Methods and compositions for treating age-related macular degeneration

Abstract

Aspects of the present disclosure relate to methods and compositions for treating certain ocular diseases and disorders, such as age-related macular degeneration (AMD). In some embodiments, the method comprises administering to a subject with AMD one or more therapeutic agents that regulate the mTORC1 pathway (or components of that pathway). The present disclosure is based in part on a method for treating AMD in a subject by administering one or more kinase inhibitors, for example, one or more serine / threonine kinase inhibitors. In some embodiments, at least one of the serine / threonine kinase inhibitors is a ribosomal protein S6 kinase beta-1 (S6K1) inhibitor.

Description

[Technical Field]

[0001] The present invention relates to methods and compositions for treating age-related macular degeneration. [Background technology]

[0002] Age-related macular degeneration (AMD) is the leading cause of blindness among elderly people in the industrialized world. The disease typically begins with the formation of lipoprotein-rich deposits called "drusen," which form between Bruch's membrane (BrM) and the retinal pigment epithelium (RPE) or between the RPE and photoreceptor (PR) outer segments. Twenty percent of individuals with drusen progress to advanced forms of the disease characterized by geographic atrophy (GA) of the RPE and inferior PR or by neovascular pathology. The only treatment available today is for neovascular pathology (also called "wet AMD"), which uses antiangiogenic antibodies to inhibit the action of vascular endothelial growth factor (VEGF). No treatments exist to prevent progression from early disease stages to advanced stages. There are also no treatments available for advanced forms of GA (often called "dry AMD"). Summary of the Invention [Problem to be solved by the invention]

[0003] Aspects of the present disclosure relate to methods and compositions for treating certain ocular diseases and disorders, such as age-related macular degeneration (AMD). In some embodiments, the method comprises administering to a subject with AMD one or more therapeutic agents that modulate the mTORC1 pathway (or components of that pathway). [Means for solving the problem]

[0004] The present disclosure is based in part on a method for treating AMD in a subject by administering one or more kinase inhibitors, for example, one or more serine / threonine kinase inhibitors. In some embodiments, at least one of the serine / threonine kinase inhibitors is a mammalian target of rapamycin complex 1 (mTORC1) inhibitor and / or a ribosomal protein S6 kinase beta-1 (S6K1) inhibitor.

[0005] Accordingly, in some aspects, the present disclosure relates to a method of inhibiting drusen formation in ocular tissue, the method comprising administering to cells of the ocular tissue one or more inhibitors of mammalian target of rapamycin complex 1 (mTORC1).

[0006] In some aspects, the present disclosure provides a method for treating age-related macular degeneration (AMD) in a subject, the method comprising administering to the subject one or more inhibitors of mTORC1.

[0007] In some aspects, the present disclosure provides a method for inhibiting drusen formation in ocular tissue, the method comprising administering to cells of the ocular tissue one or more inhibitors of ribosomal protein S6 kinase beta-1 (S6K1).

[0008] In some aspects, the present disclosure provides a method for treating age-related macular degeneration (AMD) in a subject, the method comprising administering to the subject one or more inhibitors of ribosomal protein S6 kinase beta-1 (S6K1).

[0009] In some embodiments, the ocular tissue comprises Bruch's membrane tissue, retinal pigment epithelium (RPE) tissue, macular tissue, or a combination thereof, hi some embodiments, the ocular tissue comprises photoreceptor cells, retinal pigment epithelium cells (RPE), ganglion cells, or a combination thereof.

[0010] In some embodiments, the administration comprises topical administration, intravitreal administration, subconjunctival injection, intrachoroidal injection, systemic injection, or any combination thereof. In some embodiments, the administration reduces drusen formation in ocular tissue by about 2-fold, 3-fold, 5-fold, 10-fold, 50-fold, 100-fold, or more than 100-fold compared to ocular tissue that has not been administered one or more S6K1 inhibitors. In some embodiments, the method further comprises administering an effective amount of di-docosahexaenoic acid (DHA) to the subject. In some embodiments, the DHA is administered as a dietary supplement.

[0011] In some embodiments, the at least one S6K1 inhibitor is a small molecule, peptide, protein, antibody, or inhibitory nucleic acid. In some embodiments, the inhibitory nucleic acid is a dsRNA, siRNA, shRNA, miRNA, ami-RNA, antisense oligonucleotide (ASO), or aptamer. In some embodiments, the inhibitory nucleic acid reduces or prevents the expression of S6K1 protein. In some embodiments, the inhibitory nucleic acid binds to the nucleic acid encoding S6K1 protein.

[0012] In some embodiments, the protein is a dominant negative S6K1 protein. In some embodiments, the small molecule is PF-4708671, rosmarinic acid methyl ester (RAME), A77 1726, or a salt, solvate, or analog thereof. In some embodiments, the small molecule is a selective inhibitor of S6K1. In some embodiments, the S6K1 inhibitor does not bind to or inhibit the expression or activity of mammalian target of rapamycin 1 (mTORC1).

[0013] In some embodiments, the ocular tissue is in vivo, and optionally the ocular tissue is present in the eye of a subject. [Brief explanation of the drawings]

