to induce local inflammation. Evidence for light-independent LF-mediated necroptosis includes: (i) real-time monitoring of cell death showed early impairment of membrane integrity without caspase-3 activation; (ii) LF induced dose-dependent Ser358 phosphorylation, polymerization, and plasma membrane translocation of pseudokinase mixed lineage kinase domain-like (MLKL) in the absence of light; (iii) cell death was preventable by dabrafenib and NSA; (iv) immunoprecipitation experiments revealed that LF promoted the association of MLKL with kinases, indicative of necrosome formation; (v) dark cell death could not be prevented by pan-caspase inhibitor z-VAD(OMe)-FMK or antioxidants; and (vi) Nec7 potentially prevented MLKL phosphorylation, polymerization, plasma membrane localization, and cell death by LF. See Figures 4A-4I.

[0114] LF cell death and MLKL phosphorylation / polymerization were not affected by GSK'872 (Figure 4D and Figure 4Q) and the RIP1 kinase inhibitors Nec1, Nec1s, Nec2, and Nec5 (Figure 4G and Figure 4Q-4R) and were insensitive to antioxidants (Figure 5A). Furthermore, RIPK3 was undetectable at the mRNA and protein levels in ARPE19 and hfRPE cells, even after LB treatment prior to necroptosis (Figure 4S). In cells with high levels of RIPK1 and RIPK3 expression, LF still did not induce detectable phosphorylation of these molecules, whereas the typical combination of TNFα+Smac mimetic and caspase inhibitor z-VAD (TSZ) used to induce RIPK1-RIPK3-mediated necroptosis clearly did (Figure 4T). All these combined results indicate that RIPK1 and RIPK3 are not part of the LF-induced necrosome.

[0115] Finally, aside from the deleterious effects of lipid bisretinoids, ABCA4 - / - RDH8 - / - Since mouse retinas have been suggested to die due to toxicity of all-trans-retinal (ATR) released upon illumination, the link between ATR and necroptosis was investigated. Using cell death assays, ATR was found to trigger light-independent cell death in ARPE19 cells (Figure 4W), but the mechanism was different from canonical or lipofuscin-induced necroptosis, as it was insensitive to Nec1 and Nec7 (Figure 4X). Western blots of cells treated with ATR did not show increased phosphorylation / polymerization of MLKL (Figure 4Y). Overall, these indicate that necroptosis is a specific readout of ocular lipofuscin toxicity, easily distinguishable from the toxic effects of ATR. These results indicate that dabrafenib, necrosulfonamide (NSA), necrostatin 7 (Nec7), and IRE1α inhibitors that block IRE1α dimerization are useful in methods for preventing or treating ocular diseases associated with retinal cell lipofuscin-related cytotoxicity in subjects in need thereof.

[0116] Example 5 LF triggers MLKL-induced necroptosis LF deposits are thought to induce oxidative stress (Ueda K, et al. (2018) Proc Natl Acad Sci USA 115(19):4963-4968). Thus, the protective effects of various antioxidants against LF cytotoxicity were investigated: N-acetylcysteine ​​(NAC), Trolox, L-cysteine, vitamin C, BHA, and TMB. Although NAC effectively prevented oxidative damage by H2O2, none of the tested antioxidants had any effect on RPE viability in terms of LF accumulation (Figure 5A). Furthermore, reactive oxygen species (ROS) were visualized at intracellular levels with CellROX Deep-Red®. In the dark, cellular ROS appeared to be associated only with mitochondria, not in lipofuscin granules. However, when cells were illuminated with blue light, LF granules burst and autofluorescent material diffused into the cytoplasm, becoming positive for ROS (Figure 14A). Thus, even though the method could effectively detect LF-induced ROS generation, there was no evidence of oxidative stress associated with granules during the induction of necroptosis.

