Cone photoreceptor degeneration model

Abstract

The present invention provides a method for creating a cone degeneration model, comprising the step of activating specific cell death in at least one cone photoreceptor. The invention also provides a method for isolating compounds that can protect cone photoreceptors from degeneration.

Description

[Background technology]

[0001] Introduction The retina is photosensitive nerve tissue located at the back of the eyeball. The human retina has two distinct regions. The peripheral retina has low spatial resolution and is responsible for various aspects of night vision and motion vision. The fovea (or macula) is located in the center of the retina and is responsible for high spatial resolution vision essential for reading and face recognition. Primates are the only mammals that possess a fovea. The retina is composed of three layers of cells: the outer nucleus layer (ONL), the inner nucleus layer (INL), and the retinal ganglion cell (RGC) layer. The ONL holds photoreceptors, which are specialized nerve cells that are dedicated to converting light into electrochemical signals and are therefore essential for vision. Two types of photoreceptors are distinguished: rod photoreceptors respond to weak light and enable night vision, while cone photoreceptors respond to sunlight and mediate high resolution and color vision.

[0002] Retinal neurodegeneration associated with photoreceptor dysfunction or death is a leading cause of incurable vision loss. Photoreceptor death is a common cause in many retinal diseases, including age-related macular degeneration (AMD) and retinitis pigmentosa (RP). In most of these diseases (e.g., AMD), the pathology appears to be primarily related to cone loss. Therefore, preventing the degeneration of cone photoreceptors is of particular importance.

[0003] To date, there are no effective therapies for retinal degenerative diseases, and therefore the chances of treatment remain low, making the development of new treatment strategies an urgent necessity.

[0004] In particular, diseases affecting photoreceptors, especially cone photoreceptors, are essentially untreatable. None of the available pharmacological treatments aim to prevent the degeneration of photoreceptors, especially cone photoreceptors. The only available therapies target the later stages of the disease, after the degeneration of cone photoreceptors has occurred and a large or all of the retinal cells have been lost. [Overview of the Initiative] [Problems that the invention aims to solve]

[0005] Preventing the loss of cone cells appears to be a primary goal in therapeutic strategies. This situation creates a strong need for models that can accurately reproduce photoreceptor degeneration, particularly cone photoreceptor degeneration, and enable the testing of novel therapies for retinal neuroprotection.

[0006] However, it appears that a satisfactory model has not yet been obtained. In particular, the lack of in vitro or in vivo models specific to cone degeneration hinders the isolation and testing of potential drugs for treating retinal diseases.

[0007] Therefore, there is a need for in vitro and / or in vivo models of cone photoreceptor degeneration. [Means for solving the problem]

[0008] Summary of the Invention In a first aspect, this disclosure relates to a method for constructing a model of cone degeneration. The method disclosed herein comprises the step of activating specific cell death, in particular ferroptosis, in at least one cone photoreceptor. In particular, the activation of cell death, in particular ferroptosis, is specific to cone photoreceptors; that is, cell death is activated in cone photoreceptors, but not in other retinal cell types such as rod photoreceptors and retinal pigment epithelial cells with the same concentration of cell death (in particular ferroptosis) activator.

[0009] In the methods disclosed herein, in some cases, activation of cell death, e.g., ferroptosis, results in inhibition of the Xc- system, inhibition of GPX-4, activation of lipid peroxidation, and / or an increase in intracellular iron levels at at least one cone photoreceptor. In particular, the inhibition of the Xc- system and / or GPX-4 is preferably specific, i.e., the Xc- system and / or GPX-4 are inhibited at cone photoreceptors but are substantially unaffected at the same concentration of cell death (especially ferroptosis) activator at other retinal cell types such as rod photoreceptors and retinal pigment epithelial cells. Similarly, lipid peroxidation is also preferably specifically activated and / or increased at cone photoreceptors. Finally, intracellular iron levels are preferably specifically increased at cone photoreceptors.

[0010] In one example, cell death in cone photoreceptors, such as the activation of ferroptosis, particularly the specific activation of cell death (i.e., cell death is substantially unactivated in other retinal cell types, such as rod photoreceptors and retinal pigment epithelial cells, especially with the same concentration of cell death activator), • Reduction in the viability of at least one cone photoreceptor, in particular, a specific reduction in the viability of at least one cone photoreceptor (i.e., the viability of other retinal cell types, such as rod photoreceptors and retinal pigment epithelial cells, is not substantially reduced, especially with the same concentration of the cell death activator); • Increased cell membrane permeability to the dye in at least one cone photoreceptor, in particular a specific increase in cell membrane permeability to the dye in at least one cone photoreceptor (i.e., cell membrane permeability to the dye is substantially not increased in other retinal cell types such as rod photoreceptors); • Reduction of intracellular glutathione levels in at least one cone photoreceptor, in particular, specific reduction of intracellular glutathione levels in at least one cone photoreceptor; • Increased intracellular NADP / NADPH ratio in at least one cone photoreceptor, in particular, a specific increase in the intracellular NADP / NADPH ratio in at least one cone photoreceptor; • Increased intracellular formation of lipid peroxides, particularly 5-oxo-6,8,11,14-eicosatetraenoic acid (5oxoETE), in at least one cone photoreceptor; in particular, a specific increase in intracellular formation of lipid peroxides, particularly 5-oxo-6,8,11,14-eicosatetraenoic acid (5oxoETE), in at least one cone photoreceptor; • Increased gene expression of AIFM2 (apoptosis-inducing factor 2), SLC3A2 (solute transporter family 3 member 2), HSBP1 (heat shock protein family B (Small) member 1), and Keap1 (Kelch-like ECH-related protein 1), which are particularly specific to cone photoreceptors: • Increased protein expression of ferritin and / or nuclear receptor coactivator 4 (NCOA4), which are particularly specific to cone photoreceptors. It results in one or more of the following.

[0011] In some cases, activation of cell death, such as ferroptosis, comprises contacting at least one cone photoreceptor with at least one cell death inducer. Preferably, the cell death inducer is a ferroptosis inducer. More preferably, the at least one cell death inducer, such as a ferroptosis inducer, is glutamic acid, RSL3, elastin, elastin derivatives (preferably MEII, PE, or AE), imidazole-ketone-elastin (IKE), sulfasalazine, sorafenib, sorafenib analogs (SRS13-45 and SRS13-60), artretamine, artesunate, artemisinin, dihydroatemisinin, artemether, artemisinin derivatives, ML-162 (DPI7), M The compound is selected from the group consisting of L-210 (DPI10), RSL5, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, temozolomide, MMRi62, tert-butyl hydroperoxide, FIN56, butionine sulfoximine, NDP4928, statin, Brequinal, withaferin A (WA), auranofin, almitrin, lamperizone, HG106, DPI2, DPI12, DPI13, DPI17, DPI18, DPI19, 31MEW44, and ML160.

[0012] In some examples, the methods disclosed herein include a pre-step for obtaining retinal tissue. In particular, retinal tissue can be obtained from an eye or by production from stem cells. In preferred examples, the retinal tissue comprises photoreceptors, particularly cone photoreceptors, rod photoreceptors, and / or their progenitor cells. In even more preferred examples, the retinal tissue is vertebrate-derived retinal tissue, including both human and non-human animal tissue, preferably non-human mammalian, more preferably non-human primate tissue.

[0013] In certain examples, the methods disclosed herein include a further prior step of enzymatically and / or mechanically and / or chemically dissociating retinal tissue to obtain a population of isolated retinal cells.

[0014] In certain instances, the methods disclosed herein include the further step of incubating a population of isolated retinal cells with peanut agglutinin (PNA) and recovering PNA-binding cone photoreceptors.

[0015] In another example, the retinal tissue is a retinal explant or a retinal organoid maintained in culture. For example, the retinal explant or retinal organoid is cultured in a CO2-independent medium.

[0016] In a preferred example, the methods disclosed herein include the further step of contacting the retinal tissue with a polycarbonate membrane. Preferably, the retinal tissue is contacted such that the photoreceptors face away from the membrane. According to a more preferred example, contacting at least one cone photoreceptor with at least one cell death inducer (e.g., a ferroptosis inducer) results in cone degeneration, particularly, specific cone degeneration, or microglial migration into the outer granular layer of the retinal tissue. Even more preferably, contacting at least one cone photoreceptor with at least one cell death inducer (e.g., a ferroptosis inducer) results in specific cone degeneration, i.e., degeneration of the cone photoreceptors is induced or increased, but other retinal cell types such as rod photoreceptors or retinal pigment epithelial cells are not substantially affected by the same concentration of the cell death inducer (e.g., a ferroptosis inducer).

[0017] In another example, the retinal tissue is contained within a whole eye.

[0018] In a preferred example, contacting at least one cone photoreceptor with at least one cell death inducer (e.g., a ferroptosis inducer) comprises administering at least one cell death inducer (e.g., a ferroptosis inducer) to the subretinal space, suprachoroidal space, anterior chamber, vitreous, subconjunctival space, or corneal surface. In another preferred example, administration of at least one cell death inducer (e.g., a ferroptosis inducer) can be by systemic administration (inhaler, intravenous, intramuscular or intraperitoneal) or by means of a solution, gel or implant.

[0019] According to another preferred example, contacting at least one cone photoreceptor with at least one cell death inducer (e.g., a ferroptosis inducer) results in the formation of subretinal deposits, preferably in the fovea and / or macula.

[0020] In another preferred example, contacting at least one cone photoreceptor with at least one cell death inducer (e.g., a ferroptosis inducer) results in a decrease in cone photoreceptor activity, particularly a specific decrease. More preferably, contacting at least one cone photoreceptor with at least one cell death inducer (e.g., a ferroptosis inducer) results in a decrease in cone photoreceptor activity, particularly a specific decrease, measured by ERG or mfERG.

[0021] In another aspect, the present disclosure relates to a cone degeneration model obtainable by the methods described herein.

[0022] In yet another aspect, the present disclosure provides a method for screening a compound that may exhibit protective and / or anti-degenerative properties against cone photoreceptors, · providing the above cone degeneration model, · contacting the cone degeneration model with a candidate compound, · evaluating whether the candidate compound induces a protective and / or anti-degenerative effect, particularly a specific protective and / or anti-degenerative effect, on cone photoreceptors and comprising the steps of.

[0023] Preferably, the candidate compound, with respect to cone photoreceptors, · maintains or increases the survival rate of at least one cone photoreceptor, particularly specifically maintains or increases it; · prevents an increase in cell membrane permeability to pigments in at least one cone photoreceptor, particularly specifically prevents it; · maintains or increases the intracellular glutathione level in at least one cone photoreceptor, particularly specifically maintains or increases it; • Maintaining or decreasing the intracellular NADP / NADPH ratio in at least one cone photoreceptor, in particular, specifically maintaining or decreasing it; • To maintain or reduce intracellular formation of lipid peroxides, particularly 5-oxo-6,8,11,14-eicosatetraenoic acid (5oxoETE), in at least one cone photoreceptor; in particular, to specifically maintain or reduce them; • To specifically maintain or increase the gene expression of AIFM2 (apoptosis-inducing factor 2), SLC3A2 (solute transporter family 3 member 2), HSBP1 (heat shock protein family B (Small) member 1), and Keap1 (Kelch-like ECH-related protein 1), particularly in cone photoreceptors; • To specifically maintain or decrease the protein expression of ferritin and / or nuclear receptor coactivator 4 (NCOA4), particularly in cone photoreceptors; • To prevent the proliferation, activation, or migration of microglial cells; • To prevent the loss of the extrapyramidal segment; - To prevent, and especially specifically prevent, a decrease in pyramidal activity, preferably as measured by ERG or mfERG; and • Prevent thinning of retinal thickness as shown in tissue sections or in vivo imaging by OCT. A protective and / or antidenaturing effect comprising at least one of the following, particularly one that induces a specific protective and / or antidenaturing effect. [Brief explanation of the drawing]