[0014] [Figure 1]Figure 1 shows the distribution of pathology in mice with loss of TSC1 in rods and two normal copies of S6K1 (rods TSC1- / -S6K1+ / +), mice with loss of TSC1 and loss of S6K1 in rods (rods TSC1- / -S6K1- / -), mice with loss of TSC1 and loss of one copy of S6K1 in rods (rods TSC1- / -S6K1- / +), and mice with two normal copies of TSC1 and loss of S6K1 (rods TSC1+ / +S6K1- / -). Loss of S6K1 in the setting of loss of TSC1 in rods prevents advanced pathology. [Figure 2] Fundus images and retinal pigment epithelium flat preparations. This figure shows that mice with one copy of S6K1 and loss of TSC1 (rod TSC1- / -S6K1- / +) develop fundus pathology (left) and GA (as seen in the flat preparation). In contrast, no pathology was observed in mice with loss of both TSC1 and S6K1 (rod TSC1- / -S6K1- / -). [Figure 3] Diagram showing that in the presence of TSC1 loss, S6K1 deficiency prevents the accumulation of ApoE and complement factor H (CHF), both hallmarks of early disease stage AMD. [Figure 4A] Figure 4A shows that RPE digestion of POS is altered in rod Tsc1- / - mice. Figure 4A shows the relative percentages of di-DHA PE(44:12) and PC(44:12) lipids from whole retinal extracts of the indicated genotypes at 2 months. Bars represent mean ± SEM. (n = 6-9 mice, 2 retinas per mouse; ****p<0.0001). [Figure 4B] Figure 4B shows that RPE digestion of POS is variable in rod Tsc1 − / − mice. Figure 4B shows a similar view to Figure 4A with purified POS pooled from six retinas per genotype. [Figure 4C]Figure 4C shows that RPE digestion of POS is altered in rod Tsc1- / - mice. Figure 4C shows POS clearance, expressed as the percentage of dots remaining 3 hours after peak shedding (ratio of 11 am to 8 am) in 2-month-old mice fed a DHA diet or a control diet from weaning to 2 months of age. Mean ± SEM is shown. (n = 6 RPE flat preparations; **p < 0.05; **p < 0.01). [Figure 4D] Figure 4D shows that RPE digestion of POS is altered in rod Tsc1- / - mice. Figure 4C shows a similar image from a 6-month-old mouse fed the DHA diet for only 2 weeks. Mean values ± SEM are shown. (n=6 RPE flat preparations; **p<0.01; ****p<0.0001). [Figure 4E] Figure 4D shows that RPE digestion of POS is altered in rod Tsc1- / - mice. Figure 4E shows RPE polynucleation analysis (left) and hypertrophy analysis (right) of rod Tsc1- / - mice fed a DHA diet or a control diet from weaning to 6 months of age. Bars represent mean ± SEM. (n=6 mice RPE flat preparations; *p<0.05; **p<0.01). [Figure 4F] Figure 4F shows that RPE digestion of POS is altered in rod Tsc1 − / − mice. Figure 4F shows representative fundus images of rod Tsc1 − / − mice fed a control diet (top row) or a DHA diet (bottom row) from weaning until the time indicated in the panel (M: months). [Figure 4G]Figure 4G shows that RPE digestion of POS is variable in rod Tsc1 − / − mice. Figure 4G shows AMD-associated markers on retinal sections from rod Tsc1 − / − mice fed a DHA diet or a control diet from weaning to 6 months of age. Higher magnification images of the area between the arrowheads are shown in the upper row of each panel. (Nuclei stained with DAPI; cone sheets were marked with peanut agglutinin lectin (PNA); magenta: ZO1 (marking the RPE border for ApoE and C3 panels) and phalloidin (marking the border for ApoB and CFH panels). Scale bar = 20 μm. (GCL: ganglion cell layer; RPE: retinal pigment epithelium). Images are representative of three independent experiments performed with three different animals per genotype. [Figure 4H] Figure 4H shows that RPE digestion of POS is altered in rod Tsc1- / - mice. Figure 4H shows a similar experiment to Figure 4A after mice were fed a DHA diet for 10 weeks after weaning. Bars represent mean ± SEM. (n = 3 mice, 2 retinas per mouse; *p<0.05; **p<0.01; ****p<0.0001). [Figure 5A] Figure 5A shows increased expression of PKM2 and HK2 in the PR of AMD patients. Figure 5A shows immunohistochemistry (IHC) results demonstrating increased expression of PKM2 and HK2 (purple) in retinal cross-sections. Increased expression is observed throughout the PR layer of AMD patients, particularly in the cone inner segment (arrow) and cone pedicle (arrowhead). The dotted line demarcates the boundary of a portion of the cone inner segment in a non-diseased individual. The immunohistochemistry enzyme reaction time was 6 minutes, except for the second panel from a non-diseased individual using PKM2 antibody (30 minutes; all non-diseased sections in panel A were derived from the same retina). Scale bar: 45 μm. OS: outer segment; IS: inner segment; ONL: outer nuclear layer; INL: inner nuclear layer; IPL: inner plexiform layer; GCL: ganglion cell layer. [Figure 5B]Figure 5B shows that PKM2 and HK2 expression is increased in PR in AMD patients. Figure 5B shows immunofluorescence for p-S6 (red; blue nuclei DAPI). Scale bar: 50 μm. OS: outer segment; IS: inner segment; ONL: outer nuclear layer; INL: inner nuclear layer; IPL: inner plexiform layer; GCL: ganglion cell layer. [Figure 5C] Figure 5C shows that PKM2 and HK2 expression is increased in patients with AMD. Figure 5C shows quantification of Western blots for p-S6 and PKM2 performed on retinas from 2-month-old mice (n=3) with the indicated genotypes. The top row shows representative Western images for each protein plus actin control. Results are expressed as mean ± SEM. (**P<0.01, ****P<0.0001). [Figure 5D] Figure 5D shows that PKM2 and HK2 expression is increased in patients with AMD. Figure 5D shows measurements of retinal lactate levels at 2 months for the indicated genotypes (n=4 for lactate). Results are expressed as mean ± SEM. (*P<0.05, **P<0.01). [Figure 5E] Figure 5B shows that PKM2 and HK2 expression is increased in patients with AMD. Figure 5C shows measurements of NADPH levels at 2 months for the indicated genotypes (n=8 for NADPH). Results are expressed as mean ± SEM. (*P<0.05, **P<0.01). [Figure 6A] Figure 6A shows that aged rod Tsc1 − / − mice develop GA and neovascular pathology. Figure 6A shows representative fundus images of littermate control (top row) and rod Tsc1 − / − mice (bottom row) at the indicated ages. [Figure 6B]Figure 6B shows that aged rod Tsc1 − / − mice develop GA and neovascular pathology. Figure 6B shows representative fundus fluorescein angiography (FFA; bottom row) images at 18 months of age for the indicated genotypes, along with the corresponding fundus images (top row). Rod Tsc1 + / + mice occasionally show some microglial accumulation, while all rod Tsc1 + / − mice show microglial accumulation (arrowheads). Rod Tsc1 − / − mice develop retinal folds (arrows), GA (as shown), and neovascular pathology (dotted line). [Figure 6C] Figure 6C shows that aged rod Tsc1 − / − mice develop GA and neovascular pathology. Figure 6C shows the percentage distribution of the phenotypes described in (Figure 6B) in rod Tsc1 − / − mice at the indicated ages. The last two bars show control mice with only microglial accumulation. Bars represent percentage ± MOE. Numbers in parentheses indicate the number of mice analyzed (M: months). [Figure 7A] Figure 7A shows histological analysis of advanced AMD-like pathology. Figure 7A shows RPE and corresponding retinal flat preparations from the same eye, with the corresponding areas with autofluorescent RPE cells and retinal folds marked with letters (b) and areas of GA and corresponding PR atrophy marked with letters (c). The RPE whole preparation is shown in the left half of the panel, and the corresponding retina is shown in the right half. Scale bar = 300 μm. Colors in (A–C) are indicated by labels within the panels. Color annotations for panel (A) are shown in the first two images in panel (B) (blue: nuclear DAPI; green: autofluorescence (AF) or cone sheets marked by peanut agglutinin lectin (PNA); red: RPE border marked by ZO1, cones marked by cone arrestin (CA), or microglia marked by Iba-1). [Figure 7B]Figure 7B shows a histological analysis of advanced AMD-like pathology. Figure 7B shows a higher magnification image of the area in panel (A) marked with a letter (b), depicting autofluorescent RPE cells (arrowheads, left panel) corresponding to retinal folds (arrowheads, center panel). The right panel shows a higher magnification image of a fold (different eye) stained with Iba-1 (red) to mark microglia. Scale bar = 50 μm. Colors in (A–C) are as indicated by the labels in the panels. Color annotations for panel (A) are shown in the first two images in panel (B) (blue: nuclear DAPI; green: autofluorescence (AF) or cone sheets marked with peanut agglutinin lectin (PNA); red: RPE border marked with ZO1, cones marked with cone arrestin (CA), or microglia marked with Iba-1). [Figure 7C] Figure 7C shows a histological analysis of advanced AMD-like pathology. Figure 7C shows a higher magnification image of the area of GA marked with letter (c) in panel (A), depicting the loss of RPE cells (left panel) and retinal PR (right panel; PR side facing up indicates decreased nuclear DAPI density) in grayscale. Note that no folds are visible in the area of GA (letter c) in panel A, indicating that folds are not required for GA formation. Scale bar = 50 μm. Colors in (A–C) are as indicated by the labels in the panels. Color annotations for panel (A) are shown in the first two images in panel (B) (blue: nuclear DAPI; green: autofluorescence (AF) or cone sheets marked by peanut agglutinin lectin (PNA); red: RPE border marked by ZO1, cones marked by cone arrestin (CA), or microglia marked by Iba-1). [Figure 7D] Figure 7D shows a histological analysis of advanced AMD-like pathology. Figure 7D shows a semi-thin section through intermediate stages of GA, revealing that RPE atrophy is still present along with PR. Ruptures are absent in this area of RPE atrophy. [Figure 7E]Figure 7E shows histological analysis of advanced AMD-like pathology. Sequential OCT images (same eye as shown in Figures 6A-6B, 18 months old with GA) are shown through the area of GA identified by fundus imaging, revealing disruption of the outer nuclear layer (ONL; between the dotted lines). [Figure 7F] Figure 7F shows a histological analysis of advanced AMD-like pathology. Figure 7F shows a semithin section of an eye with GA shown in (Figure 7E), demonstrating multilayered RPE (white asterisk), migration of RPE into the retina proper (arrows), RPE atrophy (between arrowheads), and retinal neovascularization (red arrow). When PR dies, retinal folds flatten where they overlap with areas of GA. Reminiscence of retinal folds is indicated by dotted lines. Scale bar = 20 μm. [Figure 7G] Figure 7G shows histological analysis of advanced AMD-like pathology. Figure 7G shows RPE multinucleation and hypertrophy analysis. The top panel shows representative RPE images with cell boundaries marked by ZO1 (red signal) and used for quantitative analysis using output from IMARIS software to identify cell shape, size, and nuclei (blue signal: nuclear DAPI). The bottom panel shows quantification of RPE multinucleation (left) and RPE cell size distribution (right). Bars represent mean ± SEM. (n = 4 RPE flat specimens; *p < 0.05;). Scale bar = 10 μm. [Figure 8A] Figure 8A shows that AMD-like pathology is dose-dependent on activated mTORC1. Figure 8A shows representative fundus images of rod Tsc1+ / + rod Raptor+ / + (top panel), rod Tsc1- / - rod Raptor+ / - (middle panel), and rod Tsc1- / - rod Raptor- / - (bottom panel) littermate mice at the indicated ages. Fundus images of rod Tsc1- / - mice are shown in Figure 2 and Figure 12. (M: months). [Figure 8B]Figure 8B shows that AMD-like pathology is dose-dependent on activated mTORC1. Figure 8B shows the distribution (%) of retinal pathology scored for mice (12-18 months old) with the indicated genotypes. Rod Tsc1+ / + is shown in Figure 6C. Graphs represent percentages ± MOE. Numbers in parentheses indicate the number of mice analyzed. [Figure 8C] Figure 8A shows that AMD-like pathology is dependent on the dose of activated mTORC1. Figure 8C shows RPE multinucleation and RPE hypertrophy analysis at 12 months for the indicated genotypes. Bars represent mean ± SEM. (n = 4 mice). [Figure 8D] Figure 8D shows that AMD-like pathology is dose-dependent of activated mTORC1. Quantification of retinal PKM2 (white) and p-S6 (gray) levels by Western blot in 2-month-old mice with the indicated genotypes is shown. Bars represent mean ± SEM. (n = 3 mice). [Figure 8E] Figure 8A shows that AMD-like pathology is dependent on the dose of activated mTORC1. Figure 8E shows retinal lactate levels at 2 months of age for the indicated genotypes. Bars represent mean ± SEM (n=4 for lactate). [Figure 8F] Figure 8A shows that AMD-like pathology is dependent on the dose of activated mTORC1. Figure 8F shows NADPH levels at 2 months of age for the indicated genotypes. Bars represent mean ± SEM (n=7 for NADPH). [Figure 8G] Figure 8G shows that AMD-like pathology depends on the dose of activated mTORC1. Figure 8G shows immunofluorescence (green signal) for ApoB, ApoE, C3, and CFH on retinal sections from 12-month-old mice with the indicated genotypes. Higher magnification images of the areas between the arrowheads are shown in the upper row of each panel. (Blue: nuclear DAPI; red: peanut agglutinin lectin (detects cone segments); magenta: ZO1 (visualizes the RPE in the ApoE and C3 panels) or phalloidin (visualizes the RPE in the ApoB and CFH panels). Images are representative of three independent experiments using three different animals. Scale bar = 20 μm. [Figure 9A] Figure 9 shows that RPE digestion of POS is altered in rod Tsc1- / - mice. Figure 9A shows representative immunofluorescence images of RPE whole preparations obtained from 2-month-old rod Tsc1- / - mice at the indicated dates, demonstrating delayed POS clearance by RPE cells (bottom row) compared to control mice (top row). POS is shown by green staining for rhodopsin, while RPE cell boundaries are shown by red staining for ZO1 expression. Scale bar = 10 μm. [Figure 9B] Figure 9B shows that RPE digestion of POS is altered in rod Tsc1- / - mice. Figure 9B shows quantification of the number of Rho-positive dots per RPE cell (obtained from immunofluorescence images shown in Figure 9A) from 2-month-old mice with the indicated genotypes over a one-day time course. Bars represent mean ± SEM (n = 6-8 RPE flat specimens; **p < 0.01; ****p < 0.0001). [Figure 9C] Figure 9C shows that RPE digestion of POS is altered in rod Tsc1- / - mice. Figure 9C shows delayed POS clearance, expressed as the percentage of dots remaining 3 hours after