[0117] Since necroptosis can be triggered by accumulation of crystalline material in cells, and atomic force microscopy revealed that the core of lipofuscin granules contains solid aggregates, the ability of lipid bisretinoids to form cell-killing crystals was investigated. In an aqueous environment, A2E (MW 592 Da) formed aggregates the size of bacteria that could not migrate through 450 nm filter pores (Figures 14B and 14C). This inability to pass was due to size exclusion and not nonspecific binding, since A2E passed through a membrane with a 3 μm cutoff of the same material. A2E accumulation was then imaged using a combination of DIC and fluorescence: A2E appeared as granules with clear edges both intracellularly or after drying on a coverslip (Figures 14D and 14E). The ability of suspected A2E crystals to damage lysosomal membranes was then investigated. A recently developed galectin-3 speckle assay for early detection of lysosomal membrane permeabilization (LMP) was used. Briefly, cytoplasmic galectin 3 becomes leaky and rapidly binds to the glycocalyx at the luminal surface of the lysosomal membrane, which is easily detectable with anti-galectin high affinity antibodies. L-leucyl-L-leucine methyl ester (LLO), a lysosomotropic peptide that triggers LMP, was used as a positive control for speckle formation (Figure 14E). 50 μM A2E accumulation also clearly triggered speckle staining and therefore LMP (Figure 14F). LMP was confirmed by showing inactivation of cathepsin D as a surrogate for lysosomal enzymes. Both LLO (Figure 14G) and A2E (Figure 14H) triggered a decrease in cathepsin D activity. Interestingly, the loss of cathepsin D was prevented by arimoclomol, a drug that promotes lysosomal integrity through upregulation of heat shock proteins, and by Nec-7, the only necrostatin that protects against lipofuscin (Figure 14I). Furthermore, arimoclomol, like Nec-7, protects against lipofuscin-induced necroptosis (Figure 14J).Also highly striking was the observation that the same unique pattern of protection with arimoclomol and Nec-7, but not Nec-1, was seen with LLO, consistent with the idea that A2E-induced necroptosis is driven by LMPs (Figure 14K).

[0118] To investigate the cellular processes leading to necroptosis, we used Ingenuity Pathway Analysis (IPA) to compare the transcriptomes of cells suffering from dark LF cytotoxicity with cells rescued by Nec7 treatment. As shown in Figure 5B, inhibition of the unfolded protein response (UPR) was by far the most important change induced by treatment with Nec7. The UPR is a conserved transcriptional response to the abnormal accumulation of misfolded proteins or lipids in the endoplasmic reticulum. The UPR is triggered by three ER endogenous sensors: PERK, IRE1α, and ATF. Visualization of the UPR in IPA revealed that the PERK and IRE1α pathways were negatively regulated by Nec7 at multiple points (Figure 5C). To assess the impact of UPR downregulation, we extracted a list of UPR transcripts inhibited by Nec7 and performed a new IPA analysis of downstream outcomes (Figure 5D). The UPR had a significant impact on cell death, along with other cellular functions and communication signals.

[0119] The causal relationship between LF accumulation and ER stress without the assistance of light was analyzed (Figure 6A). The PERK branch was significantly activated by LB. Indeed, A2E produced the strongest Ser51 phosphorylation of eIF2α, whereas ATRD produced the highest induction of ATF4 and BiP. qPCR confirmed the induction of ATF4 at the mRNA level by both LBs (Figure 6B). Activation of the IRE1α branch was visualized by qPCR. As shown in Figures 6C-6D, dose- and time-dependent induction of XBP1s by A2E and ATRD was observed in the dark.

[0120] Conventional PCR coupled with agarose gel revealed splicing of XBP1 in ARPE19 and hfRPE (Figure 6E). ATF6 also showed significant cleavage after 6 h treatment with 20 μM A2E or tunicamycin (Tn) (Figure 6F). Nec7 pretreatment completely abrogated XBP1 splicing (Figure 6G) and CHOP upregulation (Figure 6H), indicating robust inhibition of ER stress. In contrast, cells incubated with A2E and treated with RIPK3 or RIPK1 inhibitors showed normal CHOP upregulation in response to LF. Collectively, these results indicate that LB accumulation activates the three branches of the UPR without illumination and that Nec7 was able to abrogate the UPR.

[0121] To determine how the UPR is involved in LF-induced necroptosis, we individually knocked out (KO) each of the three endogenous ER stress sensors / effectors, PERK, ATF6, and IRE1α, in ARPE-19 cells and tested their sensitivity to LB in our light-free cell-death assay. Notably, IRE1α-KO showed a significant increase in survival to toxic doses of LB (Figure 6I). IRE1α is a bifunctional kinase / RNase that initiates a concatenated chain of activation events upon ER stress that begins with its dimerization, kinase activation by autophosphorylation, and culminates in the activation of its RNase function. IRE1α is a type I ER transmembrane multidomain protein with a sensor domain for the ER lumen that activates RNase at the far side of its cytoplasmic region in the presence of misfolded proteins or disordered lipid composition clusters that promote kinase activity that trans-autophosphorylates molecules, and through allosteric regulation.