[0024] [Figure 1]Figure 1. Evidence of ferroptosis development in pure cone photoreceptors. Mean and standard error of the mean are shown. A-B. Bright-field images of cone photoreceptors after 3 days of incubation with control (Ctrl=0.2% DMSO, A) and treatment solution (3 μM imidazole ketone elastin, IKE, B). C-D. Staining of cone photoreceptors with calcein to label viable cells under control (C) and treatment (D) conditions. E. Glutamate concentration-dependent cell viability of pure cone photoreceptors with or without 1 mM L-cystine (n=7, p<0.0001, IC50=4.15 μM). F. Glutathione levels in pure cone photoreceptors over 3 days in control medium, 3 μM IKE medium, and 500 μM glutamate medium (D1, D2, D3 n=18, p<0.0001). G. NADP / NADPH percentages in pure cone photoreceptors at 3 days under control and 3 μM IKE conditions (n=4, p=0.0159). H. Concentration-dependent plot of viability of pure cone photoreceptor cells after treatment with 0.01 nM to 25 nM RSL3 (n=3, IC50=8.67 nM). I. Western blot revealing the presence of 5-lipoxygenase (5LOX) and 5-lipoxygenase-activated protein (FLAP) bands in pure cone photoreceptors, but not in rod photoreceptors. J. Lipidomic analysis shows the amounts of 15-hydroxyeicosatetraenoic acid and 5-hydroxyeicosatetraenoic acid (15-HETE and 5-HETE, respectively) and 5-oxo-6,8,11,14-eicosatetraenoic acid (5oxoETE) detected in pure cone photoreceptors under control and 3 μM IKE conditions (n=6, p=0.0079(**), p=0.0159(*)). K. Survival rates of pure cone photoreceptors under control and 3 μM IKE conditions with the addition of various treatments (having anti-ferroptotic or iron chelating effects): L-cystine, diloton (ZEN), ferrostatin-1 (FST1), and deferipron (DF). [Figure 2]Figure 2. Changes in mRNA expression levels of proteins involved in ferroptosis in the control and treatment (3 μM IKE) groups of pure porcine cone photoreceptor (n=6). Solute transporter family 11 member 2 (SLC11A2) is involved in the release of Fe2+ from endosomes to the cytoplasm, and its decrease suggests dysregulation of iron metabolism (p=0.0260). Solute transporter family 3 member 2 (SLC3A2) corresponds to the heavy chain of the Xc- system (p=0.0022). Transferrin receptor (TFRC) expression was slightly increased under treatment conditions (p=0.0286). Ferroptosis suppressor protein 1 (AIFM2) is involved in the reduction of the ferroptosis mechanism (p=0.0022). Heat shock protein family B small member 1 (HSPB1) plays a role in iron uptake and may promote ferroptosis (p=0.0022). [Figure 3] Figure 3. Immunofluorescence localization of flap in retinal sections from three species (pig, rat, and non-human primate) compared with DAPI nuclear staining. In all species, flap localizes to cone photoreceptors. [Figure 4] Figure 4. Immunofluorescence of ferritin heavy chain (B, G) and NCOA4 (C, H) in isolated porcine cone photoreceptors from control groups (A-E) and treatment groups (F-J). IKE treatment increased fluorescence intensity compared to control cone photoreceptors (G, H). The IKE-treated groups (G, H) showed stronger immunolabeling than the control groups (B, C). E. Magnified image of merged labeling shows that control cells have stronger ferritin heavy chain immunolabeling than NCOA4. J. Magnified image of treated cells shows stronger NCOA4 immunolabeling than heavy chain immunolabeling. [Figure 5]Figure 5. Degeneration of cone photoreceptors and microglia migration in ex vivo retinal explants. A-H. Histological findings of retinal sections show increased ethidium permeability in the first retinal granular row, representing cone photoreceptors, in the treated explant (20 μM IKE, F) and control tissue (0.2% DMSO, B). Note the disorder of cone photoreceptors immunolabeled with Flap in the treated retina (G) compared to the control condition (C), and the presence of large amoeboid microglia cells, which are absent in the control retina (D), extending to the outer granular layer (ONL) in the treated retina (H). The granular layer is highlighted with DAPI in the control retina (A) and treated retina (E). I-M. Destruction of cone photoreceptors, indicated by loss of Flap immunopositive outer segments in flat-mounted retinal explant-treated groups (20 μM IKE), and their protection by the anti-ferroptotic agents FST-1 (L) and DF (M) compared to the control condition (J). Quantification of cone photoreceptor outer segments shows a significant reduction in treated explants and rescue by the anti-ferroptotic agents (I, n=3). N-O. Distribution of microglia cells in retinal explant thickness shows migration to the outer retinal layer in treated retinas (IKE, RSL3) and prevention of this effect by DF or FST-1 (n=3). Scale bar = 20 μM. ONL: Outer granular layer, INL: Inner granular layer, GCL: Ganglion cell layer, Iba1: Ionized calcium-binding adapter molecule 1, Ctrl: Control condition, IKE: Imidazole-ketone-elastin, OS: Outer segment, FST-1: Ferrostatin-1, DF: Deferipron [Figure 6] Figure 6. Maintenance of various retinal cell types in sections of treated porcine retinal explants (20 μM IKE) compared to control conditions (Ctrl). Rod photoreceptors were immunostained with rhodopsin, bipolar cells with PKCα, and Müller cells with vimentin. Retinal structures are highlighted with DAPI-stained nuclei, and it should be noted that the morphology of the labeled cells appears to have been preserved under the treated conditions without significant cell loss. [Figure 7]Figure 7. Effects of RSL3 on retinal explants. Ethidium permeability of RSL3 revealed dead cells in control retina (0.2% DMSO, B) and treated retina (200 nM RSL3, F). C, G. Morphological changes of Flap immunolabeled cone photoreceptors in treated retinal explants (G) compared to control explants (C), and compared to control condition (D), iba1 immunopositive microglia cells migrate toward the outer granular layer (ONL) in treated explants (H). I-L. Loss of Flap immunopositive cone outer segments (OS) in retinal explants treated with RSL3 (J) compared to control condition (I), and their preservation by FST-1 (K) or DF (L). M. Quantification of RSL3 toxicity to cone photoreceptor outer segments and their preservation by DF in retinal explants (n=3, p=0.0286). N. The distribution of microglia cells migrating toward the outer granular layer (ONL) in the presence of RSL3, compared to the control condition (see Figure 5N), along with the effect of DF or FST-1 (n=3). Scale bar = 20 μM. ONL: outer granular layer, INL: inner granular layer, GCL: ganglion cell layer, Iba1: ionized calcium-binding adapter molecule 1, Ctrl: control condition, IKE: imidazole-ketone-elastin, OS: outer segment, FST-1: ferrostatin-1, DF: deferipron. [Figure 8]Figure 8. Cone photoreceptor degeneration and microglia migration in rats after in vivo subretinal delivery of ferroptosis inducers. A. Schematic diagram and timeline of the experimental protocol. 20 μM IKE or 200 nM RSL3 (treatment group) or 0.2% DMSO (control group, Ctrl) was delivered into the subretinal space of rats. Retinas were collected on day 6 (D6) to evaluate microglia migration (K~O, n=4), or on day 30 (D30) to evaluate cone photoreceptor degeneration (H~J, n=4). B-G. Samples of in vivo retinal imaging from rats: Fundus photographs (B, C) and optical coherence tomography (OCT) (D-G) show induced retinal detachment near the optic nerve (ON) (D0, B, D), with a completely reattached retina observed at D6 (C, E); the injection site (arrow) and OCT section (dashed line) are shown in the fundus photographs; (F, G) OCT of the injection site at D6 shows hyperreflectivity points (arrow) scattered at the inner granular layer (INL) and outer plexiform layer (OPL) / outer granular layer (ONL) junctions, more frequent under the treated conditions (G). H, I. Retinal sections from the injection area taken at D30 (same rats as B-G) show a decrease in cone arrestin immunolabeled cone photoreceptors (CARs) in treated animals (I) compared to the control group (H), along with corresponding DAPI nuclear staining. The eccentricity relative to the optic nerve is the same in both cases. In J.D30, cone density (elements / mm) was quantified in retinal sections of treated and control rats (n=4). The outer segment (OS) and cell body of cones were quantified separately. In K.D6, microglia density was quantified in whole-mount retinas of treated and control rats. Three retinal planes were evaluated individually: the outer segment (OS) level, the ONL plane, and the peduncle (Ped) plane corresponding to the OPL / ONL junction. Each field of view corresponded to a 500 × 500 μm window, and repeated measurements were taken across the entire injection area. In LO.D6, the distribution of cone arrestin-positive cells and Iba1-positive microglia cells in whole-mount retinas of treated and control rats showed two different planes on the z-axis (indicated as H~I): OS (L, M) and Ped (N, O). Samples were collected in the detached area, but away from the injection site to reveal changes in cone OS morphology and infiltrating microglia cells in treated animals. The eccentricity relative to the optic nerve was the same in both cases.The eccentricity relative to the optic nerve is the same in both cases. Scale bar: 50 μm. INL: Inner granular layer, OPL: Outer plexiform layer, ONL: Outer granular layer, EZ: Ellipsoidal zone, RPE: Retinal pigment epithelium, DAPI: 4',6-diamidino-2-phenylindole, CAR: Cone arrestin, Iba1: Ionized calcium-binding adapter molecule 1, ctrl: Control. [Figure 9]Figure 9. Macular degeneration in non-human primates (NHPs) after subretinal delivery of 20 μM IKE. A. Schematic diagram of the experimental protocol. Subretinal delivery of 20 μM IKE to the right eye and DMSO diluted 1 / 500 with PBS to the left eye resulted in the detachment of the upper half of the fovea in both eyes. B-C. Fundus photographs at baseline (B), 1 month (C), and 3 months (D) after subretinal injection of 20 μM IKE. The boundary line at 1 month (point of the arrow in C) and the macular pigment deposition around the subretinal deposits at 3 months (highlighted in D) are shown. E-F. OCT vertical b scan images passing through the fovea of ​​NHPs 3 months after subretinal delivery of 1 / 500 DMSO (ctrl, E) and 20 μM IKE (F). Note the erosional ellipsoidal zone (EZ, black asterisk), punctate hyperrefractory areas (arrows), and subretinal deposits (points of arrows). G. OCT horizontal b-scan through the upper parafoveal region within the subretinal delivery of 20 μM IKE. Erosion is visible in the EZ within the boundary of the detachment area (dashed line) (black asterisk). Also note the clusters of numerous hyperrefractory lesions mainly located within the ONL and beneath the internal limiting membrane (arrows). The non-injection area (outside the dashed line) remains completely normal. H~J. OCT horizontal b-scan through a macular subretinal deposit (point of arrow) that appeared in M2 (I) and grew to M3 (J). K~L. Vertical adaptive optics imaging of the foveal photoreceptor layer at a 2-degree eccentric position within the detachment area 3 months after subretinal delivery. After 20 μM IKE delivery, the reflectivity of cones decreased, and clusters of missing cones appeared (K), but the cone mosaic on the control side maintained its regularity (L). M-N. Changes in ONL thickness in the macular region of NHP 3 months after subretinal delivery. The grayscale shows the change in ONL thickness (μm). O. Changes in cone density (cones / deg2) measured twice using adaptive optics inside and outside the injection area 3 months after subretinal delivery (n=2). P. Changes in N1 and P1 wave amplitudes as percentages relative to baseline, measured by multifocal electroretinography (n=2). Only hexagons within the injection area were analyzed. [Figure 10]Figure 10. The ferroptosis pathway elucidated in mammalian cone photoreceptors. Ferroptosis ultimately promotes ROS formation and leads to degeneration of cone photoreceptors. Three intracellular pathways must be considered in cone ferroptosis. First, the Xc- system (which can be inhibited by high extracellular glutamate concentrations induced by ferroptosis inducers (e.g., IKE)) stimulates L-cystine influx to produce glutathione. Inhibition of the glutathione redox cycle leads to inactivation of glutathione peroxidase 4 (GPX4) and accumulation of NADPH. Furthermore, the reduction in GPX4 enzyme activity leads to the production of lipid peroxides such as 5-oxo-ETE via the lipoxygenase pathway. Finally, the high ferritinophagy activity of NCOA4 causes dysregulation of iron metabolism, leading to the release of excessive iron. The Fenton reaction generates radical species, which, through interaction with lipid peroxides, enable the production of reactive oxygen species. GPX4: Glutathione peroxide 4, 12-LOX: 12-lipoxygenase, 15-LOX: lipoxygenase, 5-LOX: 5-lipoxygenase, FLAP: 5-lipoxygenase activating protein, 12-HPETE: 12-hydroxyperoxyeicosatetraenoic acid, 15-HPETE: 15-hydroxyperoxyeicosatetraenoic acid, 5-HPETE: 5-hydroxyperoxyeicosatetraenoic acid, 5-Oxo-ETE: 5-oxo-eicosatetraenoic acid, 12-HETE: 12-hydroxyeicosatetraenoic acid, 15-HETE: 15-hydroxyeicosatetraenoic acid, 5-HETE: 5-hydroxyeicosatetraenoic acid, STEAP 3: STEAP family member 3, DMT1: divalent metal transporter 1, NCOA4: nuclear receptor coactivator 4, Keap1: Kelch-like ECH-related protein 1, NFE2L2: nuclear factor (red blood cell-derived 2)-like 2, ROS: reactive oxygen species. [Figure 11]Figure 11. Cell viability after ferroptosis induction for various retinal cell types. Cone photoreceptors (cone PRs) are represented by light gray lines and circles, ARPE-19 by dark gray lines and triangles, and primary RPE cells by black lines and squares. A. Cells were treated with 0.20 μM to 25 μM IKE (imidazole ketone elastin). All cone PRs died at 25 μM, but 40% of ARPE-19 (n=3, p=0.0001) and 80% of primary RPE cells (n=3, p=0.0019) survived. B. Cells were treated with 0.01 μM to 10 μM RSL3 (Ras-selective ligand 3). All PR cone cells died at all concentrations, but 50% of ARPE-19 (n=3, p=0.0008) and primary RPE cells survived at 5 μM. Treatment of C cells with 0.001 mM to 1 mM glutamate had no effect on ARPE-19 and primary RPE cells. In contrast, all cone PR cells died at 1 mM compared to ARPE-19 cells (n=3, p=0.0050) and primary RPE cells. [Figure 12]Figure 12. Ferroptosis inhibitors reverse cone degeneration in rd1 mice at P15-P45 ex vivo and in vivo. A-D. PNA immunolabeled retinal explants of rd1 mice exposed to control solution (A) and several ferroptosis inhibitors (B-D) for 30 days during culture (P15-P45). E. Automated cone counts for retinal explants of rd1 mice exposed to various ferroptosis inhibitors (Ctrl, HTHQ, 1,3-dicaffeoylquinic acid, SRS16-86, astilbin, caffeic acid, tinoridine hydrochloride n=3; GW5074 n=6; novelgenin n=2; mangiferin n=5) during culture. White bars represent antioxidants or radical scavengers, gray bars represent molecules involved in the Xc pathway, checkered bars represent molecules acting in the lipoxygenase pathway, and vertical bars represent molecules involved in the iron pathway. F-I. PNA immunolabeled retinas of rd1 mice intraperitoneally injected daily with vehicle (Ctrl, F, and H) or 2 mg / kg GW 5074 solution (G and I) for 30 days (P15-P45). Asterisks indicate the positions of H and I. J. Automated cone counts on whole-mount retinas after daily intraperitoneal injection (n=5). Scale bars = 50 μM for A-D, H, and I; 300 μM for F and G. PNA: Peanut agglutinin, Ctrl: Control, HTHQ: 1-O-hexyl-2,3,5-trimethylhydroquinone, 1-3-DCFA: 1,3-dicaffeoylquinic acid, RA: Rosmarinic acid, SMNL: Sonic chromanol, CA: Caffeic acid, TinolysineHCl: Tinolysine hydrochloride, OH Puerarin: Hydroxy purrarain, 5-ACQ: Chlorogenic acid. [Figure 13]Figure 13. Ferroptosis inhibitors do not reverse rod degeneration in rd1 mice with P15-P45 in ex vivo and in vivo. A. Automated cone counts on retinal explants of rd1 mice exposed to various ferroptosis inhibitors (Ctrl, HTHQ, 1,3-dicaffeoylquinic acid, SRS16-86, astilbin, caffeic acid, tinoridine hydrochloride n=3; GW5074 n=6; novelgenin n=2; mangiferin n=5) that rescued cones during culture. B. Automated cone counts on whole-mount retinas after daily intraperitoneal injections (n=5). [Figure 14] Figure 14. Rods of non-human primate retinas injected with control solution and 20 μM IKE, immunolabeled with rhodopsin (Rho) and DAPI. [Modes for carrying out the invention]

[0025] explanation definition The terms “about” or “approximately” refer to the normal margin of error for a given value or range as known to those skilled in the art. Typically, this means within 20%, for example, 10%, or 5% (or 1%) of the given value or range. “About” a value or parameter as used herein includes (and describes) embodiments relating to the value or parameter itself.

[0026] As used herein, the terms “activation,” “stimulation,” or “induction,” and any grammatical derivative thereof, refer to a relative increase in a specific response (e.g., expression, enzyme activity) of a specified substance in the presence of a particular reagent. This reagent is referred to herein as the “activator.”

[0027] The term “animal” is used herein to include all animals. In some examples, non-human animals are vertebrates, particularly mammals. Examples of animals include humans, non-human primates, mice, rats, cattle, pigs, horses, chickens, ducks, geese, cats, and dogs. The term “animal” also includes individual animals at all stages of development, including the embryonic and fetal stages.

[0028] As used herein, “cell death” refers to any mechanism and / or pathway by which a cell ultimately undergoes cell death through a series of events. Cell death can be induced in individual cells as a result of many internal and external stimuli, including, but not limited to, genetic predisposition, toxic chemicals or processes, high or low temperatures, rapid environmental changes, radiation, viruses, prions, bacteria, nutritional imbalances, or exposure to byproducts from other cells undergoing cell death and signaling. “Cell death” encompasses both specific cell death and necrosis. “Specific cell death,” or “regulated cell death,” or “active cell death,” as used herein, refers to a series of processes characterized by a specific mechanism and / or pathway whose activation results in cell death. Specific cell death, as used herein, includes, but is not limited to, apoptosis or programmed cell death, necrotosis, parthanatos, entosis cell death, netosis cell death, parthanatos, lysosome-dependent cell death, autophagocytosis-dependent cell death, alkaliptosis, oxeiptosis, netosis, pyronecrosis, pyroptosis, and ferroptosis / oxyptosis. Many specific cell death pathways are known in the art; see, for example, Vanden Berghe et al. Nat Rev Mol Cell Biol. 15(2):135-47, 2014; Green. Cell. 177(5):1094-1107, 2019; Tang et al. Cell Res 29: 347-364, 2019; Lomphithak & Fadeel. Toxicol Sci. kfad008, 2023.

[0029] A "cell death inducer" or "cell death activator" refers to a drug that induces, promotes, or activates cell death.

[0030] As used herein, “contact” means bringing a compound and optionally one or more additional compounds, such as a therapeutic agent, into close proximity to a sample, such as cells, that require such adjustment. This can be achieved using prior art for drug delivery to a subject, or, in the case of in vitro, by supplying, for example, the compound and optionally other compounds, such as a therapeutic agent, to the culture medium in which the cells are placed.