peak outflow (ratio of 11 am to 8 am) in 2-month-old mice with the indicated genotypes. Bars represent mean ± SEM. (n = 6-8 RPE flat preparations; ***p < 0.001; ****p < 0.0001). [Figure 9D] Figure 9D shows that RPE digestion of POS is altered in rod Tsc1- / - mice. Figure 9D shows the relative percentages of di-DHA PE(44:12) and PC(44:12) lipids from whole retinal extracts of the indicated genotypes at 2 months. Bars represent mean ± SEM. (n = 6-9 mice, 2 retinas per mouse; ****p<0.0001). [Figure 9E] Figure 9E shows that RPE digestion of POS is variable in rod Tsc1- / - mice. Figure 9E shows a similar view to Figure 9D with purified POS pooled from six retinas per genotype. [Figure 9F] Figure 9F shows that RPE digestion of POS is altered in rod Tsc1- / - mice. POS clearance, expressed as the percentage of dots remaining 3 hours after peak outflow (ratio of 11 am to 8 am) in 2-month-old mice fed a DHA diet or a control diet from weaning to 2 months of age, is shown. Mean ± SEM (n = 6 RPE flat preparations; **p < 0.05; **p < 0.01). [Figure 9G] Figure 9G shows that RPE digestion of POS is altered in rod Tsc1- / - mice. Figure 9G shows a similar image to Figure 9F from 6-month-old mice fed the DHA diet for only 2 weeks. Mean values ± SEM are shown. (n=6 RPE flats; **p<0.01; ****p<0.0001). [Figure 9H] Figure 9H shows that RPE digestion of POS is altered in rod Tsc1- / - mice. Figure 9H shows RPE multinucleation (left) and hypertrophy (right) analysis of rod Tsc1- / - mice fed a DHA diet or a control diet from weaning to 6 months of age. Bars represent mean ± SEM. (n = 6 mouse RPE flat specimens; *p < 0.05; **p < 0.01). [Figure 9I] Figure 9I shows that RPE digestion of POS is altered in rod Tsc1- / - mice. Figure 9I shows representative fundus images of rod Tsc1- / - mice fed a control diet (top row) or a DHA diet (bottom row) from weaning until the time indicated in the panel (months). [Figure 9J]Figure 9J shows that RPE digestion of POS is variable in rod Tsc1- / - mice. Figure 9J shows AMD-associated markers on retinal sections from rod Tsc1- / - mice fed a DHA or control diet from weaning to 6 months of age. Proteins of interest are shown in green in the top row. Higher magnification images of the area between the arrowheads are shown in the top row of each panel. (Blue: nuclear DAPI; red: cone sheets marked with peanut agglutinin lectin (PNA); magenta: ZO1 (marking the RPE border for ApoE and C3 panels) and phalloidin (marking the border for ApoB and CFH panels)). Scale bar = 20 μm. (GCL: ganglion cell layer; RPE: retinal pigment epithelium). Images are representative of three independent experiments performed with three different animals per genotype. [Figure 9K] Figure 9K shows that RPE digestion of POS is altered in rod Tsc1- / - mice. Figure 9K shows a similar experiment to Figure 9D after mice were fed a DHA diet for 10 weeks after weaning. Bars represent mean ± SEM. (n = 3 mice, 2 retinas per mouse; *p<0.05; **p<0.01; ****p<0.0001). [Figure 10A] Figure 10A shows the distinct contribution of cones to disease compared with rods. Figure 10A shows representative fundus images at 12 months (top row) and the distribution (%) of pathology observed over time for the indicated genotypes (bottom graphs: microglial accumulation (top left); retinal folds (top right); GA (bottom left); and neovascularization (bottom right)). Graphs represent percentages ± MOE. (n = 10-15 mice). [Figure 10B]Figure 10B shows that the contribution of cones to disease differs from that of rods. Figure 10B shows a 12-month-old Tsc1- / - cone mouse, demonstrating PR atrophy in a retinal flat preparation with retinal microglia migrating to the injury site (left panel) and choroidal neovascularization in a corresponding RPE flat preparation of the same area (right panel). The eye corresponds to the fundus of the Tsc1- / - cone mouse shown in Figure 10A. Scale bar = 50 μm. Colors are as indicated by the labels in the panels (blue: nuclear DAPI; green: cone sheets marked by peanut agglutinin lectin (PNA) or blood vessels marked by lectin B4 (lectin B4)); red: microglia marked by Iba1 or RPE border marked by ZO1). [Figure 10C] Figure 10C shows the distinct contribution of cones to disease from rods. Figure 10C shows a semithin section image of a cone Tsc1- / - mouse at 12 months, revealing large drusen-like deposits (see inset). Bottom: EM image of the deposits and a higher magnification image of the boxed area in the EM image (right). The BrM is marked by a double arrow. The arrowhead points to the RPE basal folds, and the arrowhead points to translucent lipid vesicles. [Figure 10D] Figure 10D shows that the contribution of cones to disease is distinct from that of rods. Large drusen-like deposits (indicated by the letter (D)) in a rod and cone Tsc1- / - mouse at 12 months indicate ApoE accumulation (red signal). A bright-field magnification of the area between the arrowheads in the left image is shown on the right. Scale bar = 20 μm. Colors are as indicated by the labels in the panels (blue: nuclear DAPI; green: cone sheets marked with peanut agglutinin lectin (PNA); red: ApoE-positive deposits). [Figure 10E]Figure 10E shows the distinct contribution of cones to disease from rods. Figure 10E shows EM images of a 12-month-old Tsc1- / - cone mouse, revealing the basal eminence (asterisk: larger eminence; arrowhead: fine eminence), lipoprotein vesicles (arrows) in the BrM, dysmorphic mitochondria (M), and membranous optic disc (MD). Right: Enlarged area (arrow) of the basal eminence (representing lipoprotein vesicles in the BrM) marked with an asterisk in the left image. [Figure 10F] Figure 10F shows the contribution of cones to disease distinct from that of rods. Figure 10F shows the enlarged GA area in a rod & cone Tsc1- / - mouse at 12 months, revealing TUNEL-positive RPE cells. The left panel shows a whole RPE preparation, while the right panel shows a higher magnification image of the GA area surrounded by dysmorphic RPE cells and TUNEL-positive nuclei (arrowheads). The inset shows a higher magnification image of the TUNEL-positive nuclei. Scale bars = 300 μm (left panel) and 15 μm (right panel). Colors are as indicated by the labels in the panels (blue: nuclear DAPI; green: autofluorescence (AF) (left panel) and apoptosis marked by TUNEL (right panel); red: RPE border marked by phalloidin). [Figure 11A]Figure 11 shows that both PKM2 and HK2 expression are increased in the PR of AMD patients. Immunofluorescence (green signal) for PKM2 (Figure 11A) and HK2 (Figure 11B) expression in the PR of non-diseased human donor eyes (top row) and AMD donor eyes (bottom row). The first two columns are the donor retinas shown in Figure 5. The first column shows images with identical signal intensity between non-diseased and diseased tissue. Images in columns 2–4 show scaled signals. To better visualize the signal in the PR, PKM2 levels were increased 2-fold in the non-diseased tissue, while HK2 levels were scaled 1.5-fold in the non-diseased tissue. In both cases, there was also a slight increase in baseline signal when compared to panels showing identical intensities (compare diseased tissue in column 1 to column 2). Signal was generally stronger in the cone pedicle, cone inner segment, or throughout the outer nuclear layer in eyes from AMD patients compared to non-diseased controls. Panels in the same column in (Figure 11A) and (Figure 11B) are corresponding sections from the same donor retina (blue: nuclear DAPI; red: peanut agglutinin lectin (to visualize cone segments); green: PKM2 or HK2 (as indicated); F: female; M: male; yrs: years; individual ages are indicated in years within the panels). [Figure 11B]Figure 11 shows that both PKM2 and HK2 expression are increased in the PR of AMD patients. Immunofluorescence (green signal) for PKM2 (Figure 11A) and HK2 (Figure 11B) expression in the PR of non-diseased human donor eyes (top row) and AMD donor eyes (bottom row). The first two columns are the donor retinas shown in Figure 5. The first column shows images with identical signal intensity between non-diseased and diseased tissue. Images in columns 2–4 show scaled signals. To better visualize the signal in the PR, PKM2 levels were increased 2-fold in the non-diseased tissue, while HK2 levels were scaled 1.5-fold in the non-diseased tissue. In both cases, there was also a slight increase in baseline signal when compared to panels showing identical intensities (compare diseased tissue in column 1 to column 2). Signal was generally stronger in the cone pedicle, cone inner segment, or throughout the outer nuclear layer in eyes from AMD patients compared to non-diseased controls. Panels in the same column in (Figure 11A) and (Figure 11B) are corresponding sections from the same donor retina (blue: nuclear DAPI; red: peanut agglutinin lectin (to visualize cone segments); green: PKM2 or HK2 (as indicated); F: female; M: male; yrs: years; individual ages are indicated in years within the panels). [Figure 11C] Figure 11C shows that both PKM2 and HK2 expression are increased in the PR of AMD patients. Figure 11D shows immunofluorescence using the same PKM2 antibody in mice of different ages, demonstrating the age-related decrease in PKM2 signal. Right: Western blot and quantification of PKM2 in retinas from 3- and 36-month-old mice, showing that overall levels decline with age. (n=6 retinas) (*p<0.05). [Figure 12]Representative fundus images are shown over time for the same eye. To follow disease progression over time in the same animal, rod Tsc1− / − mice were imaged at the indicated ages. C16 and C26 mice developed GA (dotted line) and neovascular pathology. C180 and C194 developed retinal folds and had microglia migrated into the subretinal space, but did not develop advanced pathology. Rod Tsc1+ / + mice, C24, and C28, show normal fundus over time. Fundus fluorescein angiography images (shown in the right column) are fundus images from the oldest age shown. [Figure 13A] Figure 13A shows a fundus image of a 4-month-old rod Tsc1- / - mouse, showing bright spots representing retinal folds and small white spots representing microglia. [Figure 13B] Figure 13B shows a 4-month-old rod Tsc1- / - mouse. Figure 13B shows an OCT scan of the eye shown in (Figure 13A) along the green arrow in (Figure 13A). Three folds are visible (arrows) on the OCT scan. [Figure 13C] Figure 13C shows a 4-month-old rod Tsc1- / - mouse. Figure 13C shows a zoomed-in image of a retinal flat preparation, revealing folds filled with microglia (the same panel as in Figure 8B). [Figure 13D] Figure 13D shows that retinal folds are often filled with microglia. A 4-month-old rod Tsc1- / - mouse is shown. Figure 13D shows a cross section of a fold, revealing internal microglia migrating from the inner nuclear layer toward the PR layer. (C, D: Blue: nuclear DAPI; Green: peanut agglutinin lectin marking cone sheets; Red: Iba-1 marking microglia). [Figure 14A]Figure 14A shows that loss of Tsc1 in the PR does not result in rapid PR degeneration. Figure 14A shows analysis of ONL thickness at 18 months. Each symbol represents the mean ± SEM (n = 6 retinas) (*p<0.05; **p<0.01; ***p<0.0001). [Figure 14B] Figure 14B shows that loss of Tsc1 in PRs does not result in rapid PR degeneration. Figure 14B shows a time course analysis of PR function, depicting the mean a-wave amplitude of the dark-adapted response. Bars represent the mean ± SEM (n = 5, 5, 6, 4, 9 for Cre- mice and n = 8, 4, 4, 6, 5 for Cre+ mice at 2, 9, 12, 18, and >20 months, respectively) (*p<0.05; **p<0.01). [Figure 14C] Figure 14C shows that loss of Tsc1 in PRs does not result in rapid PR degeneration. Figure 14C shows a time course analysis of PR function, depicting the mean a-wave amplitude of the light-adapted response. Bars represent the mean ± SEM (n = 5, 5, 6, 4, 9 for Cre- mice and n = 8, 4, 4, 6, 5 for Cre+ mice at 2, 9, 12, 18, and >20 months, respectively) (*p<0.05; **p<0.01). [Figure 14D] Figure 14D shows that loss of Tsc1 in PRs does not result in rapid PR degeneration. Analysis of PR function over time is shown in Figure 14D, which depicts mean c-wave ERG amplitudes. Bars represent mean ± SEM (n = 5, 5, 6, 4, 9 for Cre- mice and n = 8, 4, 4, 6, 5 for Cre+ mice at 2, 9, 12, 18, and >20 months, respectively) (*p<0.05; **p<0.01). [Figure 15A]Figure 15A shows that p-S6 expression in RPE cells is independent of CRE activity and increases over time. Figure 15A shows immunofluorescence for p-S6 (red signal) on RPE flat specimens from rod Tsc1 − / − mice at 2 months (top panel) and 15 months (bottom panel). Several p-S6-positive RPE cells (arrowheads) were observed at 2 months. The right side of the bottom panel shows a higher magnification image of pS6-positive RPE cells. Scale bar = 500 μm (top left panel) and 50 μm (bottom right panel). (Green: phalloidin highlighting RPE cell boundaries). [Figure 15B] Figure 15B shows that p-S6 in RPE cells is independent of CRE activity and increases over time. Figure 15B shows a retinal section demonstrating CRE-recombinase staining (red signal) in the photoreceptor layer (left side) but not in p-S6-positive (green signal) RPE cells (arrowheads; see enlarged image on the right). Two different examples are shown. Due to the strong p-S6 signal in the RPE, the signal intensity for p-S6 was reduced in sections that also show the retina. Therefore, p-S6 in the PR appears weaker than normal. Nuclear DAPI (blue signal) and peanut agglutinin lectin (magenta signal) were removed from 50% of the panel to better visualize the red and green signals. Scale bar = 20 μm. The red signal behind the RPE is due to the nature of the anti-CRE antibody, as it is a mouse monoclonal antibody and therefore also highlights endothelial cells. [Figure 15C] Figure 15C shows that p-S6 expression in RPE cells is independent of CRE activity and increases over time. Figure 15D shows quantification of p-S6-positive RPE cells at 2 and 15 months of age for the indicated genotypes. Bars represent mean ± SEM (n = 4 mice). [Figure 16A]Figure 16A shows that rod Tsc1 − / − mice exhibit early features of AMD. Figure 16A shows immunofluorescence for ApoB, ApoE, C3, and CFH on retinal sections from 15-month-old rod Tsc1 − / − mice (green signal). Higher magnification images of the areas between the arrowheads are shown at the top of each panel. (Blue: nuclear DAPI; red: cone sheets marked with peanut agglutinin lectin (PNA); magenta: RPE border marked with ZO-1 (for ApoE and C3 panels) and phalloidin (for ApoB and CFH panels)). Scale