[0122] We assessed the critical role of IRE1α in the light-independent cell death process using small molecules that selectively blocked IRE1α at different stages of activation. A set of inhibitors selective for kinase (APY29, sunitinib) or RNase (STF-0831, 4μ8C, MKC-3946) function was tested. Surprisingly, inhibition of individual or combined kinase, RNase, or both activities did not translate to viability, suggesting that LF-mediated necroptosis does not depend on the canonical signaling pathway of IRE1α as a UPR response (Figure 6J). In contrast, blocking dimerization (e.g., KIRA3, KIRA6) was sufficient to protect against LF-induced necroptosis (Figure 6J). These results are consistent with the formation of the uprosome, a multimolecular structure in which IRE1α dimers or oligomers act as scaffolds to assemble interacting proteins that cross-circuit other pathways and provide novel functional outputs (Urra H, et al. (2018) Nat Cell Biol 20(8):942-953;Hetz C, Chevet E, Oakes SA (2015) Nat Cell Biol 17(7):829-838;Petersen SL, et al. (2015) Cell Death Differ 22(11):1846-1857;Sepulveda D, et al. (2018) Mol Cell 69(2):238-252.e7;Hetz C, Glimcher LH (2009) Mol Cell 35(5):551-561). Thus, LFs can induce the expression of adaptor proteins that assemble into the IRE1α-uplosome and bridge ER stress to necrosome formation (Figures 8A-8E; Figure 9B). These results indicate that dabrafenib, necrosulfonamide (NSA), necrostatin 7 (Nec7), arimoclomol, and IRE1α inhibitors that block IRE1α dimerization are useful in methods for preventing or treating ocular diseases associated with retinal cell lipofuscin-related cytotoxicity in subjects in need thereof.

[0123] Example 6 IRE1α-mediated necroptosis in LF-bearing retinas To examine whether the pigmented retina with LF undergoes progressive IRE1α-mediated pMLKL oligomer deposition, eyes from 2-, 12-, and 27-month-old ABCA4- / -RDH8- / - DKO and WT C57BL6 mice were dissected and their whole RPE flat-mounted and stained with anti-XBP1s (Figure 7A) or anti-phospho-Ser358MLKL (Figure 7B). XBP1s and pMLKL expression was barely detectable in the WT retina, even in 27-month-old mice. In contrast, DKO animals showed diffuse XBP1s staining at 2 months, with staining increasing in intensity in discrete clusters of RPE cells by 1 year and widespread by 27 months. Similarly, pMLKL was dim at 2 months and increasingly positive, initially intracellularly and later at the plasma membrane (Figure 7B). Staining for XBP1s and pMLKL became more intense and widespread with age, with pMLKL clearly showing association with the plasma membrane in the oldest DKOs.

[0124] The atypical necroptosis observed in cell culture was blocked by Nec7. To confirm the role of Nec7 in necroptosis detected in vivo, 1 μl of vehicle and 1 μl of Nec7 were intraocularly injected into the right and left eyes of 12-month-old DKOs, respectively, and their pMLKL levels were analyzed 1 week later. As shown in Figure 7H, membrane and cytoplasmic pMLKL labeling was reduced to undetectable levels after Nec7 treatment, confirming that the atypical necroptosis pathway is active in retinas with LF. Nec7 treatment also shows the specificity of staining with pMLKL antibody. The reduction in pMLKL levels was complete throughout the entire retina (Figure 7I). To characterize the distribution of ER stress and necroptosis markers in the retina, we performed double staining for XBP1s and pMLKL on demelanized paraffin retinal cross sections from 20-month-old DKOs. The most intense pMLKL (Fig. 7C, left) and XBP1s (Fig. 7C, right) labeling appeared in the RPE and in the small migratory population LF+, Iba-1+, CD11b+ previously described as microglia / macrophages with abundant lipofuscin content.