[0031] The terms “decreased” or “reduced,” as used herein, mean that the activity of the protein in question is at least one-fold lower than its reference value (e.g., 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1000, 10,000, or more). When “decreased” or “reduced” refers to the activity of the protein in question, it means that it is at least 5% lower than the activity of the protein in the reference sample or compared to the reference value of that protein (e.g., 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%). Furthermore, as used herein, the terms “decreased” or “reduced” mean that the level of the biomarker in question is at least one-fold lower than its reference value (e.g., 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1000, 10,000 times or more). When “decreased” or “reduced” refers to the level of the biomarker in question, it means that it is at least 5% lower than the level of the reference sample or at least 5% lower than the reference value of the marker (e.g., 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%).

[0032] As used herein, “electroretinography (ERG)” refers to a diagnostic test that measures the electrical activity of the retina in response to light stimuli. For example, ERG can record aggregate potentials from the entire retina (see, e.g., whole-field ERG). Alternatively, multifocal ERG (mfERG) assesses ERG activity in a small area of ​​the retina, and patterned ERG (pERG) assesses the activity of macular retinal ganglion cells (RGCs). ERG can be an objective indicator of retinal function that can be recorded under physiological conditions. For example, ERG can be used for the purpose of providing diagnostic information, monitoring the progression of retinal diseases and disorders, or a combination of these. ERG can also be used to determine the efficacy of any of the various therapeutic compounds disclosed herein. Examples of ERG values ​​from healthy subjects and patients can be found in Lorenz et al., Invest. Ophthalmol. Vis. Sci. 49:5235-5242, 2008.

[0033] As used herein, the term "ex vivo" refers to the process by which cells removed from a living organism are cultured outside of that organism (for example, in a cell culture plate, flask, bag, or test tube).

[0034] As used herein, “ferroptosis” refers to a form of cell death understood in the art to involve the generation of reactive oxygen species mediated by iron and characterized in part by lipid peroxidation. In ferroptosis, lethality results from the peroxidation of polyunsaturated fatty acids (PUFAs), which self-amplify unless inhibited by the lipid peroxidase glutathione peroxidase 4 (GPX4).

[0035] "Ferroptosis inducer" or "ferroptosis activator" means a drug that induces, promotes, or activates ferroptosis. As used herein, the terms "ferroptosis inducer" or "ferroptosis activator" mean any compound or means that induces ferroptosis in cells, for example, cone photoreceptors, when in contact with such cells, and / or a compound that, when administered to a subject, induces or increases ferroptotic cell death, for example, of cone photoreceptors.

[0036] As used herein, "GPX4" refers to glutathione peroxidase 4, a glutathione metabolic enzyme.

[0037] The term "GPX4 inhibitor," as used herein, refers to any agent that inhibits the activity of the glutathione peroxidase 4 (GPX4) enzyme. GPX4 inhibitors may be either direct or indirect inhibitors. GPX4 is a phospholipid hydroperoxidase that catalyzes the reduction of hydrogen peroxide and organic peroxides, thereby protecting cells from lipid peroxidation of membranes or oxidative stress. Indirect inhibitors block glutathione formation or reduce its concentration. An example, not limited to, is butionine sulfoximine (BSO). Direct inhibitors of GPX4 work by preventing the binding of either glutathione or lipid hydroperoxidase, or both, to the GPX4 active site. GPX4 has selenocysteine ​​in its active site, which is oxidized by peroxides to selenenate, producing a lipid alcohol. Glutathione reduces this selenenate (-SeOH) back to selenol (-SeH). Cell death occurs via a specific cell death process known as ferroptosis, where this catalytic cycle is disrupted. Examples of direct GPX inhibitors, though not limited to them, include RSL3 and ML162.

[0038] As used herein, the term "increase" means that the activity of a protein is at least one times its reference value (e.g., 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1000, 10,000 times or more). "Increase" also means, when referring to the activity of a protein in question, that it is at least 5% higher than the activity of the protein in the reference sample or above the reference value of that protein (e.g., 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%). Furthermore, as used herein, the term “increase” means that the level of the biomarker in question is at least one times its reference value (e.g., 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1000, 10,000 times or more). “Increase” when referring to the level of the biomarker in question means that it is at least 5% higher than the level in the reference sample or above the reference value of the marker (e.g., 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%).

[0039] As used herein, the terms “inhibit” or “suppress” refer to a relative decrease in a specific response (e.g., expression, enzyme activity) of a specified substance in the presence of a particular reagent. This reagent is referred to herein as the “inhibitor.”

[0040] As used herein, the term “isolated” means at least partially separated from a natural environment, such as the human body.

[0041] As used herein, “measurement” or “determination” refers to any qualitative or quantitative determination.

[0042] As used herein, the term “lipid peroxidation” means the result of free radical damage to lipids. Lipid peroxidation forms many oxidation products, including lipid peroxides and aldehydes, such as malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE).

[0043] As used herein, the terms “lipid peroxide” or “peroxidized lipid” refer to the oxidation products of phospholipids and polyunsaturated fatty acids (PUFAs).

[0044] As used herein, “lipoxygenase” is an enzyme that oxidizes polyunsaturated fatty acids or alkenes having two oxygen atoms to eicosanoids. This enzyme catalyzes the initial reaction of the so-called lipoxygenase pathway, which is involved in trauma and stress responses. Examples of human lipoxygenases include 15-LOX, 15-LOX-2, 12-LOX, 12R-LOX, eLOX-3, and 5-LOX. In some cases, lipoxygenase activity requires the presence of an activator. For example, activation of the 5-LOX enzyme requires the helper protein 5-LOX-activating protein (FLAP). As used herein, the term “lipoxygenase” also includes their coactivators, particularly FLAP.

[0045] As used herein, "lipoxygenase inhibitor" refers to a drug that inhibits the activity of lipoxygenase. Exemplary lipoxygenase inhibitors include PD146176 and ML351.

[0046] As used herein, “optical coherence tomography (OCT)” refers to a non-invasive imaging technique that uses light waves to capture cross-sectional images of the retina. OCT can be used for identifying retinal layers, mapping and measuring thickness, providing information for treatment decisions, providing diagnostic information, monitoring disease progression, or a combination of these. In particular, OCT can be used to quantify cone degeneration. In several embodiments, subjects not diagnosed with retinal degenerative disease have a peak cone density in the fovea of ​​approximately 200,000 cells / mm³ as measured by OCT. 2 The foveal outer granular layer thickness is approximately 100 μm when measured by OCT. Preferably, in patients diagnosed with retinal degenerative disease, the peak cone density of the fovea when measured by OCT is approximately 200,000 cells / mm². 2 It is less than [value missing]. In patients diagnosed with retinal degenerative disease, the thickness of the extrafoveal granular layer measured by OCT may be less than approximately 100 μm. Further examples of OCT values ​​in healthy subjects and patients diagnosed with retinal degenerative disease can be found in Lorenz et al., 2008.

[0047] In this specification, “organoid” refers to a microscopic, self-organizing, three-dimensional tissue culture derived from stem cells. When used herein, “organoid” typically represents a selective aspect of an organ, such as replicating most of its complexity or generating only specific cell types. Organoids can range in size from less than the thickness of a human hair to 5 millimeters. Because the human body contains a variety of tissues and organs, a potentially wide variety of organoids exist. Researchers have so far created organoids resembling the brain, retina, kidney, lung, intestine, stomach, and liver, and are working on creating many more.

[0048] As used herein, "polyunsaturated fatty acid (PUFA)" refers to a fatty acid containing at least two -CH=CH- groups, such as linoleic acid, linolenic acid, and arachidonic acid (AA).

[0049] As used herein, the term “specific” means that a compound, at a given concentration, affects one cell type (i.e., cone photoreceptors) but substantially does not affect other retinal cell types, such as rod photoreceptors or retinal pigment epithelial cells, at the same given / identical concentration. In particular, when referring to in vivo use, the term “specific” means that a compound affects one cell type (i.e., cone photoreceptors) before or without substantially affecting other retinal cell types, such as rod photoreceptors or retinal pigment epithelial cells.

[0050] As used herein, the term “substantial” refers to a qualitative state that indicates a complete or near-complete range or degree of the characteristic or property of interest. A typical technician in the biological field will understand that it is rare, if any, for biological and chemical phenomena to reach completeness and / or progress to a complete state, or to achieve or avoid absolute results. For example, “substantial” means at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or about 100%. Thus, the term “substantial” is used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.

[0051] The term "Xc-system" refers to an amino acid antiporter that mediates the exchange of extracellular L-cystine and intracellular L-glutamate across the cell plasma membrane. By exchanging intracellular glutamate for extracellular cystine, the Xc-system assists intracellular glutathione (GSH) synthesis and non-vesicular glutamate release, resulting in glutathione production and oxidative protection. The Xc-system is a heterodimeric amino acid transporter consisting of a light chain subunit xCT, encoded by the SLC7A11 gene, which confers cystine transport function, and a heavy chain subunit CD98 that localizes the Xc-system to the plasma membrane. As used herein, inhibition of the Xc-system refers to the prevention of ligand-mediated glutamate efflux and / or cystine influx. As used herein, activation of the Xc-system refers to the promotion of ligand-mediated glutamate efflux and / or cystine influx. The Xc-system is a heterodimeric amino acid transporter consisting of the xCT light chain subunit, encoded by the SLC7A11 gene, which confers cystine transport function, and the SLC3A2 heavy chain subunit, which localizes the Xc-system to the plasma membrane.

[0052] Construction of a cone photoreceptor degeneration model This disclosure provides a method for creating a cone photoreceptor degeneration model.

[0053] The inventors unexpectedly discovered that the degeneration of cone photoreceptors is caused by the activation of a specific cell death pathway. In particular, they showed that ferroptosis inducers such as glutamate, elastin, imidazole-ketone-elastin (IKE), and RSL3 cause a specific decrease in the viability of cone photoreceptors. This decrease in cone photoreceptor viability is accompanied by a decrease in glutathione (GSH) levels, an increase in lipid peroxides, and an increase in iron metabolism, especially an increase in intracellular iron levels. In stark contrast, no such effect was observed in retinal pigment epithelial cells. This specific effect was evident not only in pure cone photoreceptors in culture but also in an ex vivo retinal model, and in this case as well, microglia migrated, similar to in vivo. Furthermore, the significance of ferroptosis induction in vivo was further emphasized when it was found that administering ferroptosis inducers to the eyes of non-human primates resulted not only in cone loss but also in the formation of subretinal deposits and a decrease in electroretinogram amplitude, features remarkably similar to those exhibited by AMD patients. Surprisingly, despite the presence of ferroptosis-mediated factors such as the Xc-system and GPX-4 enzyme in both cone and rod photoreceptors (Hu et al. Eur J Neurosci. 28(8):1491-502, 2008), administration of such ferroptosis inducers to the eyes did not affect the survival rate of rod photoreceptors, further highlighting the specificity of ferroptosis to cone photoreceptors.

[0054] Therefore, cone degeneration is induced by contacting cone photoreceptors with a cell death inducer (e.g., a ferroptosis inducer). This is particularly useful because it provides a simple and reproducible method for creating a cone photoreceptor degeneration model. Furthermore, this method is not limited to the creation of in vitro models, but can also be applied to the creation of ex vivo and / or in vivo models, thus enabling a wider range of applications.

[0055] In a first aspect, the present disclosure provides a method for constructing a cone photoreceptor degeneration model. The method comprises the step of activating specific cell death in at least one cone photoreceptor, more specifically, the step of specifically activating specific cell death in at least one cone photoreceptor, i.e., in rod photoreceptors and other retinal cell types such as retinal pigment epithelial cells, the same concentration of a cell death (particularly ferroptosis) activator does not substantially activate specific cell death.

[0056] Accordingly, the present disclosure provides a method for constructing a cone degeneration model, comprising the steps of activating specific cell death in at least one cone photoreceptor, more specifically, specifically activating specific cell death in at least one cone photoreceptor (i.e., specific cell death is not substantially activated in other retinal cell types such as rod photoreceptors and retinal pigment epithelial cells with the same concentration of a cell death (particularly ferroptosis) activator).

[0057] The specific cell death described herein may be any type of regulated cell death known in the art; see, for example, Vanden Berghe et al. Nat Rev Mol Cell Biol. 15(2):135-47, 2014; Green. Cell. 177(5):1094-1107, 2019; Tang et al. Cell Res 29: 347-364, 2019; Lomphithak & Fadeel. Toxicol Sci. kfad008, 2023.

[0058] Preferably, the specific cell death activated by the methods described herein is ferroptosis. Ferroptosis is known to be regulated by many signaling pathways, including, but not limited to, the glutamate / cystine antiporter Xc-system, glutathione peroxidase-4 (GPX-4), lipid metabolic pathways, and iron metabolic pathways. In fact, inhibition of the Xc-system, inhibition of GPX-4, lipid peroxidation, and regulation of iron metabolism have each been shown to be sufficient to induce ferroptosis. Indeed, we have shown that any of these mechanisms can induce cell death and lead to pyramidal degeneration.

[0059] Accordingly, activation of cell death by the methods described herein results in inhibition of the Xc- system, inhibition of GPX-4, stimulation of lipid peroxidation, and / or an increase in intracellular iron levels, in particular in cone photoreceptors, resulting in specific inhibition of the Xc- system, specific inhibition of GPX-4, specific stimulation of lipid peroxidation, and / or a specific increase in intracellular iron levels, i.e., in other retinal cell types such as rod photoreceptors and retinal pigment epithelial cells, the Xc- system and GPX-4 are not substantially inhibited by the same concentration of the cell death activator.

[0060] In certain examples of the methods described herein, the activation of cell death, such as ferroptosis, is characterized by the following: • Reduction in the viability of at least one cone photoreceptor, in particular, a specific reduction in the viability of at least one cone photoreceptor (i.e., the viability of other retinal cell types, such as rod photoreceptors and retinal pigment epithelial cells, is not substantially reduced, especially with the same concentration of the cell death activator); • Increased cell membrane permeability to the pigment in at least one cone photoreceptor, in particular a specific increase in cell membrane permeability to the pigment in at least one cone photoreceptor (i.e., cell membrane permeability to the pigment is not substantially increased in other retinal cell types such as rod photoreceptors); • Reduction of intracellular glutathione levels in at least one cone photoreceptor, in particular, specific reduction of intracellular glutathione levels in at least one cone photoreceptor; • Increased intracellular NADP / NADPH ratio in at least one cone photoreceptor, in particular, a specific increase in the intracellular NADP / NADPH ratio in at least one cone photoreceptor; • Increased intracellular formation of lipid peroxides, particularly 5-oxo-6,8,11,14-eicosatetraenoic acid (5oxoETE), in at least one cone photoreceptor; in particular, a specific increase in intracellular formation of lipid peroxides, particularly 5-oxo-6,8,11,14-eicosatetraenoic acid (5oxoETE), in at least one cone photoreceptor; • Increased gene expression of AIFM2 (apoptosis-inducing factor 2), SLC3A2 (solute transporter family 3 member 2), HSBP1 (heat shock protein family B (Small) member 1), and Keap1 (Kelch-like ECH-related protein 1), which are particularly specific to cone photoreceptors: • Increased protein expression of ferritin and / or nuclear receptor coactivator 4 (NCOA4), which are particularly specific to cone photoreceptors. It results in one or more of the following.

[0061] These features can be assayed using any technique or method known in the art.