bar = 20 μm. Images are representative of three independent experiments in three different animals per genotype. [Figure 16B] Figure 16B shows that rod-shaped Tsc1- / - mice exhibit early features of AMD. Figure 16B shows ultrastructural images demonstrating undigested POS in the BrM, thickened BrM, neutral lipid droplets (L) within the BrM, and basal lamina deposits (BLamD). The enlarged image below shows the area between the arrowheads. [Figure 16C] Figure 16C shows that rod Tsc1- / - mice display early features of AMD. Figure 16D shows a semithin section depicting basal prominences (asterisks) of different sizes (arrows: large basal prominences). A higher magnification image of the area between the arrowheads is shown at the bottom, also depicting microprominences (asterisks). Scale bar = 20 μm. [Figure 16D] Figure 16D shows that rod Tsc1- / - mice display early features of AMD. Figure 16D shows RPE autofluorescence of the indicated genotypes at 15 months. Rod Tsc1- / - mice display greater lipofuscin accumulation (red signal). Autofluorescence was acquired using a Cy3 filter (blue: nuclear DAPI). Scale bar = 20 μm. [Figure 17A]Figure 17A shows the similarities between cone Tsc1 − / − mice, rod Tsc1 − / − mice, and cone and rod Tsc1 − / − mice. Figure 17A shows immunofluorescence (green signals) for ApoB, ApoE, C3, and CFH on retinal sections from 15-month-old cone and rod Tsc1 + / + control mice, cone Tsc1 − / − mice, and cone and rod Tsc1 − / − mice. Higher magnification images of the areas between the arrowheads are shown at the top of each panel. (Blue: nuclear DAPI; red: peanut agglutinin lectin (detects cone segments); magenta: ZO1 (visualizes the RPE in the ApoE and C3 panels) or phalloidin (visualizes the RPE in the ApoB and CFH panels). Images are representative of three independent experiments using three different animals. Scale bar = 20 μm. [Figure 17B] Figure 17A shows the similarities between cone Tsc1- / - mice, rod Tsc1- / - mice, and cone & rod Tsc1- / - mice. Figure 17B shows a summary of ApoB, ApoE, C3, and CFH expression changes observed in different genotypes and DHA feeding experiments at 15 months. Expression levels are indicated by "+" symbols. Levels are arbitrary based on visual analysis of antibody staining performed in three animals per genotype. [Figure 17C] Figure 17C shows the similarities between cone Tsc1- / - mice, rod Tsc1- / - mice, and cone & rod Tsc1- / - mice. Figure 17D shows POS clearance in the indicated genotypes at 2 months. The percentage of remaining dots at 11 am is shown. Loss of Tsc1 in cones also affects the digestion of rod outer segments when assayed with an anti-rhodopsin antibody. Bars represent mean ± SEM. (n = 6 RPE flats). [Figure 17D]Figure 17D shows the similarities between cone Tsc1- / - mice, rod Tsc1- / - mice, and cone and rod Tsc1- / - mice. Figure 17D shows the relative percentages of di-DHA-form PE (44:12) and PC (44:12) lipids from whole retinal extracts of the indicated genotypes at 2 months. Bars represent mean ± SEM. (n = 8 for cone and rod Tsc1+ / +, n = 6 for rod Tsc1- / -, n = 5 for cone Tsc1- / -, n = 3 for cone and rod Tsc1- / -, using two retinas per sample from the same animal.) [Figure 18] Diagram showing a schematic of the two-stage disease progression. In the aging eye, lipoproteins accumulate within the BrM (left side of the image) as part of the normal aging process. In some individuals, the onset of lipoprotein accumulation exceeds normal age-related accumulation, leading to the formation of a lipid barrier at the RPE-BrM interface (stage 1). This stage is accelerated by environmental risk factors, such as smoking, diet, and physical inactivity, as well as genetic risk factors that affect metabolism. Excessive thickening of the lipid barrier reduces glucose transport from the choroidal vasculature to the PR. This triggers a metabolic shift in the PR, initiating the second stage of disease. This leads to increased lipoprotein accumulation, altered expression of complement components, and a decrease in retinal di-DHA PE and PC lipids. Once this disease stage begins, new risk alleles, such as those for the complement and immune systems, are acquired. Ultimately, in some individuals, the pathology progresses to GA or choroidal neovascularization. [Figure 19A] Figure 19 shows that loss of TSC2 in rods (rod Tsc2- / -) resulted in the same overall pathology as observed for loss of TSC1 in rods. Figure 19A shows Western blot images for p-S6 (black bars) and PKM2 (white bars), demonstrating overall increased levels in rod Tsc2- / - mice. [Figure 19B]Figure 19B shows that loss of TSC2 in rods (rod Tsc2- / -) resulted in the same overall pathology as that observed for loss of TSC1 in rods. Figure 19B shows fundus pathology observed in rod Tsc2- / - mice over time. The lower arrow at 9 months represents retinal folds, and the lower arrows at 12 and 18 months represent GA or neovascular (angiogenic) pathology. [Figure 19C] Figure 19 shows that loss of TSC2 in rods (rod Tsc2- / -) resulted in the same gross pathology as that observed for loss of TSC1 in rods. Figure 19C shows that no pathology is observed in control littermates. [Figure 19D] Figure 19 shows that loss of TSC2 in rods (rod Tsc2- / -) resulted in the same overall pathology as observed for loss of TSC1 in rods. Figure 19D shows the percent distribution of pathology over time (months, M) for rod Tsc2- / - mice and at 18 months for littermate controls. Each bar represents the percent of mice ± MOE. Numbers in parentheses indicate the number of mice analyzed. [Figure 19E] Figure 19 shows that loss of TSC2 in rods (rod Tsc2- / -) resulted in the same overall pathology as that observed for loss of TSC1 in rods. Figure 19E shows that fundus (left) and RPE flat specimen (right; ZO1: upper right panel) images show that rod Tsc2- / - mice exhibited distinct GA formation at 12 months: mild to intermediate GA (top), severe ring formation of GA (middle), and irregular patches of GA (bottom). Arrows indicate GA sites. [Figure 19F]Figure 19F shows that loss of TSC2 in rods (rod Tsc2- / -) resulted in the same overall pathology as observed for loss of TSC1 in rods. Figure 19F shows immunofluorescence for ApoB, ApoE, C3, and CFH on retinal sections from 12-month-old mice with the indicated genotypes. Similar to loss of TSC1 in rods, loss of TSC2 leads to the accumulation of lipoproteins (ApoE, ApoB), complement factor H (CFH), and loss of complement factor C3. Higher magnification images of the areas between the arrowheads are shown in the upper panel of each panel. (Scale bar: 50 μm). [Figure 20A] Figure 20A shows that loss of TSC2 in rods (rod Tsc2- / -) resulted in the same overall pathology as that observed with loss of TSC1 in rods. Figure 20A shows representative images of RPE flat preparations at 8 am and 11 am, demonstrating the accumulation of shed POS (rhodopsin and ZO-1; scale bar = 50 mm) in both rod Tsc2+ / + and rod Tsc2- / - mice at 2 months. At 11 am, more POS are still present in rod Tsc2- / - mice. [Figure 20B] Figure 20B shows that loss of TSC2 in rods (rod Tsc2- / -) resulted in the same overall pathology as that observed in loss of TSC1 in rods. Figure 20B shows quantification of remaining POS / RPE cells at 8 am and 11 am. [Figure 20C] Figure 20 shows that loss of TSC2 in rods (rod Tsc2- / -) resulted in the same overall pathology as that observed with loss of TSC1 in rods. Figure 20C shows the percentage of phospholipids in retinal lipid profiling, demonstrating a reduction in the DHA-dual-containing PE and PC lipids in rod Tsc2- / - mice. Similar data were observed with loss of TSC1 in rods. [Figure 20D]Figure 20A shows that loss of TSC2 in rods (rod Tsc2- / -) resulted in the same overall pathology as that observed with loss of TSC1 in rods. Figure 20D shows ERG recordings demonstrating an increase in scotopic rod responses in rod Tsc2- / - mice, similar to that observed in rod Tsc1- / - mice. Light-adapted ERG recordings are unchanged between rod Tsc2+ / + and rod Tsc2- / - mice. Bars represent mean a-wave amplitude (μV) ± SEM. (n = 8 and 14 mice, respectively). [Figure 21A] Figure 21A shows that loss of TSC2 and HK2 in rods (rod Tsc2- / - rod HK2- / -) still results in global pathology similar to that seen with loss of TSC2 in rods. Figure 21A shows lactate assays using 2-month-old mice, demonstrating that retinal lactate levels return to normal in rod Tsc2- / - rod HK2- / - mice or in mice in which mTORC1 activity is blocked (loss of Raptor: rod Tsc2- / - rod Raptor- / -). Each bar represents the relative fold change ± SEM compared to the respective wild-type littermate control. (N = 4-6 mice). [Figure 21B] Figure 21B shows that loss of TSC2 and HK2 in rods (rod Tsc2- / - rod HK2- / -) still results in global pathology similar to that seen with loss of TSC2 in rods. Figure 21B shows the percent distribution of pathology in 12- and 18-month-old rod Tsc2- / - rod HK2- / - and littermate controls. Each bar represents the percentage of mice ± MOE. Numbers in parentheses indicate the number of mice analyzed. [Figure 21C]Figure 21C shows that loss of TSC2 and HK2 in rods (rod Tsc2- / - rod HK2- / -) still results in overall pathology similar to that seen with loss of TSC2 in rods. Figure 21D shows examples of GA and neovascular pathology in rod Tsc2- / - rod HK2- / - mice. The first panel shows the fundus. The second panel shows fundus fluorescein angiography (FFA) to detect neovascular pathology. The third panel shows optical coherence tomography (OCT) of an area of blood leakage, revealing sub-RPE edema and neovascularization invading the retina. The final panel shows a higher magnification image of an RPE flat preparation from the same eye. The developing blood vessels were marked with IB-4 and appear in red. [Figure 21D] Figure 21D shows that loss of TSC2 and HK2 in rods (rod Tsc2- / - rod HK2- / -) still results in similar overall pathology as loss of TSC2 in rods. Figure 21D shows examples of ApoE-positive drusen-like deposits seen in bright field and fluorescence in rod Tsc2- / - rod HK2- / - mice. [Figure 21E] Figure 21E shows that loss of TSC2 and HK2 in rods (rod Tsc2- / - rod HK2- / -) still results in global pathology similar to that seen with loss of TSC2 in rods. Figure 21E shows immunofluorescence for ApoE, C3, and CFH on retinal sections from aged mice with the indicated genotypes. Similar to Figures 19A-19F, rod Tsc2- / - rod HK2- / - mice still showed accumulation of ApoE, CFH, and reduced C3. Higher magnification images of the area between the arrowheads are shown in the top of each panel. (Scale bar: 50 μm). [Figure 21F]Figure 21F shows that loss of TSC2 and HK2 in rods (rod Tsc2- / - rod HK2- / -) still results in global pathology similar to that seen with loss of TSC2 in rods. Figure 21F shows a photoreceptor outer segment (POS) digestion assay as shown in Figures 20A-20D. At 11 am (3 hours after peak POS efflux), there was a 37% increase in undigested POS in rod Tsc2- / - rod HK2- / -. Interestingly, fewer outer segments were effluxed in rod Tsc2- / - rod HK2- / - than in Cre- littermate control mice (black bars). [Figure 21G] Figure 21A shows that loss of TSC2 and HK2 in rods (rod Tsc2- / - rod HK2- / -) still results in global pathology similar to that seen when TSC2 is lost in rods. Figure 21G shows dark- and light-adapted electroretinograms in rod Tsc2- / - rod HK2- / - mice, demonstrating that the increases observed in rod Tsc2- / - rod mice are reversed in rod Tsc2- / - rod HK2- / - mice. Bars represent mean a-wave amplitude (μV) ± SEM. (n = 9 and 11 mice, respectively). [Figure 22A] Figure 22A shows that loss of TSC1 and Rictor in rods (rod Tsc1- / - rod Rictor- / -) still resulted in the same overall pathology seen when TSC1 was lost in rods. Figure 22A shows an example fundus image from an 18-month-old mouse. Genotype is indicated in each fundus image. [Figure 22B] Figure 22B shows that loss of TSC1 and Rictor in rods (rod Tsc1- / - rod Rictor- / -) still resulted in the same overall pathology seen when TSC1 was lost in rods. Figure 22B shows the percent distribution of pathology in rod Tsc1- / - rod Rictor- / - mice and heterozygous (rod Tsc1- / - rod Rictor- / +) littermate control mice at 18 months of age. Heterozygous and homozygous Rictor loss-of-function mice still develop similar pathology at a frequency similar to that of rod Tsc1- / - mice. [Figure 23A] Distribution of pathologies (GA and CNV: neovascularization) observed in rod Tsc1- / -S6K1- / - and matched littermate controls at 12 months of age. Figure 23A shows examples of fundus images of the indicated genotypes. [Figure 23B] Figure 23A shows the distribution of pathologies (GA and CNV: angiogenesis) observed in rod Tsc1- / -S6K1- / - and matched littermate controls at 12 months of age. Figure 23B shows the distribution (%) of pathologies (GA and CNV: angiogenesis) observed in rod Tsc1- / -S6K1- / - and matched littermate controls at 12 months of age. [Figure 24] Figure 1 shows accumulation of ApoE and CFH and loss of C3 expression in the RPE and BrM of 15-month-old mice with the indicated genotypes. Higher magnification images of the area between the arrowheads are shown in the top row of each panel. (See text for details.) [Figure 25] Figure showing the percentage distribution of di-DHA-containing phospholipids in PE and PC for the indicated genotypes. Measurements were performed in 2-month-old mice (**P<0.01; ***P<0.001). [Figure 26] Figure 1 shows the percentage distribution of di-DHA phospholipids in PE and PC in mice fed a DHA-enriched diet for 10 weeks after weaning. In mice lacking TSC1 in rods, feeding with DHA did not alter the levels of di-DHA PE and PC lipids. Note: There are slight differences in baseline levels between rod Tsc1- / - mice (Figure 8) and rod Tsc1- / -S6K1- / - mice (Figure 25), which may be due to differences in genetic background. [Figure 27]Figure showing p-S6 staining in retinal cross sections from a non-diseased individual and a diseased individual with AMD. A significant overall increase was observed in the AMD patient's retina, particularly in the photoreceptor layer (P). The strongest staining was observed in the inner segment region. The photoreceptor segment region is marked with (S). The area marked with (S) includes the inner and outer segments with the most intense p-S6 staining. The arrowheads point to drusen deposits in this AMD patient. Each panel represents a different individual. DETAILED DESCRIPTION OF THE INVENTION