[0125] Phospho-MLKL staining was particularly evident in microglia / macrophages that infiltrated the subretinal space and appeared attached to the RPE in flat mounts from 20-month-old DKOs (Figure 7J). Phospho-MLKL labeling in areas of RPE shedding large fragments of RPE cells showed more intense staining notably around the lesion than in the lesion itself, suggesting that the staining was primarily derived from microglia / macrophages encapsulating the sloughed RPE cells (Figure 7K). XBP1s was also positive around the main body, inner segment, and most of the outer segment of the PR. To further confirm the activation status of microglia, we analyzed the phenotype of Iba-1+ cells that infiltrated into the subretinal space. RPE flat mounts from 25-27 month old DKOs were prepared and double stained with Iba-1 / XBP1s or Iba-1 / pMLKL (Figure 7D). Iba-1+ cells displayed an activated morphology and stained positive for ER stress and necroptosis markers.

[0126] Very striking was the finding of extensive phospho-pMLKL staining around areas of the neural retina where RPE cells were migrating (Figures 7E-7F), indicating the induction of a halo of necroptosis in the surrounding area. This could represent adjacent photoreceptor cells undergoing necroptosis or the dispersion of neurodestructive activated macrophages / microglia piggy-bagged to the migrating RPE. In either case, as shown in Figures 2C and 2H, intense phospho-MLKL staining appears to precede and correlate well with exacerbated photoreceptor loss in areas of the ONL bordering the migrating RPE.

[0127] To establish the therapeutic potential to inhibit the atypical necroptosis pathway, 2 μl of necrostatin or vehicle was intraocularly injected into the eyes of 26-month-old DKO mice. One week later, their retinal status was assessed by staining RPE and neural retinal flat mounts with anti-phospho-MLKL antibody. We confirmed that Nec7, but not Nec1, reduced membrane and cytoplasmic phospho-MLKL staining to undetectable levels (Figure 7L), supporting the idea that the atypical necroptosis pathway detected in our in vitro system is active in lipofuscin-loaded retinas. Figure 7I shows the phospho-MLKL mean fluorescence expression values ​​of four vehicle-treated and four Nec7-treated eyes measured at every 0.1 mm interval from the ONH in flat-mounted RPE of the lower half of the retina from a 24-month-old DKO. Staining became negative from the center to the periphery in the Nec7-treated eyes, but remained strongly positive in the other mock-treated eyes. To analyze the effects of treatment on photoreceptors, we compared phospho-MLKL expression in neural retinal flat mounts from control and Nec7-treated eyes. Necroptosis labeling was significantly reduced by Nec7 (Figure 7M). Similarly, Nec7 reduced infiltration of CD11b cells in the subretinal space, as shown by the reduction in macrophages / microglia on RPE flat mounts from 25-27 month old DKO mice (Figure 7N). In summary, the results show that disruption of necroptosis with Nec7 significantly reduces signs of retinal degeneration. Based on these observations, we generated a model that summarizes these data and explains the mechanism underlying light-independent lipofuscin cytotoxicity (Figure 9B). According to this working model, lipofuscin accumulation triggers LMP, which induces the assembly of atypical necrosomes, which then mediate MLKL phosphorylation / polymerization. Phospho-MLKL oligomeric pores progressively insert into the lysosomal and plasma membranes, triggering more LMP, which then promotes more phospho-MLKL deposition on the plasma membrane, creating a dangerous loop until the number of phospho-MLKL pores per cell undergoes necroptotic collapse.

[0128] These results indicate that dabrafenib, necrosulfonamide (NSA), necrostatin 7 (Nec7), arimoclomol, and IRE1α inhibitors that block IRE1α dimerization are useful in methods for preventing or treating ocular diseases associated with retinal cell lipofuscin-related cytotoxicity in subjects in need thereof.

[0129] Example 7 Efficacy of IRE1α inhibition in LF-bearing retinas To establish the therapeutic potential to inhibit IRE1α-driven cell death in pigmented retina with LF accumulation, IRE1α inhibitor, KIRA6, or vehicle were intraocularly injected into eyes of 26-month-old DKO mice. One week later, their retinal status was assessed by staining whole RPE flat mounts with specific antibodies. Compared to mock-treated eyes, XBP1s staining was reduced from the center to the periphery by KIRA6 treatment. These results indicate that KIRA6 is effective in blocking IRE1α signaling in vivo and that XBP1s detection is specific (Figure 8A).