[0062] In particular, the viability of cone photoreceptor cells can be measured by any means known to those skilled in the art. For example, the viability of these cells can be assayed in vitro using the cell membrane permeability to dyes, such as calcein AM, MTT, Alamar Blue, ethidium, and CellTiterGlo. Alternatively, cone viability can be assayed in an ex vivo retinal model by measuring the number of cones after labeling retinal tissue with reagents specific to the outer segments or cones (e.g., anti-FLAP antibody, anti-opsin antibody, peanut agglutinin lectin, anti-cone arrestin antibody). In yet another example, cone viability can be assayed in vivo by optical coherence tomography (OCT) or adaptive optics retinal imaging. For example, OCT is widely used clinically to characterize potential retinal degeneration in patients because it allows for the identification of the location and nature of changes in the retina and surrounding structures and enables objective assessment of the thickness of the retina and surrounding structures. Therefore, the prevention of cone degeneration reduction can be measured by OCT. All these techniques are well known to those skilled in the art. Cone cell viability can also be indicated in histological samples by measuring retinal thickness, measuring the number of nuclei in the ONL (e.g., after DAPI staining), or determining the number of cones after immunolabeling the retinal tissue with reagents specific to the outer segments or cones (e.g., anti-FLAP antibody, anti-opsin antibody, peanut agglutinin lectin, or anti-cone arrestin antibody). Those skilled in the art will understand that each of these methods can be adapted as needed depending on the situation to effectively measure the viability of cone photoreceptors.

[0063] Those skilled in the art will know that many commercially available assays for measuring glutathione levels are available, including those used in the examples. Similarly, many commercially available assays for measuring the NADP / NADPH ratio are available, including those used in the examples. Quantitative analysis of lipid peroxides in biological samples can be performed using several methods known to those skilled in the art, including liquid chromatography-mass spectrometry (LC-MS / MS) and ultra-high-performance liquid chromatography (UPLC)-MS / MS (Chhonker et al. Bioanalysis. 10(24):2027-2046, 2018).

[0064] When expression levels are measured at the polynucleotide level, this can be done using well-known techniques, particularly high-throughput sequencing, quantitative PCR, or nucleic acid microarray technologies (including cDNA and oligonucleotide microarrays). These techniques are currently used conventionally by those skilled in the art and therefore do not need to be described in detail here. Alternatively, tissue microarrays combined with fluorescence in situ hybridization can also be used.

[0065] When expression levels are measured at the protein level, this can be done using well-known techniques, particularly with specific antibodies, such as Western blotting, ELISA or ELISPOT, antibody microarrays, or tissue microarrays combined with immunohistochemistry.

[0066] Preferably, cell death, such as activation of ferroptosis, results in one of the above features. More preferably, cell death, such as activation of ferroptosis, results in two, three, four, five, six, or seven of those features. These features can be assayed by any technique or method known to those skilled in the art. Those skilled in the art will refer to the examples, in particular, for specific examples of such assays.

[0067] In certain specific examples, the methods disclosed herein include contacting at least one cone photoreceptor with a cell death inducer (e.g., a ferroptosis inducer) to specifically activate cell death (e.g., ferroptosis) in the cone photoreceptor. Cell death inducers as described herein may act through any pathway leading to cell death. For example, as used herein, cell death inducers include Xc-system inhibitors, GPX-4 inhibitors, lipid peroxidation inducers, and iron metabolism modulators that result in increased intracellular iron levels.

[0068] The cell death inducers of this disclosure, such as ferroptosis inducers, may act, for example, by inhibiting the Xc-system, a transmembrane cystine-glutamate antiporter that transports cystine into cells. Cystine, or cysteine ​​disulfide, is required for the biosynthesis of glutathione (GSH), a cofactor and auxiliary substrate of GPX4. Depletion of GSH leads to decreased GPX4 activity, resulting in lethal lipid peroxide accumulation and ferroptotic cell death. Glutamate, elastin and its more potent analogs imidazole-ketone elastin (IKE) and piperazine elastin (PE), as well as clinically used drugs sulfasalazine and sorafenib, belong to this type of cell death inducer.

[0069] In some other examples, cell death inducers, such as ferroptosis inducers, work by directly inhibiting GPX4. (1S,3R)-RSL3 (hereinafter, RSL3) covalently interacts with GPX4, inhibiting its enzymatic activity and leading to ferroptotic cell death.

[0070] In other examples, cell death inducers, such as ferroptosis inducers, promote lipid peroxidation. For example, ferroptosis inducer 56 (FIN56) and caspase-independent lethality inducer 56 (CIL56) deplete the GPX4 protein and mevalonate-derived coenzyme Q10 (an endogenous lipophilic antioxidant that inhibits lipid peroxidation).

[0071] In some other examples, cell death inducers, such as ferroptosis inducers, work by modulating iron metabolism, for example, by promoting the accumulation of iron, particularly iron oxide, within cells. For example, the ferroptosis inducer endoperoxide (FIN02) works by oxidizing iron, promoting lipid peroxidation, and indirectly inactivating the enzymatic function of GPX4 in cells.

[0072] Many compounds have been described as having activity as inducers or promoters of specific cell death, such as ferroptosis inducers, and such compounds known to those skilled in the art should be included in this disclosure.

[0073] Agonists or inducers of cell death, such as ferroptosis inducers, may be small molecules, for example. Such inducers are well known to those skilled in the art and are disclosed, for example, in Hassannia et al. Cancer Cell. 35(6):830-849, 2019; Du & Guo. Cell Death Discov. 8(1):501, 2022; Nie et al. Cancer Cell Int. 22(1):12, 2022; Zhang et al. Front Nutr. 9:844757, 2022; Cai et al. Front Oncol. 13:1119369, 2023; and Yu et al. Nanoscale Adv. 5(5):1271-1290, 2023.

[0074] Preferably, the cell death inducer is glutamic acid, RSL3, elastin, elastin derivatives (preferably MEII, PE, or AE), imidazole ketone elastin (IKE), sulfasalazine, sorafenib, sorafenib analogs (SRS13-45 and SRS13-60), altretamine, artesunate, artemisinin, dihydroatemisinin, artemether, artemisinin derivatives (e.g., artesunate, artemisinin, and di Hydroartemisinin (DHA), ML-162 (DPI7), ML-210 (DPI10), RSL5, 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid, Temozolomide, MMRi62, tert-Butyl Hydroperoxide, FIN56, CIL56, β-Element, Butionine Sulfoximine, NDP4928, Statins, Brequinal, Withaferin A (WA), Auranofin, Almitrin, Lamperizone, Albinoside A, CDDO-Im, BAY 11-7085, Withaferin A, NSC-207895, Bigerovin, Bufotalin, Ruscogenin, Pifislin-μ, Bardoxolone Methyl, Omeveloxolone, Erianin, Brussatol, KRA-533, Muruberofran G, RGB-286638, Xanthanthine, Dodecyl Gallate, Piperlongmin, Octyl Gallate, Phenylethyl β-Isothiocyanate (PEITC), Tancinone II A, Cryptotancinone, Ungeremin, Erianin, VAS It is a small molecule selected from the group consisting of 3947, nordihydroguaiyaretinic acid, GPX4-IN-3, HG106, DPI2, DPI12, DPI13, DPI17, DPI18, DPI19, 31MEW44, ML160, and ML162.

[0075] In other examples, the cell death inducers disclosed herein (e.g., ferroptosis inducers) are nanomaterials. Such nanomaterials capable of inducing specific cell death include, in particular, iron-based and non-ferrous nanomaterials. Preferably, nanomaterials usable in the methods of the present disclosure include, for example, fermoxyl, Cornell dots (ultra-small poly[ethylene glycol]-coated silica nanoparticles), C' dots, SPION, GA-Fe(II), UPDATED-PEG, WS2, MoS2, CPMNS, LDL-DHA nanoparticles, ChA CQD, and zinc oxide nanoparticles.

[0076] Furthermore, the cell death inducer (e.g., ferroptosis inducer) may be a compound selected from antibodies, other antigen-binding compounds, cytokines, T cell receptors, or nucleic acids. For example, cell death inducers (e.g., ferroptosis inducers) include anti-TfR1 antibodies, such as 3F3 anti-ferroptosis membrane antibody (3F3-FMA), anti-TfR1 3B8 2A1 antibody, and anti-TfR1 FI68.4 antibody. Alternatively, the cell death inducer (e.g., ferroptosis inducer) may be interferon-γ.

[0077] In some cases, the cell death inducer is a cellular molecule involved in regulating ferroptosis, such as a nucleic acid expressing a protein. For example, this nucleic acid may result in an increase in the expression of a protein that activates ferroptosis. Such proteins include, for example, p53, BAP1, PKCβII, and NCOA4. Vectors for controlling the expression of transgenes are well known in the art. Such vectors can be introduced into cone photoreceptors by any means known to those skilled in the art.

[0078] In some cases, agonists or inducers of specific cell death (e.g., ferroptosis) may be inhibitory nucleic acids that target the expression of cellular molecules involved in regulating ferroptosis. Nucleic acids such as shRNAs and antisense nucleic acids can target cellular components including cysteine-glutamate antiporters (Xc system), glutathione peroxidase 4 (GPX4), NOX, and ALOX15. Examples of shRNAs and inhibitory RNAs for such cellular targets include shRNAs such as miR-672-3p and miR-129-5p, which are described in more detail in US2015 / 0079035, US2015 / 0175558, US2010 / 0081654, WO2015 / 051149, WO2015 / 084749, WO2013 / 152039, and WO2015 / 109009. Other cell targets and corresponding inhibitory nucleic acids are also described in Gao et al. Cell Res. 26:1021-1032, 2016.

[0079] It is understood that at least one cone photoreceptor used in the methods disclosed herein can be provided in a variety of ways. In particular, the cone photoreceptor used in these methods may be part of retinal tissue.

[0080] In this example, the present disclosure includes a prior step of providing retinal tissue.

[0081] Retinal tissue may be complete, i.e., containing all retinal cell types, or partial, i.e., containing only some of the retinal cell types. In some other examples, cone photoreceptors are isolated cone photoreceptors (i.e., in vitro models). Such isolated cone photoreceptors are available as a substantially purified population of cones obtained from retinal tissue. Retinal tissue may be organoids or retinal explants (i.e., ex vivo models). Alternatively, retinal tissue may be contained in a complete eye, preferably a complete eye of a living subject (i.e., an in vivo model), more preferably a complete eye of a living non-human subject, such as a living non-human primate. Each of these models has its own advantages and applications, as will be further detailed below.

[0082] Advantageously, the retinal tissue may be adult tissue. Preferably, the retinal tissue can be obtained from any vertebrate. Preferably, the retinal tissue is mammalian retinal tissue (primate or non-primate retinal tissue), e.g., human retinal tissue, or retinal tissue from a non-human animal, e.g., pig, rat, or mouse retinal tissue. In some examples, the retinal tissue is of human origin. In some other examples, the retinal tissue is retinal tissue from a non-human primate.

[0083] In vitro model of cone photoreceptor degeneration In certain respects, the cone degeneration model obtained by the method of this disclosure is an in vitro model. In vitro models of cone degeneration are particularly useful because they allow for easy screening of therapeutic compounds, such as those that maintain or restore the viability of cone photoreceptors after specific cell death (e.g., ferroptosis) has been induced in cells, and especially compounds that specifically maintain or restore this viability.

[0084] Advantageously, a method for creating an in vitro model of cone degeneration involves the isolation of a substantially pure population of adult cone photoreceptor cells.

[0085] Accordingly, the method disclosed herein comprises a prior step of providing retinal tissue. Advantageously, the retinal tissue comprises photoreceptors. In particular, the retinal tissue comprises cone photoreceptors. The retinal tissue may also comprise rod photoreceptors. The retinal tissue may also comprise cone progenitor cells and / or rod photoreceptors. Furthermore, the retinal tissue may further comprise other cell types, such as bipolar cells, ganglion cells, horizontal cells, and amacrine cells, and / or adjacent supporting tissue, such as retinal pigment epithelium.

[0086] Retinal tissue can be obtained, in particular, from stem cells. Methods for producing cone photoreceptors and / or rod photoreceptors from stem cells have been described and can be readily used by those skilled in the art to obtain retinal tissue (WO2018 / 154295;WO2020 / 178222;Khalili et al. Stem Cell Res. 33:215-227, 2018).

[0087] Preferably, retinal tissue is used to obtain a substantially pure population of adult cone photoreceptor cells. The process for isolating a substantially pure population of adult cone photoreceptor cells has been previously described; see, e.g., WO2005 / 103232A2; Balse et al. Invest Ophthalmol Vis Sci. 46(1):367-74, 2005.

[0088] In the first step, the retinal tissue is dissociated from the extracellular matrix. Dissociation of the extracellular matrix of retinal tissue or retinal tissue fragments can be achieved by any means available to those skilled in the art, insofar as it is possible to dissociate the retinal cells without causing lysis of cone cells. Preferably, the dissociation is carried out enzymatically (e.g., using papain and / or trypsin) and / or mechanically and / or chemically. This step yields a population of isolated retinal cells.

[0089] After dissociation, cone photoreceptors can be identified by their morphology, specific markers, or characteristic properties, such as the specific binding of peanut germ agglutinin (PNA) to cone photoreceptors or specific binding by specific antibodies.

[0090] Advantageously, the method comprises a further step of isolating cone photoreceptors from a population of isolated retinal cells to obtain a substantially pure population of cone photoreceptors. Preferably, the isolation step comprises incubating the isolated population of retinal cells with an antibody or lectin, preferably PNA, and recovering the cone photoreceptors bound to the PNA.

[0091] Advantageously, PNA is conjugated to a solid support, for example, via an anti-PNA antibody. Anti-PNA antibodies are commercially available. Any solid support suitable for cell conjugation can be used, such as cell culture plates, coverslips, or magnetic beads conjugated with an anti-PNA antibody.

[0092] Cone photoreceptors can be isolated using PNA from retinal tissue dissociated by any means suitable for lectin-cell binding, such as panning, MACS (magnetically activated cell sorting), or FACS (fluorescence-activated cell sorting). Preferably, cone photoreceptors are isolated from the dissociated retinal tissue by panning.

[0093] In another example, the method of the present disclosure comprises a further step of growing substantially pure cone photoreceptors by cell-conditioning. Müller glial cells synthesize and release extracellular nutrients that promote the survival and development of cone cells. Therefore, preferably, the method advantageously comprises a further step of growing substantially pure cone photoreceptors in vitro in a cell-conditioned culture medium obtained by recovering the culture medium after in vitro culture of Müller glial cells.

[0094] Ex vivo model of cone photoreceptor degeneration In vitro models are suitable for isolating antidenaturing compounds and testing their effects on isolated cells. However, in vitro models cannot examine the effects of these compounds on an entire tissue, which consists of complex interactions between different cell types located in specific structural and tissue units. Information obtained from a substantially pure population of a specific cell type, e.g., a substantially pure population of cone photoreceptors, is limited to that specific cell type, e.g., cone photoreceptors. In particular, under these conditions, it is difficult to evaluate the effects of compounds that prevent cone degeneration on other cell types, such as rod photoreceptors or RPE cells. On the other hand, in the ex vivo model used in the example, we were able to show that microglia migrate toward the outer granular layer (ONL) after induction of specific cell death at cone photoreceptors. Microglial migration is well known to those skilled in the art and can be determined by any means known in the art (Rashid et al. Front Immunol. 10:1975, 2019).

[0095] In certain respects, the cone degeneration model obtained by the method of this disclosure is an ex vivo model. In this respect, the retinal tissue is, for example, a cultured organoid or retinal explant.