[0015] Aspects of the present disclosure relate to methods and compositions for treating certain ocular diseases and disorders, such as age-related macular degeneration (AMD).The present disclosure is based in part on a method for treating AMD in a subject by administering one or more kinase inhibitors, for example, one or more serine / threonine kinase inhibitors.In some embodiments, at least one of the serine / threonine kinase inhibitors is a mammalian target of rapamycin complex 1 (mTORC1) inhibitor.In some embodiments, at least one of the serine / threonine kinase inhibitors is a ribosomal protein S6 kinase β-1 (S6K1) inhibitor.

[0016] The mammalian target of rapamycin (mTOR) pathway plays a crucial role in regulating energy, nutrient, and growth factor availability to control key biological processes, including cell proliferation, metabolism, and protein synthesis, through phosphorylation of the downstream ribosomal protein, S6 kinase 1 (S6K1). mTOR regulates the activity of two key translation regulators, ribosomal S6 kinases (S6K1 and S6K2), following changes in various cellular events (e.g., amino acid levels and energy sufficiency, as well as hormone and mitogen stimulation). These mTOR-regulated effectors (e.g., S6K1) control cell size and contribute to efficient G1 cell cycle progression. Inappropriate regulation of S6K1 contributes to oncogenesis in cells with loss-of-function mutations in tumor suppressors (e.g., PTEN, TSC1 / 2, or LKB) or gain-of-function mutations in many growth factor receptors, phosphatidylinositol 3-kinase (PI3K), or Akt (protein kinase B). In addition, inappropriate mTOR signaling can contribute to metabolic diseases such as diabetes and obesity.