[0130] Furthermore, KIRA6 abrogated necroptosis, as indicated by the loss of intracellular and plasma membrane labeling with pMLKL antibodies from the center to the periphery (Figure 8B). Comparative analysis of pMLKL vs. XBP1s dual color dot-plots obtained by confocal microscopy of demelanized paraffin cross sections from 20-month-old DKOs shows a reduction in double-positive cells, i.e., RPE and microglial cells, from 33% to 4% by IRE1α inhibitors (Figure 8C). To determine whether the reduction in double-positive microglia was caused by a reduction in the infiltration of Iba1+ cells or by downregulation of the expression of XBP1s / pMLKL markers, RPE flat mounts from KIRA6-treated and mock-treated eyes were stained with Iba-1 and XBP1s antibodies. In sequential images taken from the center to the periphery of the retina, no Iba-1+ cells were detected attached to the apical portion of the RPE, indicating that KIRA6 treatment acted to reduce infiltration of activated microglia and consequently retinal inflammation (Figure 8D). To confirm the reduction of retinal degeneration (EDN2, FGF2), inflammation / angiogenesis (GFAP, SERP, VEGF, CXCL15), ER stress (XBP1s, SCAND1, CEBPA) and necroptosis (HMGA), total RNA from whole retina (neural retina + RPE) of KIRA6 and control injected eyes was isolated and mRNA levels were quantified by qPCR. Results showed a generalized reduction in all tested markers of retinal degeneration, inflammation / angiogenesis, ER stress and necroptosis (Figure 8E).

[0131] Taken together, these results indicate that ER stress and necroptosis were exacerbated in retinas with LF. As in RPE cultures, treatment with Nec7 reduced pMLKL staining, indicating that the labeling was specific and that necroptosis in the retina was inhibited by Nec7. Histology of retinas with high LF content revealed that IRE1α and pMLKL membrane deposits were increasingly present in the RPE and infiltrating microglia, and that both IRE1α and pMLKL tended to colocalize in the same cells undergoing LF-driven degeneration. The type of necroptosis seen in the retina was amenable to inhibition by Nec7, suggesting that it represents the same type of atypical necroptosis observed in cultured cells. Treatment with KIRA6 normalized the levels of IRE1a activation, pMLKL oligomerization, and Iba1+ microglial infiltration as well as multiple markers of ongoing retinal degeneration detected by qPCR (Figure 8E). Staining of retinal cross sections revealed that RPE as well as microglial (CD11b+, Iba-1+) LF+ cells were positive for XBP1s and phospho-MLKL Ser345. Treatment with KIRA6 eliminated XBP1s and phospho-MLKL Ser345 labeling from all cell types.

[0132] Figures 15A-15E and Figures 16A-16B show comparative proteomic analysis between ARPE-19 cells with or without lipofuscin, revealing proteins regulated in RPE cells to survive lipofuscin accumulation. As shown in Figure 17, the above anti-necroptosis pathways, eIF2a, eIF4, mTOR, and UPS, identified by proteomic methods in cultured cells, are involved in ABCA4. - / - RDH8 - / - In the RPE of eyes of double knockout (DKO) mice, which appears to be increased with lipofuscin, the protective effect of inducers of the eIF2a, eIF4 or mTOR pathway against a lethal dose of lipofuscin was evaluated.

[0133] Only Salvulinal (SAL) and SAL003, which target both cellular eIF2α phosphatases, composed of PP1 bound to either catalytic subunit GADD34 or CreP, protected against lipofuscin. In contrast, guanabenz, which disrupts PP1-GADD34 association or only eIF4 and mTOR activators, did not confer significant protection (Figures 18A-18B). Thus, not all inhibitors of eIF2a, eIF4 or mTOR pathways can confer protection from lipofuscin cytotoxicity. Figures 18C-18D show that SAL does not protect against phototoxic degradation of lipid bisretinoids, but can protect cells even when their cytosol contains a large amount of lipofuscin. Figures 19A-19B show that SAL requires PERK, but not ATF4, to exert protection against lipofuscin. SAL inhibits IRE1a signaling and therefore prevents lipofuscin-induced RPE necroptosis. Knockdown of IRE1a (a triple sensor of ER stress), but not PERK or ATF6, prevents lipofuscin-induced necroptosis. See Figures 20A-20B.