[0096] In the first example, retinal tissue is an organoid. As those skilled in the art know, organoids are three-dimensional in vitro culture models originating from self-organizing stem cells. Organoids can mimic the structural and functional specificity of body organs in vivo. With respect to the retina, stem cells, particularly induced pluripotent stem cells (iPS cells), can differentiate into RPE cells and neuroretinal cells, including rods and cones. The process of collecting somatic cells, reprogramming them into virus-free human iPSCs (hiPSCs), and then into retinal organoids has been standardized in various laboratories (WO2020 / 178222; Sharma et al. Int J Mol Sci. 21(22):8484, 2020; Cobb et al., Transl Vis Sci Technol. 10(10):9, 2021).

[0097] Alternatively, the retinal tissue is a retinal explant. The retinal explant of this disclosure comprises living cone photoreceptor cells. Preferably, the retinal explant comprises, in addition to living cone photoreceptors, other living cells such as rods, retinal pigment epithelial cells, ganglion cells, bipolar cells, amacrine cells, horizontal cells, astrocytes, and / or Müller cells.

[0098] Preferably, the retinal explant of this disclosure comprises rod- and cone layers.

[0099] In certain cases, the retinal explant comprises, in addition to the rod- and cone layers, a nerve fiber layer, a ganglion cell layer, an internal plexiform layer, an internal plexiform layer, an external plexiform layer, an external plexiform layer, and / or an external limiting membrane. In some cases, the retinal explant comprises, in addition to the nerve fiber layer, a ganglion cell layer, an internal plexiform layer, an internal plexiform layer, an internal plexiform layer, an external plexiform layer, an external plexiform layer, an external limiting membrane, and / or retinal pigment epithelium. In some cases, the retinal explant comprises all of the nerve fiber layer, ganglion cell layer, internal plexiform layer, internal granular layer, external plexiform layer, external granular layer, external limiting membrane, rod- and cone layer, and / or retinal pigment epithelium.

[0100] Retinas of any origin are usable in this disclosure. In some examples, the retina is a human retina. In other examples, the retina is of non-human origin, e.g., from non-human primates, cattle, pigs, or rodents. Human retinas are readily available from corneal banks, which are typically discarded after corneal dissection. Adult retinas have a large surface area (approximately 1100 mm²). 2 ), it can be easily divided into multiple experimental subregions.

[0101] Organoids and / or retinal explants are maintained in culture. Several culture media suitable for the proliferation of organoids and / or retinal explants have been described to date (see, for example, Fradot et al. Hum Gene Ther. 22(5):587-93, 2011; Belhadj et al. J Vis Exp. 2020 Nov 25;(165); Marie et al. Cell Death Dis. 2020 Aug 29;11(8):711, 2020). Those skilled in the art will refer to examples for specific examples of media that can be used to maintain organoids or retinal grafts alive.

[0102] Advantageously, the method comprises a further step of bringing retinal tissue, i.e., organoids or retinal explants, into contact with a polycarbonate membrane. The retinal tissue may be in either orientation when in contact with the membrane, i.e., the photoreceptors may face toward the membrane, or the photoreceptors may face away from the membrane. In some examples, it is advantageous for the photoreceptors to face away from the membrane.

[0103] In vivo model of cone photoreceptor degeneration In another respect, retinal tissue is included in a complete eye. Preferably, the eye is that of a living animal, more preferably a non-human animal.

[0104] Such in vivo models are particularly useful for studying the physiological significance of drugs aimed at preventing cone photoreceptor degeneration. This is especially true when healthy eyes must be considered during the research phase toward translational therapy. This allows for the study of not only the effects of the therapy on the cone photoreceptors themselves, but also on other retinal cell types and even more complex interactions with other organs. Such research is necessary before proceeding to clinical application. Therefore, this in vivo cone photoreceptor degeneration model is particularly useful for characterizing potential drugs for retinal degenerative pathologies.

[0105] In fact, the inventors have shown that this in vivo model faithfully reproduces the characteristics of retinal degenerative conditions such as AMD. In particular, when a specific cell death inducer, such as a ferroptosis inducer, was injected into the eyes of non-human primates, a loss of cones, especially a significant loss of the outer cone segments, was observed. Furthermore, the loss of cone receptors in this model was accompanied by an increase in the number of microglia cells in the outer retinal layer. The retinal thickness was reduced, as shown in tissue sections or OCT (optical coherence tomography). Subretinal deposits were detected in the fovea and / or macula. Furthermore, these histological findings correlated with physiological results indicating decreased retinal cone activity when assayed using electroretinography (ERG) or multifocal electroretinography (mfERG); see, e.g., Asanad S, Karanjia R. Multifocal Electroretinogram. [Updated 2022 Oct 9]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2023 Jan.

[0106] Therefore, in a method for creating an in vivo model of cone photoreceptor degeneration as disclosed herein, specific cell death, such as activation of ferroptosis, • Proliferation, activation, and / or migration of microglia cells to the ONL; Loss of the extrapyramidal segment; • Formation of subretinal deposits, preferably in the fovea and / or macula; • A decrease in cone activity in the retina, preferably cone activity measured by ERG or mfERG; and • Thinning of retinal thickness as shown in tissue sections or in vivo imaging by OCT It results in one or more of the following.

[0107] The methods disclosed herein involve contacting a specific cell death inducer, such as a ferroptosis inducer, with at least one cone photoreceptor. In the in vivo models described herein, such contact can be made by administering the cell death inducer (e.g., a ferroptosis inducer) to an animal. The administration is performed topically to the ocular or adnexal tissue. Preferably, the composition is administered intravitreously, subretinally, or topically. Topical ophthalmic formulations include eye drops. Preferably, the method does not involve systemic administration. Topical ocular administration has several advantages because the eye is an immune-privileged environment, and compounds administered to the eye act locally with little to no systemic diffusion.

[0108] Depending on the circumstances, this method may further include the administration of a pharmaceutically acceptable carrier.

[0109] The term "pharmaceutically acceptable" means, within the bounds of sound medical judgment, a composition, polymer, other material, and / or dosage form that is suitable for use in contact with human and animal tissues, without excessive toxicity, irritation, allergic reactions, or other problems or complications, and in proportion to a reasonable benefit-to-risk ratio.

[0110] A "pharmaceutically acceptable carrier" refers to any pharmaceutically acceptable material, composition, or vehicle involved in transporting any supplement or composition, or its components, from one organ or body part to another, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulant. Each carrier must be "acceptable" in the sense that it is compatible with the other components of the supplement and is not harmful to the patient. In some cases, a pharmaceutically acceptable carrier is nonpyrogenic. Examples of materials that can serve as pharmaceutically acceptable carriers include: (1) sugars such as lactose, sucrose, and sucrose; (2) starches such as corn starch and potato starch; (3) cellulose and its derivatives, e.g., sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients such as cocoa butter and suppository wax; (9) oils such as peanut oil, cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil, and soybean oil; (10) p Examples include glycols such as polypropylene glycol; (11) polyols such as glycerin, sorbitol, mannitol, and polyethylene glycol; (12) esters such as ethyl oleate and ethyl laurate; (13) agar; (14) buffers such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer; and (21) other non-toxic and suitable substances used in pharmaceutical formulations.

[0111] Cone photoreceptor degeneration models and their uses In another aspect, this disclosure relates to a cone photoreceptor degeneration model that can be obtained by the methods disclosed herein. This model may be any of the models disclosed herein, namely an in vitro model, an ex vivo model, or an in vivo model.

[0112] The model disclosed herein is particularly useful for screening and / or testing compounds with potential therapeutic activity. In fact, preliminary screening of a compound library using this in vitro model yielded several compounds capable of restoring cell viability, demonstrating the usefulness of the pyramidal degeneration model described herein.

[0113] Therefore, in another aspect, this disclosure relates to a method for screening compounds that may exhibit protective and / or anti-denaturing properties with respect to cone photoreceptors. Preferably, the method is a) To provide a pyramidal degeneration model described herein, b) Contacting the cone degeneration model with the candidate compound. c) Evaluate whether the candidate compound induces protective and / or antidenaturing effects on cone photoreceptors. It consists of including.

[0114] The protective and / or antidenaturing effects can be evaluated by any means known to those skilled in the art. Preferably, the protective and / or antidenaturing effects on cone photoreceptors are induced by the candidate compound, and when a cone degeneration model is brought into contact with the candidate compound, • Maintenance or increase of the survival rate of at least one cone photoreceptor, in particular, specific maintenance or increase; • Maintenance or increase of intracellular glutathione levels in at least one cone photoreceptor, in particular, specific maintenance or increase of intracellular glutathione levels; • Maintenance or decrease of the intracellular NADP / NADPH ratio in at least one cone photoreceptor, in particular, specific maintenance or decrease of the intracellular NADP / NADPH ratio; • Maintenance or reduction of intracellular formation of lipid peroxides, particularly 5-oxo-6,8,11,14-eicosatetraenoic acid (5oxoETE), in at least one cone photoreceptor, especially specific maintenance or reduction; • Maintenance or increase in gene expression of AIFM2 (apoptosis-inducing factor 2), SLC3A2 (solute transporter family 3 member 2), HSBP1 (heat shock protein family B (Small) member 1), and Keap1 (Kelch-like ECH-related protein 1), particularly those specific to cone photoreceptors; • Maintenance or reduction of ferritin and / or nuclear receptor coactivator 4 (NCOA4) protein expression, particularly specific to cone photoreceptors; • Prevention of increased cell membrane permeability to pigments, particularly those specific to cone photoreceptors. At least one of these will be achieved.

[0115] This effect can also be expressed as any combination of the above properties. In particular, the effect on the degeneration of cone photoreceptors may result in at least one, at least two, at least three, at least four, at least five, or at least six of the above properties. Preferably, the effect on the degeneration of cone photoreceptors may result in all seven properties.

[0116] As described herein, the various pyramidal degeneration models (in vitro, ex vivo, in vivo) described herein provide various levels of analysis.

[0117] For example, the screening methods described herein can be used to identify one or more compounds having protective and / or antidenatural properties using an in vitro model, i.e., a pyramidal degeneration model in which specific cell death is induced in a substantially pure population of pyramidal cells. The compounds thus identified can be further screened in an ex vivo model, according to the methods described herein, to identify compounds that still exhibit an effect against pyramidal photoreceptor degeneration in an entire organ. This effect manifests as increased migration of microglial cells in the ONL, or one of the following: • Maintenance or increase of the survival rate of at least one cone photoreceptor, in particular, specific maintenance or increase of the survival rate; • Maintenance or increase of intracellular glutathione levels in at least one cone photoreceptor, in particular, specific maintenance or increase of intracellular glutathione levels; • Maintenance or decrease of the intracellular NADP / NADPH ratio in at least one cone photoreceptor, in particular, specific maintenance or decrease of the intracellular NADP / NADPH ratio; • Maintenance or reduction of intracellular formation of lipid peroxides, particularly 5-oxo-6,8,11,14-eicosatetraenoic acid (5oxoETE), in at least one cone photoreceptor, especially specific maintenance or reduction; • Maintenance or increase in gene expression of AIFM2 (apoptosis-inducing factor 2), SLC3A2 (solute transporter family 3 member 2), HSBP1 (heat shock protein family B (Small) member 1), and Keap1 (Kelch-like ECH-related protein 1), particularly those specific to cone photoreceptors; • Maintenance or reduction of ferritin and / or nuclear receptor coactivator 4 (NCOA4) protein expression, particularly specific to cone photoreceptors; • Prevention of increased cell membrane permeability to pigments.

[0118] This effect can also be expressed as any combination of the above properties. In particular, the effect on cone photoreceptor degeneration may result in at least one, at least two, at least three, at least four, at least five, at least six, or at least seven of the above properties. Preferably, the effect on cone photoreceptor degeneration results in all eight properties.

[0119] Finally, this in vivo model can be used in the screening method of this disclosure to identify compounds that exhibit therapeutic effects.

[0120] Preferably, protective and / or antidenaturing effects on cone photoreceptors are induced in vivo by the candidate compound, and when a cone degeneration model is brought into contact with the candidate compound, • Maintenance or increase of survival in at least one cone photoreceptor, in particular, specific maintenance or increase of survival; • Maintenance or increase of intracellular glutathione levels in at least one cone photoreceptor, in particular, specific maintenance or increase of intracellular glutathione levels; • Maintenance or decrease of the intracellular NADP / NADPH ratio in at least one cone photoreceptor, in particular, specific maintenance or decrease of the intracellular NADP / NADPH ratio; • Maintenance or reduction of intracellular formation of lipid peroxides, particularly 5-oxo-6,8,11,14-eicosatetraenoic acid (5oxoETE), in at least one cone photoreceptor, in particular, specific maintenance or reduction of intracellular formation of lipid peroxides; • Maintenance or increase in gene expression of AIFM2 (apoptosis-inducing factor 2), SLC3A2 (solute transporter family 3 member 2), HSBP1 (heat shock protein family B (Small) member 1), and Keap1 (Kelch-like ECH-related protein 1), particularly those specific to cone photoreceptors; • Maintenance or reduction of ferritin and / or nuclear receptor coactivator 4 (NCOA4) protein expression, particularly specific to cone photoreceptors; • Prevention of increased cell membrane permeability to pigments, particularly those specific to cone photoreceptors; • Prevention of proliferation, activation, or migration of microglial cells; • Prevention of loss of the pyramidal exopartum; • Prevention of a decrease in cone activity in the retina, preferably measured by ERG or mfERG; and • Prevention of retinal thinning as shown in tissue sections or in vivo imaging by OCT. It brings about at least one of the following.

[0121] This effect can also be expressed as any combination of the above properties. In particular, the effect on the degeneration of cone photoreceptors may result in at least one, at least two, at least three, at least four, at least five, at least six, or at least seven, at least eight, at least nine, or at least ten of the above properties. Preferably, the effect on the degeneration of cone photoreceptors may result in all eleven properties.

[0122] The present invention is further illustrated in its entirety by the following embodiments. However, these embodiments are not intended to limit the scope of the invention, which is defined by the appended claims. [Examples]

[0123] Example 1 method Data Availability Animal model: rat Eight-week-old Long Evans wild-type rats were obtained from Janvier Labs (ISO 9001 certified). On day 0 (D0), the control group and the treatment group were each administered either 0.2% DMSO (D2650, Millipore Sigma, Burlington, MA) in PBS (14190-094 Life Technologies Europe BV, Bracewijk, Netherlands) or 0.2% 20 μM IKE in a DMSO solution diluted in PBS by subretinal injection.