[0017] In some embodiments, mTOR activates S6K1 in response to cellular energy status, nutrient levels, and mitogens, and S6K1 activation is initiated by mTOR / raptor-mediated phosphorylation of T389, which requires a TOS motif located at the N-terminus of S6K.

[0018] inhibitors The present disclosure relates in part to agents that inhibit the expression or activity of one or more proteins in the mTORC1 pathway, such as mTORC1 or ribosomal protein S6 kinase beta-1 (S6K1). Inhibitors of mTORC1 and / or S6K1 can be peptides, proteins, antibodies, small molecules, or nucleic acids.

[0019] As used herein, the term "inhibitor" or "repressor" refers to any agent that inhibits, suppresses, represses, or reduces gene expression (e.g., reducing transcription or translation from genes such as MTOR, Raptor, MLST8, PRAS40, DEPTOR, or RPS6KB1) or that inhibits, represses, or reduces a specific activity, such as the activity of mTORC1 and / or S6K1 proteins. In some embodiments, the inhibitor selectively inhibits mTORC1 or S6K1 activity. As used herein, "selectively inhibits" refers to inhibiting only a specific target protein or gene (e.g., MTOR, RPS6KB1, mTOR protein, S6K protein, etc.) without inhibiting other genes or proteins. In some embodiments, the inhibitor is a direct inhibitor of S6K1 (e.g., an inhibitor that binds to or interacts with S6K1 protein or a nucleic acid encoding S6K1, causing inhibition of S6K1 expression levels and / or activity). In some embodiments, the direct S6K1 inhibitor is a peptide, protein, or antibody that directly binds to S6K1 and inhibits its activity.In some embodiments, the direct S6K1 inhibitor is a small molecule inhibitor that directly binds to S6K1 and inhibits its activity.In some embodiments, the direct S6K1 inhibitor is an inhibitory nucleic acid that directly binds to S6K1 protein or S6K1 mRNA and inhibits the expression level and / or activity of S6K1.

[0020] mTORC1 (also known as mammalian target of rapamycin complex 1) is a protein complex that includes mTOR, mTOR regulatory associated protein (Raptor), mammalian lethal with SEC13 protein 8 (MLST8), PRAS40, and DEPTOR. In some embodiments, mTOR is encoded by the MTOR gene comprising the sequence set forth in NCBI Reference SEQ ID NO: NM_004958.4. In some embodiments, the inhibitor binds directly to the mTOR protein. In some embodiments, the inhibitor binds to a nucleic acid (e.g., DNA, mRNA, etc.) encoding the mTOR protein.

[0021] Ribosomal protein S6 kinase beta-1 (S6K1), also known as p70S6 kinase (p70S6K, p70-S6K), is a protein kinase that in humans is encoded by the RPS6KB1 gene. In some embodiments, the inhibitor binds directly to the S6K1 protein. In some embodiments, the inhibitor binds to a nucleic acid (e.g., DNA, mRNA, etc.) encoding the S6K1 protein (e.g., RPS6KB1 or an mRNA encoded from such a gene). In some embodiments, the nucleic acid encoding the S6K1 protein comprises the sequence set forth in NCBI Reference SEQ ID NO: NM_003161.4.

[0022] In some embodiments, delivery of an inhibitor to a cell results in a decrease in the level of expression and / or activity of a gene (e.g., MTOR, RPS6KB1, etc.) by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, or 500% compared to the level of expression and / or activity of the gene in control cells to which the inhibitor has not been delivered. In some embodiments, delivery of an inhibitor to a cell results in a decrease in the level of expression and / or activity of a gene (e.g., MTOR, RPS6KB1, etc.) by 10% to 50%, 10% to 100%, 10% to 200%, 50% to 500%, or more compared to the level of expression and / or activity of the gene in control cells to which the inhibitor has not been delivered. Methods for measuring gene expression and / or activity are known in the art and include, for example, quantitative PCR (qPCR), Western blot, mass spectrometry (MS) assay, substrate assay, etc.

[0023] In some embodiments, the inhibitor (e.g., an inhibitor of mTOR or S6K1) is a small molecule. In some embodiments, the term "small molecule" refers to a synthetic or naturally occurring chemical (e.g., a peptide or oligonucleotide), a natural product, or any other low molecular weight (often less than about 5 kilodaltons) organic, bioinorganic, or inorganic compound of natural or synthetic origin, which may be optionally derivatized. Such small molecules may be therapeutically deliverable substances or may be further derivatized to facilitate delivery. In some embodiments, the inhibitor inhibits S6K1 but not mTOR. In some embodiments, the inhibitor is a small molecule inhibitor of mTOR. Examples of mTOR inhibitors include, but are not limited to, rapamycin, everolimus, sirolimus, temsirolimus, deforolimus, KU-0063794, and salts, solvates, and analogs thereof. Examples of small molecule inhibitors of S6K1 include, but are not limited to, PF-4708671, rosmarinic acid methyl ester (RAME), A77 1726, and salts, solvates, and analogs thereof. In some embodiments, the inhibitor is a small molecule inhibitor of S6K1, such as the S6K1 inhibitors described in U.S. Patent No. 10,144,726, U.S. Patent No. 10,730,882, Korean Patent No. 102106851, International Publication No. 2016170163, International Publication No. 2005019829, and International Publication No. 2005019829, each of which is incorporated herein by reference.

[0024] In some embodiments, the inhibitor is a protein. In some embodiments, the protein is a dominant-negative mutant of S6K1. In some embodiments, the dominant-negative mutant of S6K1 is S6K-DN as described in Zhang et al., J Biol Chem. 2008 Dec. 19;283(51):35375-35382. In some embodiments, the inhibitor is a nucleic acid encoding the dominant-negative mutant of S6K1.

[0025] In some embodiments, the inhibitor is an antibody that targets S6K1. Antibody, as used herein, refers to at least one immunoglobulin variable domain or a polypeptide comprising at least one antigenic determinant, e.g., a paratope, that specifically binds to an antigen. In some embodiments, the antibody is a full-length antibody (e.g., an anti-S6K1 antibody). In some embodiments, the antibody is a chimeric antibody (e.g., an anti-S6K1 antibody). In some embodiments, the antibody is a humanized antibody (e.g., an anti-S6K1 antibody). However, in some embodiments, the antibody is a Fab fragment, a Fab' fragment, a F(ab')2 fragment, an Fv fragment, or an scFv fragment (e.g., a Fab fragment, a Fab' fragment, a F(ab')2 fragment, an Fv fragment, or an scFv fragment that targets S6K1). In some embodiments, the antibody is a nanobody derived from a camelid antibody or a nanobody derived from a shark antibody (e.g., an anti-S6K1 nanobody). In some embodiments, the antibody is a diabody (e.g., an anti-S6K1 diabody). In some embodiments, the antibody comprises a framework having human germline sequences. In another embodiment, the antibody comprises a heavy chain constant domain selected from the group consisting of IgG, IgG1, IgG2, IgG2A, IgG2B, IgG2C, IgG3, IgG4, IgA1, IgA2, IgD, IgM, and IgE constant domains. Non-limiting examples of S6K1 antibodies include antibody clones R.566.2, B12H16L8, B12HCLC, OTI6B2, and the like.

[0026] In some embodiments, the inhibitor is an inhibitory oligonucleotide. Inhibitory oligonucleotides can interfere with gene expression, transcription, and / or translation. Generally, inhibitory oligonucleotides bind to a target polynucleotide through a region of complementarity. For example, binding of an inhibitory oligonucleotide to a target polynucleotide can induce RNAi pathway-mediated degradation of the target polynucleotide (in the case of dsRNA, siRNA, shRNA, etc.) or block the translation machinery (e.g., antisense oligonucleotides). In some embodiments, the inhibitory oligonucleotide has a region of complementarity that is complementary to at least 8 (e.g., 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or more) nucleotides of the mRNA encoded by the MTOR gene or RPS6KB1 gene. Inhibitory oligonucleotides can be single-stranded or double-stranded. In some embodiments, the inhibitory oligonucleotide is DNA or RNA. In some embodiments, the inhibitory oligonucleotide is a hairpin-forming RNA selected from the group consisting of an antisense oligonucleotide, an artificial miRNA (AmiRNA), an siRNA, an shRNA, and an miRNA. Generally, a hairpin-forming RNA is configured as a self-complementary "stem-loop" structure that includes a single nucleic acid encoding a stem portion with a double-stranded portion including a sense strand (e.g., a passenger strand) connected to an antisense strand (e.g., a guide strand) by a loop sequence. The passenger strand and the guide strand share complementarity. In some embodiments, the passenger strand and the guide strand share 100% complementarity. In some embodiments, the passenger strand and the guide strand share at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% complementarity. The passenger strand and the guide strand may lack complementarity due to base pair mismatches.In some embodiments, the passenger strand and guide strand of a hairpin-forming RNA have at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 mismatches. Generally, the first 2 to 8 nucleotides of the stem (relative to the loop) are referred to as "seed" residues and play an important role in target recognition and binding. The first residue of the stem (relative to the loop) is referred to as the "anchor" residue. In some embodiments, hairpin-forming RNAs have mismatches at the anchor residue. Hairpin-forming RNAs are useful for translational repression and / or gene silencing via the RNAi pathway. Due to their common secondary structure, hairpin-forming RNAs share the characteristic of being processed by proteins (Drosha and Dicer) before being loaded into the RNA-induced silencing complex (RISC). The length of the double-stranded portion between hairpin-forming RNAs can vary. In some embodiments, the double-stranded portion is about 19 nucleotides to about 200 nucleotides in length. In some embodiments, the double-stranded portion is about 14 to about 35 nucleotides in length. In some embodiments, the double-stranded portion is about 19 to 150 nucleotides in length. In some embodiments, the hairpin-forming RNA has a double-stranded portion region that is 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides in length. In some embodiments, the double-stranded portion is about 19 to 33 nucleotides in length. In some embodiments, the double-stranded portion is about 40 to 100 nucleotides in length. In some embodiments, the double-stranded portion is about 60 to about 80 nucleotides in length.