[0134] These results indicate that dabrafenib, necrosulfonamide (NSA), necrostatin 7 (Nec7), salubrinal, SAL003, arimoclomol, and IRE1α inhibitors that block IRE1α dimerization are useful in methods for preventing or treating ocular diseases associated with retinal cell lipofuscin-related cytotoxicity in subjects in need thereof.

[0135] equivalent The present technology is not limited to the specific embodiments described in this application, but is intended as a single description of each aspect of the technology. As will be apparent to those skilled in the art, many modifications and variations of the present technology can be made without departing from its spirit and scope. In addition to those listed herein, functionally equivalent methods and devices within the scope of the present technology will be apparent to those skilled in the art from the preceding description. Such modifications and variations are intended to fall within the scope of the present technology. It should be understood that the present technology is not limited to specific methods, reagents, compounds, compositions, or biological systems, which may of course vary. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only, and is not intended to be limiting.

[0136] Moreover, when features or aspects of the disclosure are described in terms of the Markush group, those skilled in the art will recognize that the disclosure is also described in terms of any individual members or subgroups of members of the Markush group.

[0137] As will be understood by those of skill in the art, for any and all purposes, particularly with respect to providing a written description, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof. It can be readily recognized that any range listed is fully descriptive and that the same range can be broken down into at least two, three, four, five, ten, etc. As a non-limiting example, each range described herein can be readily broken down into a lower third, a middle third, and an upper third, etc. As will also be understood by those of skill in the art, all language such as "up to," "at least," "greater than," "less than," etc., includes the recited number and then refers to a range that can be broken down into subranges as described above. Finally, as will be understood by those of skill in the art, a range includes each individual member. Thus, for example, a group having 1-3 cells refers to a group having 1, 2, or 3 cells. Similarly, a group having 1-5 cells refers to a group having 1, 2, 3, 4, or 5 cells, etc.

[0138] All patents, patent applications, provisional applications, and publications mentioned or cited in this specification are incorporated by reference in their entirety, including all figures and tables, to the extent not inconsistent with the explicit teachings of this specification.

Claims

1. 1. A composition for preventing or treating an ocular disease associated with retinal cell lipofuscin-associated cytotoxicity in a subject in need thereof, comprising an effective amount of at least one therapeutic agent selected from the group consisting of dabrafenib, necrosulfonamide (NSA), arimoclomol, kinase-inhibiting RNase attenuator (KIRA) compounds, salubrinal, SAL003, and any pharma- ceutical acceptable salt thereof, wherein the ocular disease associated with retinal cell lipofuscin-associated cytotoxicity is autosomal recessive retinitis pigmentosa (RP), Stargardt's disease (STGD), Best's disease (BD), cone-rod dystrophy, or ABCA4 mutation age-related macular degeneration (AMD).

2. The composition of claim 1 , wherein the KIRA compound is KIRA3, KIRA6, KIRA7, or KIRA8.

3. The composition of claim 1 or 2, wherein the subject comprises a mutation in ABCA4 and / or RDH12.

4. The composition of claim 3 , wherein the mutation in ABCA4 and / or RDH12 is homozygous or heterozygous.

5. The composition of any one of claims 1 to 4, wherein the effective amount of at least one therapeutic agent is effective to prevent the progression of lipofuscin-associated cytotoxicity in retinal cells in a subject.

6. 1. A composition for preventing or treating an ABCA4 mutant ocular disease associated with retinal cell lipofuscin-associated cytotoxicity in a subject in need thereof, comprising an effective amount of necrostatin 7 (Nec7) or a pharma- ceutically acceptable salt thereof, wherein the ABCA4 mutant ocular disease associated with retinal cell lipofuscin-associated cytotoxicity is autosomal recessive retinitis pigmentosa (RP), cone-rod dystrophy, or age-related macular degeneration (AMD).

7. 7. The composition of claim 6, wherein an effective amount of Nec7, or a pharma- ceutically acceptable salt thereof, is effective in preventing the exacerbation of lipofuscin-associated cytotoxicity in retinal cells in a subject.