[0124] Surgical and anatomical procedures in rats Rats were anesthetized by intraperitoneal (IP) injection of 40 mg / kg ketamine (Ketamidor 100 mg / ml, Axience SAS, Pantan, France) and 0.14 mg / kg medetomidine (Domitor 0.85 mg / ml, Vetoquinol SA, Paris, France). Local anesthesia was performed with oxybuprocaine eye drops (1.6 mg / 0.4 ml, Thea, Clermont-Ferrand, France). 0.5% tropicamide eye drops (Mydriaticum, Thea, Clermont-Ferrand, France) were used to dilate the left eye. Tear gel (Lubrithal, Dechron, Shrewsbury, UK) was used at the lens-eye interface throughout the procedure. Using an ophthalmic microscope (Lumera 700, Carl Zeiss, Oberkochen, Germany), conjunctival-scleral channels were created with a 30-gauge (G) needle (BD Microlance 3, Becton, Dickinson SA, Fraga, Spain). Visualization of the retinal plane was possible using flat contact lenses (Cover slips, mini, 8mm, World Precision Instruments, Sarasota, FL). 9 μL of control solution or IKE solution was delivered subretinally via the nasal side using a 30G cannula with a non-bevel metal tip attached to a 10 μL syringe (Hamilton, Rino, NV) mounted on a microinjector (Micro 4, World Precision Instruments, Sarasota, FL). Postoperatively, patients received chloramphenicol / retinol ophthalmic ointment (Ophtalon 10 mg / g, TVM, Lembodes, France), 1 mL of 5% glucose monohydrate (Osalia, Paris, France) via intracellular injection, and 0.9 mg / kg of atipamezole (Antidorm 4.27 mg / ml, Axience SAS, Pantan, France) via subcutaneous injection. In vivo anatomical examinations, namely optical coherence tomography (OCT, Bioptigen, Durham, NC) and fundus photography (Micron IV, Phoenix-Micron, Inc., Bend, OR), were performed to confirm subretinal delivery on day 0 and to confirm retinal reattachment on day sacrificial death.In one group of animals (n=4), sacrificial death was performed on day 6 (D6) to assess microglial migration, and in another group of animals (n=8), on day 30 (D30) to assess cone degeneration, using intracardiac injection of 1 ml / kg pentobarbital (Exagon, Axience SAS, Pattin, France). The left eye was harvested and immediately immersed in 4% paraformaldehyde (PFA, J61899.AP ThermoFisher Scientific, Waltham, MA) for 2 hours, then stored in PBS at +4°C. The eyeball was dissected and the entire retina was harvested and immersed in a 24-well plate (#351147 Corning Inc., Corning, NY) for immunohistochemical analysis. Furthermore, optic cup sections labeled (with CAR and Iba1) were prepared for evaluation of other quantitative analyses. For this purpose, the eyeball was harvested from the rats and the cornea was removed. Subsequently, the eyeballs were immersed in 4% PFA for 1 hour, followed by a stepwise sucrose bath from 10% to 30%. Finally, the lenses were carefully removed, and the optic cups were placed in a freezing medium in liquid nitrogen. These were stored at -20°C until 12 μm sections were prepared using a cryostat (CM3050 S, Leica, Wetzlar, Germany).

[0125] Mammalian eyeball The pig eyeballs were obtained from a local slaughterhouse in accordance with an agreement with local regulatory authorities and veterinarians from the French Ministry of Agriculture (agreement number FR75105131).

[0126] Pure cone photoreceptors The retina was obtained by dissecting the pig eyeball and shredded. It was then digested with 4 U / mL papain (LSO 3124, Worthington) and L-cysteine ​​(5.5 mM, Sigma-Aldrich) at 37°C for 20 minutes. Enzyme activity was inactivated using Neurobasal-A (NBA) medium (10888022, ThermoFisher Scientific, Waltham, MA, USA) containing 5% fetal bovine serum (FBS) and 15 μg / mL DNase I (D4263, Millipore Sigma, Burlington, MA). A series of short centrifuges (30 seconds, 5-6 times) were performed, and the supernatant was collected, except for the first supernatant which mainly contained rod photoreceptors. The pellet was resuspended using P1000 to disperse any remaining cells between centrifuges. The cell suspension was then centrifuged at 800 rpm for 10 minutes, and the pellet was resuspended in NBA medium containing 1% L-glutamine (G3126, Millipore Sigma, Burlington, MA) and 1% B27 supplement (17504044, ThermoFisher Scientific, Waltham, MA, USA).

[0127] Pyramid PRs were purified by lectin panning sorting, as previously described (Balse et al. (2005) IOVS 46:3672), with some modifications to obtain millions of pyramids in suspension. The cells were then seeded at 3000 cells / well in 384-well plates (781091, Greiner, Uris, France) using a robot (Viaflo Assist, Integra-Biosciences, Saint-Ouen, France) and incubated at 37°C and 5% CO2 for 3 days.

[0128] retinal explant Pig and non-human primate retinas were cultured as previously described (Greferath et al. (2016) Ophthalmology 123:1320). After isolating the retina in CO2-independent medium, tissue samples were collected from the posterior optic nerve region using a 2 mm biopsy punch and placed on a polycarbonate membrane (140652, Thermo, Waltham, Massachusetts) with the photoreceptors facing upward. The explants were cultured in a 37°C CO2 incubator for 3 days.

[0129] treatment To induce ferroptosis, the following components were used: 1 μM to 500 μM glutamate (49621, Sigma Aldrich, Missouri, USA) in vitro; 3 μM imidazole ketone elastin (IKE) (HY-114481, MedChemExpress, Monmouth, USA) in purified cone photoreceptors or 20 μM in ex vivo and in vivo experiments; and 20 nM, 200 nM, and 10 μM RSL3 (HY-114481, MedChemExpress, Monmouth, USA) in cells, ex vivo, and in vivo experiments, respectively. To inhibit ferroptosis, ferrostatin-1 (FST-1, HY-100579, MedChemExpress, Monmouth, USA) was used at 50 nM in purified cones or 400 nM in retinal explants. Furthermore, deferipron (HY-B0568, MedChemExpress, Monmouth, USA) was effective at 50 μM in pure cones, and Zillowton (HY-14164, MedChemExpress, Monmouth, USA) was effective at 20 μM in purified cones.

[0130] Measurement of cell viability After a 3-day incubation period, cell viability was measured using calcein (C1430, Thermo, Waltham, Massachusetts) diluted 1 / 4000 and incubated at 37°C for 1 hour. The number of viable cells was counted using an automated fluorescence microscope, ArrayScan (Cellomics ArrayScan VTI HCS reader, Thermo, Waltham, MA).

[0131] Screening in primary cells The purified cone photoreceptors were transferred to a transparent, flat-bottomed 384-well plate (781091, Greiner, Uris, France) using a robot equipped with a 384-well head (BRAVO, Assist, Integra-Biosciences, Saint-Ouen, France) for 4 * 10 3 Cells were seeded to a cell / well ratio. In the first two rows, cells were treated with DMSO (D2650, Millipore Sigma, Burlington, MA) alone, and in the rest of the plate, cells were treated with either RSL3 (HY-114481, MedChemExpress, Monmouth, USA) to a final 20 nM or IKE (HY-114481, MedChemExpress, Monmouth, USA) to a final 3 μM. Seventeen hours after induction of cytopathy, the ferroptosis library compound (HY-L051, MedChemExpress, Monmouth, USA) was added to the cells using a robot with a 384-well head (BRAVO, Assist, Integra-Biosciences, Saint-Ouen, France) to a final concentration of 10 μM of the compound. Each treatment solution was diluted in Neurobasal medium (10888022, ThermoFisher Scientific, Waltham, MA, USA) and 1 / 100 L-glutamine (G3126, Millipore Sigma, Burlington, MA). The cells were then incubated at 37°C and 5% CO2 for a further 2 days. Cell viability was then measured on day 3 after seeding.

[0132] The results were analyzed using TIBCO Spotfire® software (California, USA). A robust Z' factor was calculated for each plate, and those with a value greater than 0.45 were selected. Next, the percentage of viable cells from the positive control was calculated for each plate. The average number of cells counted was calculated and reported as 100%. This positive control ratio was reported for all molecules in the plate. Next, all plates treated with the same method (IKE or RSL3) were displayed, and compounds with cell viability exceeding 50% were selected and extracted into Excel.

[0133] Lipidomics Analysis Purified cone photoreceptors treated with or not treated with 3 μM IKE were washed three times with PBS, crushed in 100 μL, rapidly frozen in liquid nitrogen immediately after collection, and stored at -80°C until extraction. Eicosanoid extraction and analysis were performed at the Lipidomics Facility Core: MetaToul-Lipidomique (I2MC, Inserm, Toulouse, France), MetaboHUB-ANR-1 1-INBS-0010.

[0134] Western blot Cells were collected and separated from the culture medium by centrifugation, and total cellular proteins were extracted in lysis buffer (10 mM HEPES [pH 7], 100 mM NaCl, 2 mM EDTA, 0.5% NP-40, and a protease inhibitor cocktail (Millipore Sigma, Burlington, MA)). After centrifugation at 13000 rpm, the supernatant was maintained for further WB analysis. Next, the samples were loaded onto an SDS-PAGE in 4%–15% Mini-PROTEAN TGX Tris-glycine buffer and transferred to a 0.2 μm Trans-Blot Turbo nitrocellulose membrane (Bio-Rad, California, USA). The membrane was blocked at room temperature for 1 hour in 1× Tris-buffered saline (TBS, 10 mM Tris-HCl [pH 8], 150 mM NaCl) supplemented with 5% (w / v) dried skim milk powder. Primary antibody incubation was performed overnight at 4°C. The presence of 5LOX and FLAP at their expected molecular weights in the total lysis solution was confirmed by Western blotting using anti-5LOX (1:200; Abcam AB169755) and anti-FLAP (1:500; Abcam AB85227). The secondary HRP-conjugated antibody used for the detection of both 5LOX and FLAP was goat anti-rabbit (1:20,000; 111-035-003, Jackson ImmunoResearch Laboratories, Pennsylvania). Between and after antibody incubation, the membranes were thoroughly washed with TBS containing 2.5% Tween-20 (TBS-T). Western blots were visualized using enhanced chemiluminescence (ECL Prime detection reagent, Amersham, UK).

[0135] NADPH assay The NADP / NADPH assay kit (ab176724, Abcam, Cambridge, UK) was used according to the manufacturer's instructions to quantify the levels of NADP and NADPH.

[0136] Glutathione (GSH) assay GSH levels were quantified according to the manufacturer's instructions for the GSH assay kit (V6912, Promega, Madison, WI).

[0137] Cryostat section After a 3-day incubation, retinal explants were fixed in 4% paraformaldehyde (15714, Electron Microscopy Sciences, Hatfield, Pennsylvania) at room temperature for 1 hour and washed three times with PBS. The retinal explants were then either stored at +4°C or prepared as cross sections. The sections were cryopreserved in sequential 10%, 20%, and 30% sucrose baths. Subsequently, the samples were frozen in tissue freezing medium (72592, Electron Microscopy Sciences, Hatfield, Pennsylvania) immersed in liquid nitrogen until the tissue freezing medium hardened. The samples were stored at -20°C until 10 μm cross sections were prepared using a cryostat (CM3050 S, Leica, Wetzlar, Germany). The retinal cross sections were stored at -20°C.

[0138] immunolabeling First, the samples (fixed retinal explants and retinal cross-sections) were permeabilized with 0.5% Triton 100X (T8787, Sigma-Aldrich, Missouri, USA) diluted in PBS 1×. Next, nonspecific regions were blocked with a saturated buffer consisting of 10% normal donkey serum (S30-100ML, Millipore Sigma, Burlington, MA) diluted in PBS 1×. The primary and secondary antibodies used in this study are listed in Table 1.

[0139] [Table 1]

[0140] PNA lectin (L21409, Thermo, Waltham, Massachusetts) diluted 1 / 50 and rabbit FLAP antibody (ab85227, Abcam, Cambridge, UK) at an effective concentration of 5 μg / mL target antigenic sites on the extracellular side of the cell membrane. No permeabilization step was performed for these labels.

[0141] Finally, the retinal explants were washed three times in PBS and flat-mounted for fluorescence microscopy on Cellvis plates (P12-1.5HN, IBL, Geräsdorf, Austria) using Permafluore (TA-030-FM, Thermo, Waltham, Massachusetts). Retinal cross sections and whole-mount retinas were also mounted on coverslips or on 6-well plates (P06-1.5HN, Cellvis, Mountain View, CA) using Permafluore, respectively.

[0142] Confocal microscope Retinal images were acquired using a laser scanning confocal microscope (Fluoview V-1000, Olympus, Tokyo, Japan). In rat retinas, cone counting (outer segment and cell body / mm) was performed manually on retinal sections.

[0143] CQ1 Imaging Images of rat whole-mount retinas and flat-mount porcine explants were acquired using a CQ1 confocal microscope (Yokogawa Electric Corporation, Tokyo, Japan). In the injectable rat retinas, the inferior nasal and inferior temporal visual fields were manually observed in four z-axis planes defined by cone labeling: the outer segment (OS) plane, the cone nucleus (CN) plane, the internal fiber (IF) plane, and the cone copedal (CP) plane. Emphasis was placed on microglia migration. Representative samples were extracted from the CQ1 software. Z-stack images were analyzed using Cell Pathfinder software (Yokogawa Electric Corporation, Tokyo, Japan), which enables 2D, 3D, or section quantification. Algorithms (for porcine explants and rat whole-mount retinas) were generated to quantify cone cell bodies or outer segments and microglia cells along the Z-axis. Extracted quantitative data were analyzed using TIBCO Spotfire® (California, USA).

[0144] Non-human primates (NHPs) This study used two non-human primates (NHPs): a 5-year-old male and a 15-year-old female. The animals were born in captivity and originated from AAALAC-certified authorized suppliers (SARL Bioprim, Bazièges, France; Cynologics-Silabe, Niederhausen, France). Ethical approval was obtained from the MIRCen local ethics committee CETEA n°44, followed by the French Ministry of Education and Research. A detailed description of rearing and perioperative care can be found in Dentel et al. (2023) Ophthalmol. Sci. 3:100316.

[0145] The toxicity of IKE in NHP was evaluated by subretinal delivery of 20 μM IKE solution to one eye and a control solution (0.2% DMSO diluted in PBS) to the contralateral eye. A comprehensive description of anesthesia, surgery, and data acquisition can be obtained from Dentel et al. Briefly, subretinal injections were performed to detach the upper half of the fovea centralis in each eye. Anatomical examinations (slit-lamp examination, optical coherence tomography (OCT), adaptive optics (AO)) and functional examinations (full-field and multifocal electroretinograms, ffERG and mfERG, respectively) were performed at baseline (within 1 week before surgery), on day 3 (to confirm completion of retinal reattachment), 1 month, and then monthly until the 4th month. Cone mosaic parameters by adaptive optics were quantified using the built-in software of rtx1 (AOdetect, Imagine Eyes, Orsay, France). Additionally, the power spectrum spacing method, which was additionally used to quantify the cone mosaic in NHP, is detailed in Dentel et al.

[0146] Analysis and Statistics Analyses, graphs, and figures were created using GraphPad Prism 8.4.0 software (GraphPad Software Inc., San Diego, CA). Treatment groups and control groups were statistically compared by the Mann–Whitney t-test, and considered significant when p < 0.05 ( * ), p < 0.01 ( ** ), p < 0.001 ( *** ), or p < 0.0001 ( **** ).

[0147] In the figures, data were normalized to the control group and presented as mean + / − standard error, whenever possible

[0148] The control and treatment groups were compared in in vitro and ex vivo analyses using the Mann-Whitney t-test. In rats, differences in cone density and microglia density were compared using multiple t-tests, and statistical significance was determined using the Holm-Sidak method. In NHP, differences in cone density, regularity, interval values, and ERG amplitude waveforms were compared using two-sided t-tests. For mfERG in NHP, binocular symmetry was considered, and each hexagon was compared with its corresponding hexagon on the contralateral side.

[0149] result Glutamate-induced ferroptosis in purified cone photoreceptors Photoreceptors release the neurotransmitter glutamate at synaptic terminals, but they only express glutamate transporters (they are not classical ion channel receptors). When glutamate was added to purified porcine cone photoreceptors (Figure 1A-D), the survival rate decreased by 20% at 10 μM, and by up to 82% at the highest test concentration of 500 μM (Figure 1E). This glutamate range is physiologically encountered, as indicated by the glutamate affinity of the receptor and transporter (Gielen (2010) medecine / sciences 26:65). This toxicity has been reported in cone photoreceptors by immunohistochemistry (Dun et al. (2006) Cell Tissue Res 324:189; Bridges et al. (2004) Ophthalmol. Vis. Sci. 45:2906-2914)Xc - To define whether it may be related to the expression of the antiporter system, glutamate was added in the presence of 1 mM L-cystine. At all glutamate concentrations, L-cystine prevented glutamate toxicity (n=7, p<0.0001, IC50=4.15 μM) (Figure 1E). - Because this transporter has pharmacological properties that are very different from other glutamate transporters or receptors, we investigated how different agonists and antagonists of this transporter affect pyramidal survival. -Two functional inhibitors of the system, elastin and imidazole-ketone elastin (IKE), induced toxicity to isolated cone photoreceptors, similar to glutamate, but at lower dose ranges due to their higher affinity (Figures 1A-D, F). L-cystine also rescued cones exposed to IKE (n=6, p=0.0022) (Figure 1K).