[0027] In some embodiments, the hairpin-forming RNA targeting S6K1 is an artificial microRNA (AmiRNA). As used herein, "artificial miRNA" or "amiRNA" refers to miRNA and miRNAs, as described, for example, by Eamens et al. (2014), Methods Mol. Biol. 1062:211-224. * (e.g., passenger strand of miRNA duplex) sequence is * AmiRNA refers to an endogenous pri-miRNA or pre-miRNA (e.g., a miRNA scaffold, which is a miRNA precursor capable of generating a functional mature miRNA) that has been replaced with a sequence (which directs highly effective RNA silencing of the targeted gene). In some embodiments, the AmiRNA scaffold is derived from a pri-miRNA selected from the group consisting of pri-MIR-21, pri-MIR-22, pri-MIR-26a, pri-MIR-30a, pri-MIR-33, pri-MIR-64, pri-MIR-122, pri-MIR-155, pri-MIR-375, pri-MIR-199, pri-MIR-99, pri-MIR-194, pri-MIR-155, and pri-MIR-451.

[0028] In some embodiments, inhibitory nucleic acids targeting S6K1 include any inhibitory nucleic acids known in the art, such as inhibitory nucleic acids targeting S6K2, such as those described in U.S. Patent Application Publication No. 20030083284 and U.S. Patent Application Publication No. 20070191259, each of which is incorporated herein by reference.

[0029] In some embodiments, the inhibitory oligonucleotide is a modified nucleic acid. The term "nucleotide analog" or "modified nucleotide" or "modified nucleotide" refers to a non-standard nucleotide, including non-naturally occurring ribonucleotides or deoxyribonucleotides. In some embodiments, the nucleotide analog is modified at any position to change certain chemical properties of the nucleotide while still retaining the ability of the nucleotide analog to perform its intended function. Examples of nucleotide positions that can be derivatized include the 5-position, such as 5-(2-amino)propyluridine, 5-bromouridine, 5-propyneuridine, 5-propenyluridine, etc.; the 6-position, such as 6-(2-amino)propyluridine; and the 8-position for adenosine and / or guanosine, such as 8-bromoguanosine, 8-chloroguanosine, 8-fluoroguanosine, etc. Nucleotide analogs also include deazanucleotides, such as 7-deaza-adenosine; O- and N-modified (e.g., alkylated, e.g., N6-methyladenosine, or otherwise known in the art) nucleotides; and other heterocyclically modified nucleotide analogs, such as those described in Herdewijn, Antisense Nucleic Acid Drug Dev., August 2000, 10(4):297-310.

[0030] Nucleotide analogs may also include modifications to the sugar portion of the nucleotide. For example, the 2'OH group may be replaced with a group selected from H, OR, R, F, Cl, Br, I, SH, SR, NH2, NHR, NR2, and COOR (wherein R is a substituted or unsubstituted C1-C6 alkyl, alkenyl, alkynyl, aryl, etc.). Other possible modifications include those described in U.S. Patent Nos. 5,858,988 and 6,291,438. Locked nucleic acids (LNAs) are modified RNA nucleotides, often referred to as inaccessible RNAs. The ribose moiety of an LNA nucleotide is modified with an extra bridge connecting the 2' oxygen and the 4' carbon.

[0031] The phosphate groups of the nucleotides can also be modified, for example, by replacing one or more of the oxygens of the phosphate group with sulfur (e.g., phosphorothioates), or by making other substitutions that allow the nucleotide to perform its intended function, as described, for example, in Eckstein, Antisense Nucleic Acid Drug Dev. 2000 April 10(2):117-21; Rusckowski et al., Antisense Nucleic Acid Drug Dev. 2000 October 10(5):333-45; Stein, Antisense Nucleic Acid Drug Dev. 2001 October 11(5):317-25; Vorobjev et al., Antisense Nucleic Acid Drug Dev. 2001 April 11(2):77-85, and U.S. Pat. No. 5,684,143. Certain of the above-cited modifications (e.g., modifications of phosphate groups) preferably reduce the hydrolysis rate of polynucleotides containing the analogs in vivo or in vitro, for example. In some embodiments, the inhibitory oligonucleotides are modified inhibitory oligonucleotides. In some embodiments, the modified inhibitory oligonucleotides comprise locked nucleic acids (LNAs), phosphorothioate backbones, and / or 2'-O-Me modifications.

[0032] method Aspects of the present disclosure relate to methods of inhibiting drusen formation in ocular tissue, comprising administering to cells of the ocular tissue one or more inhibitors of mammalian target of rapamycin complex 1 (mTORC1), such as MTOR or RPS6KB1 (or proteins encoded by such genes). In some embodiments, the cells are in vitro. In some embodiments, the cells are in a subject (e.g., the cells are in vivo).

[0033] In some embodiments, the present disclosure provides a method for treating age-related macular degeneration (AMD) in a subject, the method comprising administering to the subject one or more inhibitors of mTORC1 (e.g., MTOR or RPS6KB1, or proteins encoded by such genes).

[0034] Age-related macular degeneration (AMD) is one of the leading causes of visual impairment in the elderly. The disease is multifactorial, involving both genetic and non-genetic risk factors. Among non-genetic risk factors, smoking and diet have been shown to be the most important modifiable risk factors. A diet rich in omega-3 fatty acids, particularly docosahexaenoic acid (DHA), has been shown to reduce disease risk. Similarly, high DHA plasma levels correlate with reduced disease risk. Additionally, individuals with AMD have a 30% reduction in retinal DHA levels.

[0035] As used herein, "subject" is interchangeable with "subject in need thereof," and both can refer to a subject with age-related macular degeneration (AMD) or a subject at increased risk of developing such a disorder compared to the general population (e.g., a subject with one or more gene mutations associated with AMD, such as complement factor H (CFH)). A subject in need thereof can be a subject exhibiting one or more signs or symptoms of AMD. In some embodiments, a subject (e.g., a subject with AMD or at increased risk of having AMD) has overactive S6K1 or an increased risk of overactivating S6K1 (e.g., constitutive activation of S6K1) compared to a subject not at risk. In some embodiments, loss of TSC1 and / or TSC2 (e.g., loss of expression or function of TSC1 and / or TSC2) causes overactivation of S6K1. In some embodiments, a subject with overactivated S6K1 is TSC1 deficient (e.g., loss of expression or function of TSC1). In some embodiments, the subject in which S6K1 is overactivated is TSC2 deficient (e.g., has a loss of TSC2 expression or function). In some embodiments, the subject in which S6K1 is overactivated is TSC1 and TSC2 deficient (e.g., has a loss of TSC1 and / or TSC2 expression or function). The subject may be a human, a non-human primate, a rat, a mouse, a cat, a dog, or other mammal.

[0036] As used herein, the terms "treatment," "treating," and "therapy" refer to therapeutic treatment and prophylactic or preventative manipulation. The terms further include alleviating existing symptoms, preventing additional symptoms, alleviating or preventing the underlying cause of symptoms, and preventing or reversing the cause of symptoms, such as those associated with age-related macular degeneration (AMD). Thus, the terms refer to the production of beneficial results for a subject with a disorder (e.g., AMD) and at risk of developing such a disorder. Furthermore, the term "treatment" is defined as the application or administration of an agent (e.g., a therapeutic agent or therapeutic composition) to a subject who may have a disease, a symptom of a disease, or a susceptibility to a disease, or to an isolated tissue or cell system derived from a subject, with the aim of correcting, curing, alleviating, mitigating, altering, relieving, ameliorating, improving, or influencing the disease, the symptom of a disease, or the susceptibility to a disease. The "development" or "progression" of a disease refers to the initial symptoms and / or subsequent progression of the disease. The onset of disease can be detected and evaluated using standard clinical techniques that are well known in the art.However, onset also refers to the progression that may be undetectable.For the purpose of this disclosure, onset or progression refers to the biological process of symptoms." onset "includes occurrence, recurrence and onset.As used herein, the "onset" or "onset" of disease (for example, AMD).

[0037] The present disclosure is based in some aspects on a method for treating AMD, comprising administering to a subject di-docosahexaenoic acid (DHA) in addition to one or more inhibitors.In some embodiments, DHA is administered as a dietary supplement (e.g., administered orally).

[0038] The therapeutic agent or composition may comprise a pharmaceutically acceptable form of a compound that prevents and / or reduces the symptoms of a particular disease (e.g., AMD). For example, the therapeutic composition may be a pharmaceutical composition that prevents and / or reduces the symptoms of AMD. The therapeutic composition of the present invention may be provided in any suitable form. The form of the therapeutic composition depends on several factors, including the mode of administration, as described herein. The therapeutic composition may contain diluents, adjuvants, and excipients, among other ingredients, as described herein.

[0039] Pharmaceutical compositions containing inhibitors and / or other compounds can be administered by any suitable route for administering pharmaceuticals. A variety of administration routes are available. The particular mode selected will, of course, depend on the particular agent or agents selected, the particular condition being treated, and the dosage required for therapeutic effect. The methods of the present disclosure can generally be practiced using any medically acceptable mode of administration, i.e., any mode that produces a therapeutic effect without causing clinically unacceptable side effects. Various modes of administration are discussed herein. When used in therapy, an effective amount of inhibitors and / or other therapeutic agents can be administered to a subject by any mode that delivers the agent to the desired surface, e.g., a mucosal surface, a systemic surface.

[0040] In some embodiments, inhibitory oligonucleotides can be delivered to cells via an expression vector engineered to express inhibitory oligonucleotides. An expression vector is a vector into which a desired sequence can be inserted, for example, by restriction enzyme cleavage and ligation, so that it is operably linked to a control sequence and can be expressed as an RNA transcript. Expression vectors generally contain an insert that is the coding sequence for a protein or an inhibitory oligonucleotide, such as shRNA, miRNA, or miRNA. The vector may further contain one or more marker sequences suitable for use in identifying cells, and the marker sequence may or may not be transformed or transfected together with the vector. Markers include, for example, genes encoding proteins that increase or decrease antibiotic resistance or sensitivity, or genes encoding other compounds, enzymes (the activity of which can be detected by standard assays or fluorescent proteins, etc.).