8. The composition of any one of claims 1 to 7, wherein the eye disease is genetic, non-genetic, or age-related.

9. The composition according to any one of claims 1 to 8, wherein the AMD is dry AMD.

10. 1. The method of claim 1, wherein the cone-rod dystrophy is an autosomal recessive cone-rod dystrophy.

9. The composition according to any one of claims 1 to 8.

11. The subject is ABCA4 D2177N, ABCA4 G1961E, ABCA4 G863A, ABCA4 1847delA, ABCA4 L541P, ABCA4 T2028I, ABCA4 N247I, ABCA4 E1122K, ABCA4 W499 * 11. The composition of any one of claims 1 to 10, having at least one ABCA4 mutation selected from the group consisting of ABCA4 A1773V, ABCA4 H55R, ABCA4 A1038V, ABCA4 IVS30+1G→T, ABCA4 IVS40+5G→A, ABCA4 IVS14+1G→C, and ABCA4 F1440del1cT.

12. The subject is RDH12 G127 * , RDH12 Q189 * , RDH12 Y226C, RDH12 A269Gfs * , RDH12 L274P, RDH12 R65 * 12. The composition of claim 1, having at least one RDH12 mutation selected from the group consisting of RDH12 H151D, RDH12 T155I, RDH12 V41L, RDH12 R314W and RDH12 V146D.

13. 13. The composition of any one of claims 1 to 12, wherein dabrafenib, NSA, arimoclomol, KIRA compound, salubrinal, SAL003, Nec7, or a pharma- ceutically acceptable salt thereof is formulated for topical, intravitreal, intraocular, subretinal, or subscleral administration.

14. The composition of any one of claims 1 to 13, wherein dabrafenib, NSA, arimoclomol, KIRA compound, salubrinal, SAL003, Nec7, or a pharma- ceutically acceptable salt thereof is effective to reduce or eliminate lipofuscin bisretinoid (LB) lipid-induced phosphorylation and / or polymerization of MLKL.

15. The composition of any one of claims 1 to 14, wherein dabrafenib, NSA, arimoclomol, KIRA compound, salubrinal, SAL003, Nec7, or a pharma- ceutically acceptable salt thereof is effective to reverse LB lipid-induced translocation of phosphorylated MLKL (pMLKL) to the plasma membrane of retinal pigment epithelial cells.

16. The composition of claim 14 or 15, wherein the LB lipid is selected from the group consisting of N-retinylidene-N-retinylethanolamine (A2E), A2E isomers, oxidized derivatives of A2E, and all-trans retinal dimer (ATRD).

17. 17. The composition of any one of claims 1 to 16, wherein dabrafenib, NSA, arimoclomol, KIRA compound, salubrinal, SAL003, Nec7, or a pharma- ceutically acceptable salt thereof is effective for reducing the mRNA or protein levels of one or more genes associated with retinal degeneration, inflammation / angiogenesis, ER stress and / or necroptosis.

18. 18. The composition of claim 17, wherein the one or more genes associated with retinal degeneration, inflammation / angiogenesis, ER stress and / or necroptosis are selected from the group consisting of EDN2, FGF2, GFAP, SERP, VEGF, CXCL15, XBP1s, SCAND1, CEBPA and HMGA.

19. The composition of any one of claims 1 to 18, wherein dabrafenib, NSA, arimoclomol, KIRA compound, salubrinal, SAL003, Nec7, or a pharma- ceutically acceptable salt thereof is effective for inhibiting or reducing lipofuscin-induced necroptosis.

20. The composition of any one of claims 1 to 19, wherein dabrafenib, NSA, arimoclomol, KIRA compound, salubrinal, SAL003, Nec7, or a pharma- ceutically acceptable salt thereof is effective for reducing infiltration of activated microglia / macrophages in retinal pigment epithelial cells.

21. 21. The composition of any one of claims 1 to 20, wherein dabrafenib, NSA, arimoclomol, KIRA compound, salubrinal, SAL003, Nec7, or a pharma- ceutically acceptable salt thereof is conjugated to an agent that targets retinal pigment epithelial cells.

22. Drugs that target retinal pigment epithelial cells include tamoxifen, chloroquine (CQ) / hydroxychloroquine (HCQ), ethambutol (EMB), or sodium iodate (NaIO 3 22. The composition of claim 21 .

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