[0150] To evaluate the molecular mechanisms underlying this glutamate-induced cone photoreceptor toxicity, GSH levels in cones exposed to 500 μM glutamate and imidazole ketone elastin (IKE3 μM) were measured. Compared to the control condition, a 50% decrease in GSH was observed from day 1 onward under both conditions (Figure 1F, n=18, p<0.0001). Therefore, cone photoreceptor cell death is associated with Xc - This is due to dysregulation of intracellular GSH levels induced by dysfunction of the system. Given that GSH is involved in the redox cycle in which its oxidized form is reduced by hydrogen atoms carried by NADPH (Dixon et al. (2019) Annu. Rev. Cancer Biol. 3:35), the NADPH / NADP ratio was measured in cones treated with 3 μM IKE. This condition increased the amount of NADPH from 25% of the control to 85% of the treated culture (n=4, p=0.0159) (Figure 1G). The intracellular NADPH / NADP ratio is indirectly related to the activity of glutathione peroxidase 4 (GPX4) in the redox process of glutathione. Inactivation of GPX4 may inactivate this cycle and induce this accumulation of NADPH, which is related to Xc - This suggests that inhibition of the system leads to downregulation of the redox cycle (Azuma et al. (2022) J. Biol. Chem. 298:101824). To investigate whether GSH is essential for cone survival, we used Ras-selective lethal 3 (RSL3), a specific inhibitor of GPX4. This inhibitor showed toxic effects on cone photoreceptors at extremely low concentrations (n=3, IC2). 50(=8.67nM) (Figure 1H), highlighting the important role of GPX4 in cone cell survival. Next, RNA extraction was performed from pure cone cell photoreceptors in the treatment and control groups, showing altered expression of proteins involved in the regulation of the glutathione redox cycle and downstream mechanisms (Figure 2).

[0151] GPX4 is also known to prevent the formation of lipid peroxides that react with free radical species (Xie et al. (2016) Cell Death Differ. 23:369; Dixon et al. 2019). Lipid peroxides are formed by lipoxygenases such as 5-lipoxygenase (5LOX), which is activated by 5-lipoxygenase-activating protein (FLAP). To investigate the presence of these mechanisms in cone photoreceptors, we performed Western blotting and found the presence of both 5LOX and FLAP in cones, which are not present in rod photoreceptors (Figure 1I). Specific immunostaining of retinal tissues from various species revealed the localization of this specific FLAP in cone photoreceptors (Figure 3). To evaluate the role of this lipid peroxide pathway in glutamate-induced cell death, cone cell extracts were analyzed by lipidomics after incubation with 3 μM IKE for 2 days. Analysis revealed an increase in the amount of lipid peroxides, such as 5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE), a metabolite of 5LOX (n=6, p=0.0079). ** ), p=0.0159( * (Figure 1J). To demonstrate that this lipid peroxidation contributes to the observed glutamate-induced toxicity, the effect of Ziloton (ZEN), an enzymatic antagonist of 5LOX, was investigated in the presence of IKE. ZEN rescued 80% of cone photoreceptors (Figure 1K, n=3, p=0.0238), suggesting that lipid peroxidation plays a crucial role in glutamate-induced cone degeneration.

[0152] Lipid peroxidation triggers alterations in iron metabolism during ferroptosis. The ferritinophagy process can be highlighted by immunolabeling of ferritin and the ferritinophagy-inducing factor nuclear receptor coactivator 4 (NCOA4). Pyramid cells treated with IKE showed increased immunolabeling intensity for both ferritin heavy chain and NCOA4. NCOA4 appeared to be slightly stronger than ferritin heavy chain, suggesting increased iron metabolic activity in treated pyramid cells (Figure 4). The ferritinophagy process can be inhibited by ferrostatin-1 (FST1: a specific ferroptosis inhibitor) and deferlipron (DF: an iron chelator that prevents the Fenton reaction by limiting the intracellular accumulation of free iron). Both rescued pyramid cells from IKE-induced toxicity, with rescue rates of 68.5% (n=8, p=0.0047) for FST1 and 77.2% (n=5, p=0.0079) for DF (Figure 1K). Furthermore, other anti-ferroptosis molecules targeting different components involved in ferroptosis activation were tested after treatment with 20 nM RSL3 and 3 μM IKE (Table 2). Thus, pure cone photoreceptors exposed to ferroptosis inducers (e.g., IKE) appear to be salvageable by anti-ferroptosis treatment, highlighting the role of ferroptosis in cone survival.

[0153] [Table 2] JPEG2026524899000003.jpg240165

[0154] Ferroptosis of cone cells in an ex vivo retinal model To demonstrate that cone photoreceptors can be degenerated by ferroptosis in an integrated retinal model lacking retinal pigment epithelium, IKE was applied to newly prepared porcine retinal explants. The specificity and selectivity of ferroptosis in the retina were evaluated by examining the cell membrane permeability to ethidium stains in damaged cells after 3 days of incubation. Under control conditions, ethidium hardly labeled scattered cells, but in IKE-treated explants, the anterior row of photoreceptor nuclei was ethidium-labeled (Figure 5B, F). This prominent layer corresponds to the nuclei of cone photoreceptors in porcine retina, as shown by flap immunolabeling (Pattnaik et al. (2000) J. Neurosci. 29:6789) (Figure 5C, G). Figure 5G shows the morphological changes of cone photoreceptors induced by 20 μM IKE. Other retinal cell types were also labeled, but no significant qualitative changes were observed (Figure 6). Subsequently, in situ cytotoxicity in cone photoreceptors was evaluated by automatically counting (using CellPath software for CQ1®) at the level of immunolabeled outer segments (OS) with Flap (Figures 5J-M). 20 μM IKE induced a 50% reduction in cone OS (n=3, p=0.0286) (Figure 5I). 1 μM FST-1 and 100 μM DF rescued 84% and 78.4% of OS, respectively (n=3, p=0.1000 and p=0.0286, respectively) (Figure 5I). Similarly, when cone degeneration was induced in retinal explants by 200 nM RSL3 (Figure 7), 100 μM DF rescued 78.5% of OS (n=3, p=0.0286) (Figure 7M). These data suggest that ferroptosis is an effective and selective pathway for cone photoreceptor degeneration in integrated retinal tissue.

[0155] Furthermore, in retinal tissue exposed to ferroptosis inducers, migration of microglia from the inner retina (normally localized) to the outer retina (where microglia are normally absent) appeared to be induced. Microglial cell activation was suggested by increased volume and changes in shape (expansion of cells surrounding the cones) (Figure 5H). Automated quantitative analysis revealed significant intraretinal microglia displacement to the outer granular layer (ONL) induced by both 20 μM IKE and 200 nM RSL3 (n=3) (Figure 5N). This migration was partially inhibited by ferroptosis inhibitors (FST-1 and DF) (n=3) (Figure 5O; Figure 7N). This morphological change of microglia and migration to the photoreceptor layer provides further evidence of ferroptosis-induced cone degeneration.

[0156] Pyramidal degeneration and microglia migration in rats To evaluate whether ferroptosis inducers enable the initiation of degenerative and inflammatory processes in living retinas, ferroptosis inducers were injected into the subretinal space of Long Evans rats (20 μM IKE, see Figure 8A). On day 6, optical coherence tomography (OCT) revealed numerous punctate hyperreflectivity suggestive of microglia in rats injected with ferroptosis inducers (arrows in Figure 8G). Subsequently, the same retinas were confirmed by immunostaining for cones and microglia. On day 30, the density of cone photoreceptors was assessed in retinal sections passing through the injection area. Overall survival (OS) and cell body size were significantly reduced in animals treated with ferroptosis inducers via subretinal delivery (Figures 8H-I). Quantitative analysis in retinal sections showed that ferroptosis inducers reduced cell body size by approximately one-third and OS by almost half (Figure 8J). Quantification of microglia migration within the injection area in whole-mount retina of D6 rats revealed increased OS levels and microglia at cone synaptic terminals (Figure 8K). Figures 8L-O show an example of microglia densely dispersed in the photoreceptor layer on day 6. These data suggest that induction of ferroptosis in living rat retina leads to cone cell degeneration and microglia migration to the photoreceptor layer.

[0157] Macular degeneration in non-human primates The ferroptosis inducer was similarly applied to the retinas of non-human primates living in vivo. Subretinal delivery of 20 μM IKE to one eye and a control solution (1 / 500 DMSO) to the contralateral eye was performed, followed by 4 months of observation (see Figure 9A). Previous studies have shown that injection of vehicle solutions into the subretinal space does not have functional or anatomical adverse effects on the eye or systemic effects in NHPs (to date, two NHPs have been followed for 6 months), but it does result in transient changes in photoreceptors confirmed by adaptive optics fundus examination, which have completely recovered after 4 months (Dentel et al. 2023).

[0158] In eyes injected with 20 μM IKE, structural and functional changes in the retina were observed in vivo. First, macular pigment deposition around the foveal subretinal deposits occurred within 3 months (Figure 9B-D). OCT showed erosion of the ellipsoidal zone (i.e., the regions of the inner and outer segments of the photoreceptor) and scattered punctate hyperreflectivity in the ONL only within the area detached by 20 μM IKE (Figure 9E-G). Multiple subretinal deposits were observed at the foveal level (Figure 9F) at 1 month and at the macular level (Figure 9H-J) at 2 months. Adaptive optics imaging of the multiple subretinal deposits (a technique that enables visualization of cone photoreceptors in vivo) was identical to that seen in patients with age-related macular degeneration (AMD), indicating a significant loss of cones within the area detached by 20 μM IKE (Figure 9K-L). Embedded OCT software, which enables segmentation of the ONL through the injection area, showed a significant change in ONL thickness within the area injected with 20 μM IKE (Figure 9M, N).

[0159] Adaptive optics was also used to quantify cone density at the injection site. The effect of standard subretinal injection of 0.2% DMSO on cone reflectance in adaptive optics imaging has already been reported (Dentel et al. 2023). In eyes injected with 20 μM IKE, cone loss was observed, with a decrease in cone cell density in the 2-degree superior eccentric region to 37.50 cone cells / mm² at 4 months. 2 For comparison, 661.00 cones / deg2 The result was (n=2, p<0.001). No change was observed with a 2-degree downward eccentricity (i.e., outside the injection area).

[0160] Functional changes in cone photoreceptors were assessed using multifocal electroretinography (mfERG): changes in N1 wave amplitude (an indicator of hyperpolarization response of photoreceptors to light) and P1 wave amplitude (indicating the functionality of the inner retinal layer) from baseline were analyzed. N1 wave amplitude was significantly reduced in eyes injected with 20 μM IKE (-52.19%) compared to eyes injected with the control solution (-2.95%) (n=2, p=0.0396). A non-significant decrease was also observed in P1 wave amplitude (-33.03% and -3.83%, n=2, p=0.0874). Subretinal delivery of ferroptosis-inducing agents at the macular level in non-human primates resulted in changes that strongly resembled the clinical features observed in macular degeneration.

[0161] Example 2 Ferroptosis does not affect primary retinal pigment epithelial cells. material and method Purification of porcine cone photoreceptors Pig eyeballs were dissected, and the retinas were collected. These were then chopped and digested at 37°C for 20 minutes with 4 U / ml papain (LSO 3124, Worthington) and L-cysteine ​​(5.5 mM, Sigma-Aldrich). The enzymatic reaction was stopped by adding Neurobasal-A (NBA) medium (10888022, Thermo Fisher Scientific, Waltham, MA, USA) containing 5% fetal bovine serum (FBS) and 15 μg / ml DNase I (D4263, Millipore Sigma, Burlington, MA). Several short centrifuges (30 seconds at 110xg, 5-6 times) were performed, and the supernatant was collected, excluding the supernatant from the first centrifuge which mainly contained rod photoreceptors. The pellet was resuspended using P1000 to disperse any remaining cells between centrifuges. The cell suspension was then centrifuged at 110xg for 10 minutes, and the pellet was resuspended in NBA medium containing 1% L-glutamine (G3126, Millipore Sigma, Burlington, MA) and 1% B27 supplement (17504044, ThermoFisher Scientific, Waltham, MA, USA).

[0162] Cone photoreceptors were purified by lectin panning, a slightly modified version of a previously described method (Balse et al. IOVS 2005), to obtain millions of cones in suspension. These cells were then seeded at a density of either 3,000 or 4,000 cells / well in clear 384-well plates (781091, Greiner, Ulis, France) or white 384-well plates (781080, Greiner, Ulis, France) using a robot (Viaflo Assist, Integra-Biosciences, Saint-Ouen, France), and incubated for 3 days at 37°C in air containing 5% CO2.

[0163] Purification of porcine retinal pigment epithelial (RPE) cells Pig eyeballs were dissected, the retina was removed, and optic cups containing RPE cells were collected from the fundus. Preheated 0.25% trypsin (25200-056, Thermo Fisher Scientific, Waltham, MA, USA) was added to the optic cups and incubated at 37°C for 1 hour. The cells were then collected from the optic cups by rinsing with a pipette and transferred to tubes containing DMEM medium (41966-029, Thermo Fisher Scientific, Waltham, MA, USA) and 20% FBS (A31605-01, Thermo Fisher Scientific, Waltham, Massachusetts) (=D20). After centrifugation at 110xg for 5 minutes, the pellet was resuspended in D20, the cells were seeded in 60mm petri dishes, and incubated at 37°C and 5% CO2. The medium was changed the following day.

[0164] Subsequently, these cells were seeded at a rate of 4,000 cells / well in white 384-well plates (781080, Greiner, Ulis, France) using a robot (Viaflo Assist, Integra-Biosciences, Saint-Ouen, France), and incubated for 3 days at 37°C in air containing 5% CO2.

[0165] ARPE-19 cell line This cell line was obtained from ATCC and supplied by LGC. These cells were used in phases P15-P30 and seeded in D20 medium.

[0166] Next, these cells were seeded at a rate of 4,000 cells / well in white 384-well plates (781080, Greiner, Ulis, France) using a robot (Viaflo Assist, Integra-Biosciences, Saint-Ouen, France), and incubated for 3 days at 37°C in air containing 5% CO2.

[0167] treatment Ferroptosis was induced using the following compounds: glutamate (49621, Millipore Sigma, Burlington, MA) (concentrations of 1 μM to 1 mM in vitro), IKE (HY-114481, MedChemExpress, Monmouth, USA) (concentrations of 200 nM to 25 μM, such as 3 μM in purified cone photoreceptors and 20 μM in ex vivo and in vivo experiments), and RSL3 (HY-100218A, MedChemExpress, Monmouth, USA) (concentrations of 10 nM to 10 μM, such as 20 nM, 200 nM, and 10 μM in cells, ex vivo, and in vivo experiments). For ferroptosis inhibition, FST-1 (HY-100579, MedChemExpress, Monmouth, USA) was used at a concentration of 50 nM in purified cones or 400 nM in retinal explants. DF (HY-B0568, MedChemExpress, Monmouth, USA) was effective in pure cones at a concentration of 50 μM, and ZEN (HY-14164, MedChemExpress, Monmouth, USA) was effective in purified cones at 20 μM (see Figure 1K, 5I, and 7M).

[0168] CellTiter Glo (CTG) Cell viability assays were performed using RPE cells, ARPE-19 cells, and cone photoreceptors with CellTiter Glo (G7573, Promega, Madison, WI). Viability was measured according to the manufacturer's instructions.

[0169] result IKE and RSL3 do not alter primary RPE cells in vitro. The inventors further investigated the sensitivity threshold of retinal pigment epithelial cells (RPE) after administration of ferroptosis-inducing agents. It is well known from the literature that RPE cells can undergo ferroptosis-mediated cell death (Lee et al. (2022) Oxid. Med. Cell. Longev. 2022:1792894). However, identifying the cell type most sensitive to cone light PR remains a challenge. Therefore, the inventors used the ARPE-19 cell line, which has been used in all studies to characterize RPE cells, and tested IKE, RSL3, and glutamate within a certain concentration range. When cells were treated with IKE, ARPE-19 cells showed a weaker effect at 25 μM, with 34.3% surviving cells compared to 0% for cones (n=3, p=0.0001) (Figure 11A). Similarly, treatment of ARPE-19 cells with RSL3 resulted in a higher mortality rate, but at a considerably higher concentration than that used to kill cones. At 10 μM RSL3, only 5.3% of ARPE-19 cells remained viable, and at 5 μM, only 50% of cells survived (n=3, p=0.0008) (Figure 11B). However, the EC50 of RSL3 in cones was 1.58 nM, demonstrating that cones are far more sensitive to GPX4 inhibition. Finally, glutamate treatment had no effect on ARPE-19, unlike cone PR, which was completely destroyed at 1 mM (n=3, p=0.0050) (Figure 11C).

[0170] However, the inventors were also interested in comparing the effects of ferroptosis-inducing molecules in the ARPE-19 cell line and primary porcine RPE cells. Significant differences were observed with IKE treatment, and even at 25 μM, there was no effect on primary cells (n=3, p=0.0019) (Figure 11A). However, the survival rate trends were similar for RSL3 and glutamate treatment (Figures 11B, C).

[0171] conclusion These results indicate that cone photoreceptors are more sensitive to ferroptosis inducers than RPE cells. Therefore, lower levels of oxidative stress may induce cell death of cone photoreceptors without causing cell death of RPE cells. Furthermore, it should be noted that studies on ferroptosis in RPE cells have used cell lines that divide readily and are therefore immortal (Kozlowski et al. (2015) Curr. Eye Res. 40:501). However, there is sufficient documentation on ferroptosis in RPE cells.

[0172] Example 3 Ferroptosis rescues RD1 mice from degeneration both in ex vivo and in vivo. material and method Animal model: Mouse The experiments and procedures on mice were conducted in an approved facility affiliated with the Institut de la Vision (Paris, France), in accordance with European Directive 2010 / 63 / UE, with the approval of the local animal ethics committee (Charles Darwin CEEACD #5). All experimental work was carried out in accordance with the institution's biosecurity and safety procedures. The mice were housed in a controlled environment, under conditions where the dark / light cycle reversed every half day, and were given free access to food and water except during surgery.

[0173] The C3H / HeNRj strain (rd1, obtained by crossing a Bagg Albino female with a DBA male) was purchased from Janvier Laboratories (Le Genest Saint-Isle, France, ISO 9001 certified) for production and breeding purposes.

[0174] Retinal explants of rd1 mice Eyeballs were collected at P15, washed, and incubated for 20 minutes at 37°C with 5% CO2 in a CO2-independent medium containing glucose (6.5 g / L) diluted 1 / 50 with papain (LSO 3124, Worthington), in a 1 / 10 L-cysteine ​​(Millipore Sigma, Burlington, MA) solution of 3.5 mg / 10 mL. The eyeballs were then immersed on ice for 5 minutes in Neurobasal medium (10888022, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 1% L-glutamine (G3126, Millipore Sigma, Burlington, MA) = NBAg, further supplemented with 10% SVF (A31605-01, Thermo Fisher Scientific, Waltham, Massachusetts). Dissection was performed at +4°C to collect the entire retina, including the RPE. Whole-mount retinas were spread flat on a polycarbonate membrane (140652, Thermo Fisher Scientific, Waltham, Massachusetts). The test components were diluted to 10 μM with NBAg and 1 / 50 B27 supplement (17504044, Thermo Fisher Scientific, Waltham, MA, USA) and replaced twice weekly for 30 days (until P45 was reached). The explants were then fixed with 4% PFA (15714, Electron Microscopy Sciences, Hatfield, PA) for 1 hour. Finally, PNA 488 immunolabeling (L21409, Thermo Fisher Scientific, Waltham, MA) was performed, and the explants were mounted on Cellvis plates (P12-1.5HN, IBL, Geräsdorf, Austria) using an imaging mounting medium.

[0175] Intraperitoneal injection in rd1 mice Rd1 mice were intraperitoneally injected daily from P15 to P45 with either 2 mg / kg GW 5074 (HY-10542, MedChemExpress, Monmouth, USA) diluted in 40% PEG300 (HY-Y0873, MedChemExpress, Monmouth, USA) or 5% Tween80 (HY-Y1891, MedChemExpress, Monmouth, USA) in PBS (14190144, Thermo Fisher Scientific, Waltham, MA, USA), or vehicle only (control group). Eyeballs were harvested at P45 and fixed with 4% PFA (15714, Electron Microscopy Sciences, Hatfield, PA) for 1 hour. Retinas were harvested and immunostained with PNA 488 (L21409, Thermo Fisher Scientific, Waltham, MA). Images of the retina of rd1 mice were obtained at 10x magnification using a CQ1(registered trademark) confocal microscope (Yokogawa Electric Corporation, Tokyo, Japan).

[0176] Imaging and Statistics Using Cell PathFinder analysis software, the number of PNA-labeled objects was quantified in the hand-drawn central region of retinal explants in ex vivo experiments, and in whole-mount retina in in vivo experiments. The number of detected objects (=cones) was quantified across the entire region, and then mm 2 I reported it as correct.

[0177] The difference in cone density between retinal explants and injected mice was evaluated using the Mann-Whitney U test.

[0178] result Various ferroptosis inhibitors were screened in isolated porcine cones after initial exposure to 20 nM RSL3 and 3 μM IKE to search for protective components that prevent cone loss in retinal degeneration.

[0179] The inventors then investigated the neuroprotective effects of some of these components in the rd1 mouse (Leveillard et al. (2004) Nat. Genet. 36:755), a well-studied model of retinal degeneration. In in vivo rd1 mice, cone degeneration begins early at P5, followed by loss of overall survival (OS) at P15-P20, with only half of the cell body remaining at P45. Retinas were harvested from rd1 mice at P15, and retinal explants were exposed to ferroptosis inhibitors during culture up to P45. Several treatments showed cone protection (Figures 12A-E). However, while this model induces rapid degeneration, the inventors may have identified molecules that could rescue cone photoreceptor degeneration in more slowly degenerative diseases such as AMD.

[0180] Thanks to automated cone counting, the inventors identified five antioxidant molecules with significant neuroprotective effects: 1,3-dicaffeoylquinic acid (n=3, p=0.0055), HTHQ (1-O-hexyl-2,3,5-trimethylhydroquinone, n=3, p=0.0176), rosmarinic acid (n=4, p=0.0205), novelgenin (n=2, p=0.0256), and tinoridine hydrochloride (n=3, p=0.0055). On the other hand, the inventors identified five molecules that inhibit the ferroptosis process while also having significant neuroprotective effects: Two of these molecules act directly on 5-LOX (SRS16-86 (n=3, p=0.0001) and caffeic acid (n=3, p=0.0055)), and two of these play a role in iron metabolism (mangiferin (n=5, p=0.0005) and astilbin (n=3, p=0.0055)). The last molecule involved in specific ferroptosis inhibition (via the Xc pathway) showed a 138.92% neuroprotective effect against pure cone photoreceptors and was also proven effective against retinal explants, hence this molecule GW In vivo injections using 5074 were performed (n=6, p=0.0001). This molecule was administered intraperitoneally at a dose of 2 mg / kg daily from P15 to P45. Immunolabeling of the retina collected at P45 showed that rd1 mice injected with GW 5074 showed +81.48% cone rescue (1597 vs. 880 cones / mm²) compared to a control group injected with vehicle alone daily. 2 (n=5, p=0.0079) (Figure 12J).

[0181] These findings demonstrate that ferroptosis inhibitors can reverse cone degeneration in animal models of retinal degeneration, both ex vivo and in vivo.

[0182] Example 4 Ferroptosis inhibitors do not rescue rods in RD1 mice. material and method Similar to the cones, rods were evaluated in rd1 mouse retinal explants after exposure to ferroptosis inhibitors at P15-P45 during culture, as described in Example 3 above. The inventors also performed a similar evaluation after intraperitoneal injection of GW 5074 as described in Example 3 above.

[0183] The retina was labeled with Rho, imaged using CQ1, and rods were counted using Cell Pathfinder.

[0184] Cone cells were plotted using "magnification change," but the results for rods were not sufficient to account for the change from baseline in each retina (the amount of change sometimes exceeded the total amount of rods), so density (mm 2 The data was plotted using rods (percentage).

[0185] result There were no significant differences between the control group and the ferroptosis inhibitor group, either in retinal explants or in vivo (Figure 13).

[0186] Example 5 Ferroptosis inducers do not alter rods in non-human primates. material and method Similar to cone labeling in NHP injected with 20 μM IKE into the subretinal space, rods were labeled with rhodopsin antibody.

[0187] result No significant changes were observed in the rod outer segment level in the retina injected with 20 μM IKE compared to the control group (Figure 14).

[0188] References JPEG2026524899000004.jpg241170JPEG2026524899000005.jpg239170JPEG2026524899 000006.jpg234170JPEG2026524899000007.jpg243170JPEG2026524899000008.jpg49170

Claims

1. A method for creating a cone degeneration model, comprising the step of activating specific cell death in at least one cone photoreceptor.

2. The method according to claim 1, wherein activation of cell death results in inhibition / activation of the Xc- system, inhibition of GPX-4, lipid peroxidation, and / or modulation of iron metabolism.

3. The activation of cell death - Reduction in the survival rate of at least one cone photoreceptor, - Increased cell membrane permeability to the pigment, - Reduction of intracellular glutathione levels in at least one cone photoreceptor; - Increased intracellular NADP / NADPH ratio in at least one cone photoreceptor; - Increased intracellular formation of lipid peroxides, particularly 5-oxo-6,8,11,14-eicosatetraenoic acid (5oxoETE), in at least one cone photoreceptor; Increased gene expression of AIFM2 (apoptosis-inducing factor 2), SLC3A2 (solute transporter family 3 member 2), HSBP1 (heat shock protein family B (Small) member 1), and Keap1 (Kelch-like ECH-related protein 1); - Increased protein expression of ferritin and / or nuclear receptor coactivator 4 (NCOA4) The method according to claim 1 or 2, which provides one or more of the following.

4. The method according to any one of claims 1 to 3, wherein the activation of cell death comprises contacting at least one cone photoreceptor with at least one cell death inducer.

5. At least one cell death inducer is glutamic acid, RSL3, elastin, elastin derivatives (preferably MEII, PE, or AE), imidazole ketone elastin (IKE), sulfasalazine, sorafenib, sorafenib analogs (SRS13-45 and SRS13-60), altretamine, artesunate, artemisinin, dihydroartemisinin, artemether, artemisinin derivatives, ML-162 (DPI7), ML-210 (DPI10), RSL5, 4, 4 The method according to claim 4, wherein the compound is selected from the group consisting of '-diisothiocyanostilbene-2,2'-disulfonic acid, temozolomide, MMRi62, tert-butylhydroperoxide, FIN56, butionine sulfoximine, NDP4928, statin, brequinal, withaferin A (WA), auranofin, almitrin, lamperizone, HG106, DPI2, DPI12, DPI13, DPI17, DPI18, DPI19, 31MEW44, and ML160.

6. The method according to any one of claims 1 to 5, comprising a preliminary step of obtaining retinal tissue comprising photoreceptors, particularly cone photoreceptors, rod photoreceptors, or their precursor cells, from an eye or from stem cells.

7. The method according to claim 6, wherein the retinal tissue is retinal tissue derived from a vertebrate, including both human and non-human animal tissue, preferably non-human mammalian, and more preferably non-human primate tissue.

8. The method according to claim 7, further comprising a further pre-step of enzymatically and / or mechanically and / or chemically dissociating retinal tissue to obtain a population of isolated retinal cells.

9. The method according to claim 8, further comprising a further pre-step of incubating a population of isolated retinal cells with peanut agglutinin (PNA) to recover PNA-bound cone photoreceptors.

10. The method according to claim 6, wherein the retinal tissue is a retinal explant or a retinal organoid maintained in culture.

11. The method according to claim 10, further comprising the step of bringing retinal tissue into contact with a polycarbonate film such that the photoreceptors face away from the film.

12. The method according to any one of claims 6 to 11, wherein contact of at least one cone photoreceptor with at least one cell death inducer results in cone degeneration or microglia migration to the outer granular layer of retinal tissue.

13. The method according to claim 6, wherein the retinal tissue is contained in a complete eye.

14. The method according to claim 13, wherein contacting at least one cone photoreceptor with at least one cell death inducer comprises administering the at least one cell death inducer to the subretinal space, suprachoroidal space, anterior chamber, vitreous humor, subconjunctival space, or corneal surface, or administering it systemically (by inhaler, intravenously, intramuscularly, or intraperitoneally), or administering it as a solution, gel, or implant.

15. Contacting at least one cone photoreceptor with at least one cell death inducer is • Proliferation, activation, and / or migration of microglial cells; Loss of the extrapyramidal segment; - Formation of subretinal deposits, preferably in the fovea and / or macula; - A decrease in cone activity in the retina, preferably cone activity as measured by ERG or mfERG; and - Thinning of retinal thickness as shown in tissue sections or in vivo imaging by OCT The method according to any one of claims 13 or 14, which results in one or more of the following.

16. A model of cone dystrophy or macular degeneration that can be obtained by the method described in any one of claims 1 to 15.

17. A screening method for compounds that may exhibit protective and / or antidenaturation properties for cone photoreceptors, To provide a model of cone dystrophy or macular degeneration according to claim 16, Contacting a model of cone dystrophy or macular degeneration with the candidate compound, Evaluate whether the candidate compound induces protective and / or antidenaturing effects on cone photoreceptors. A method that includes the following:

18. The candidate compound affects the cone photoreceptor, - To maintain or increase the survival rate of at least one cone photoreceptor; - To maintain or increase intracellular glutathione levels in at least one cone photoreceptor; - To maintain or decrease the intracellular NADP / NADPH ratio in at least one cone photoreceptor; - To maintain or reduce intracellular formation of lipid peroxides, particularly 5-oxo-6,8,11,14-eicosatetraenoic acid (5oxoETE), in at least one cone photoreceptor; - To maintain or increase the gene expression of AIFM2 (apoptosis-inducing factor 2), SLC3A2 (solute transporter family 3 member 2), HSBP1 (heat shock protein family B (Small) member 1), and Keap1 (Kelch-like ECH-related protein 1); - To maintain or decrease the protein expression of ferritin and / or nuclear receptor coactivator 4 (NCOA4); - To prevent increased cell membrane permeability to the pigment; - To prevent the proliferation, activation, or migration of microglial cells; - To prevent the loss of the pyramidal extrasegments; - To prevent a decrease in pyramidal activity, preferably as measured by ERG or mfERG; and - Prevent thinning of retinal thickness as indicated by tissue sections or in vivo imaging by OCT. The screening method according to claim 17, comprising at least one of the following to induce a protective and / or antidenaturing effect.

Images