[0041] As used herein, a coding sequence (e.g., a protein-coding sequence, miRNA sequence, shRNA sequence) and a regulatory sequence are said to be "operably" linked when the sequences are covalently linked such that the expression or transcription of the coding sequence is under the influence or control of the regulatory sequence. When it is desired that the coding sequence be translated into a functional protein, two DNA sequences are said to be operably linked if induction of a promoter in a 5' regulatory sequence causes transcription of the coding sequence, and if the nature of the linkage between the two DNA sequences does not (1) cause the introduction of a frameshift mutation, (2) interfere with the ability of the promoter region to direct transcription of the coding sequence, or (3) interfere with the ability of the corresponding RNA transcript to be translated into a protein. Thus, a promoter region is operably linked to a coding sequence if it is capable of enabling transcription of the DNA sequence so that the resulting transcript can be translated into the desired protein or polypeptide. It is recognized that a coding sequence may encode a miRNA, shRNA, or miRNA.

[0042] The precise nature of the regulatory sequences required for gene expression may vary between species or cell types, but generally must include, as necessary, 5' non-transcribed and 5' non-translated sequences involved with initiation of transcription and translation, respectively, such as the TATA box, capping sequence, CAAT sequence, etc. Such 5' non-transcribed regulatory sequences include promoter regions containing promoter sequences for controlling transcription of an operably linked gene. Regulatory sequences may also include enhancer sequences or upstream activator sequences, as desired. The vectors of the present disclosure may optionally include a 5' leader or signal sequence.

[0043] In some embodiments, the viral vector for delivering nucleic acid molecules is selected from the group consisting of adenovirus, adeno-associated virus, poxvirus, including vaccinia virus and attenuated poxvirus, Semliki Forest virus, Venezuelan equine encephalitis virus, retrovirus, Sindbis virus, and Ty virus-like particles. Examples of viruses and virus-like particles that have been used to deliver exogenous nucleic acids include replication-deficient adenovirus, modified retrovirus, non-replicating retrovirus, replication-deficient Semliki Forest virus, canarypox virus, and highly attenuated vaccinia virus derivatives, non-replicating vaccinia virus, replicating vaccinia virus, Venezuelan equine encephalitis virus, Sindbis virus, lentiviral vectors, and Ty virus-like particles. Another virus useful for certain applications is adeno-associated virus. Adeno-associated virus has the ability to infect a wide range of cell types and species and can be engineered to be replication-deficient. Adeno-associated viruses have additional advantages, such as heat and lipid solvent stability, high transduction frequencies in cells of various lineages, including hematopoietic cells, and lack of superinfection inhibition, thus enabling serial multiple transductions. Adeno-associated viruses can integrate into human cellular DNA in a site-specific manner, thereby minimizing the possibility of insertional mutagenesis and variability in inserted gene expression. In addition, wild-type adeno-associated virus infection has been tracked in tissue culture for over 100 passages in the absence of selective pressure, suggesting that adeno-associated virus genomic integration is a relatively stable event. Adeno-associated viruses can also function in an extrachromosomal manner.

[0044] Other useful viral vectors are generally based on non-cytopathic eukaryotic viruses in which non-essential genes have been substituted for the gene of interest. Non-cytopathic viruses include certain retroviruses, whose life cycle involves reverse transcription of genomic viral RNA into DNA followed by proviral integration into host cell DNA. Retroviruses are generally replication-deficient (e.g., capable of directing the synthesis of desired transcripts but unable to produce infectious particles). Such genetically modified retroviral expression vectors have general utility for highly efficient gene transduction in vivo. Standard protocols for generating replication-defective retroviruses (including the steps of incorporating foreign genetic material into a plasmid, transfecting a packaging cell line with the plasmid, generating recombinant retrovirus by the packaging cell line, collecting viral particles from tissue culture medium, and infecting target cells with the viral particles) are provided in Kriegler, M., Gene Transfer and Expression, A Laboratory Manual, W.H. Freeman Co., New York, USA (1990), and Murry, E.J. Ed., Methods in Molecular Biology, vol. 7, Humana Press, Inc., Clifton, New Jersey, USA (1991).

[0045] Depending on whether the nucleic acid molecule is introduced into a host in vitro or in vivo, various techniques can be employed to introduce the nucleic acid molecules of the present disclosure into cells, including transfection of nucleic acid molecule-calcium phosphate precipitates, transfection of nucleic acid molecules associated with DEAE, transfection or infection with the above-mentioned viruses containing the nucleic acid molecule of interest, liposome-mediated transfection, etc. Other examples include the N-TER™ Nanoparticle Transfection System by Sigma-Aldrich, FECTOFLY™ Transfection Reagent for Insect Cells by Polyplus Transfection, Polyethylenimine "Max" by Polysciences, Inc., Unique, Non-Viral Transfection Tool by Cosmo Bio Co., Ltd., LIPOFECTAMINE™ LTX Transfection Reagent by Invitrogen, SATISFECTION™ Transfection Reagent by Stratagene, LIPOFECTAMINE™ Transfection Reagent by Invitrogen, FUGENE® HD Transfection Reagent by Roche Applied Science, Polyplus Transfection Reagent by Polyplus Examples of suitable transfection reagents include the GMP-compliant IN VIVO-JETPEI™ transfection reagent from Eppendorf Transfection, and the insect GENEJUICE® transfection reagent from Novagen.

[0046] The S6K1 inhibitor (for example, any one of the S6K1 inhibitors described herein, or a combination thereof) can be delivered to a mammalian subject by, for example, intramuscular injection or by administration into the bloodstream of the mammalian subject. Administration into the bloodstream can be by injection into a vein, artery, or any other blood vessel. In some embodiments, the S6K1 inhibitor (for example, any one of the S6K1 inhibitors described herein, or a combination thereof) is administered into the bloodstream by isolated limb perfusion, a technique well known in the surgical arts, which allows a person skilled in the art to essentially separate a limb from systemic circulation before administering the S6K1 inhibitor (for example, any one of the S6K1 inhibitors described herein, or a combination thereof). In addition, in certain cases, it may be desirable to deliver the S6K1 inhibitor (for example, any one of the S6K1 inhibitors described herein, or a combination thereof) to the ocular tissue of a subject. The S6K1 inhibitor (e.g., any one of the S6K1 inhibitors described herein, or a combination thereof) can be delivered directly to the eye by injection, for example, subretinal or intravitreal administration. In some embodiments, the S6K1 inhibitor as described in the present disclosure (e.g., any one of the S6K1 inhibitors described herein, or a combination thereof) is administered by intravenous injection. In some embodiments, the S6K1 inhibitor (e.g., any one of the S6K1 inhibitors described herein, or a combination thereof) is administered by intrathecal injection. In some embodiments, the S6K1 inhibitor (e.g., any one of the S6K1 inhibitors described herein, or a combination thereof) is delivered by intramuscular injection.

[0047] Aspects of the present disclosure relate to compositions comprising an S6K1 inhibitor (e.g., any one of the S6K1 inhibitors described herein, or a combination thereof). In some embodiments, the composition further comprises a pharmaceutically acceptable carrier. As used herein, "carrier" includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspending agents, colloids, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Supplementary active ingredients can also be incorporated into the composition. The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce an allergic or similar adverse reaction when administered to a host.

[0048] The compositions of the present disclosure may contain one S6K1 inhibitor alone (e.g., an siRNA targeting S6K1) or in combination with one or more other S6K1 inhibitors (e.g., an S6K1 antibody or a polypeptide targeting S6K1). In some embodiments, the compositions contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different S6K1 inhibitors.

[0049] Suitable carrier can be easily selected by those skilled in the art, taking into consideration the indication that S6K1 inhibitor (for example, any one of the S6K1 inhibitors described herein, or their combination) is intended to target.For example, one suitable carrier includes physiological saline (can be formulated with various buffer solutions, for example, phosphate-buffered saline).Other exemplary carriers include sterile physiological saline, lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, peanut oil, sesame oil, and water.The selection of carrier is not a limitation of the present disclosure.

[0050] Optionally, the compositions of the present disclosure may contain, in addition to the S6K1 inhibitor and carrier(s), other conventional pharmaceutical ingredients, such as preservatives or chemical stabilizers. Suitable exemplary preservatives include chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, parabens, ethyl vanillin, glycerin, phenol, parachlorophenol, and poloxamers (non-ionic surfactants), such as Pluronic® F-68. Suitable chemical stabilizers include gelatin and albumin.

[0051] S6K1 inhibitor or its composition is administered in a sufficient amount to provide the cell of desired tissue (for example, ocular tissue) with sufficient level to inhibit S6K1 without excessive side effects.Traditional pharmaceutically acceptable administration route includes but is not limited to direct delivery to selected organ (for example, intraportal delivery to liver), oral, inhalation (including intranasal and intratracheal delivery), intraocular, intravenous, intramuscular, subcutaneous, intradermal, intratumoral, oral administration and other parenteral administration route.Administration route can be combined if desired.

[0052] The formulation of pharmaceutically acceptable excipient and carrier solutions is well known to those skilled in the art, as is the development of suitable administration and treatment regimens for use with the particular compositions described herein in various treatment regimens.

[0053] Generally, such formulations will contain at least about 0.1% or more of the active compound, although the percentage of active ingredient(s) can, of course, vary and can conveniently be from about 1% or 2% to about 70% or 80% or more by weight or volume of the total formulation. Naturally, the amount of active compound contained in each therapeutically useful composition can be adjusted so that a suitable dosage is obtained in any given unit dose of the compound. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, etc., as well as other pharmacological considerations, will be considered by those skilled in the art when preparing such pharmaceutical formulations, and various dosages and treatment regimens may be desired accordingly.

[0054] In certain circumstances, it may be desirable to deliver the S6K1 inhibitor (e.g., any one of the S6K1 inhibitors described herein, or a combination thereof) contained in a suitably formulated pharmaceutical composition disclosed herein subretinal, intravitreal, subcutaneous, intrapancreatic, intranasal, parenteral, intravenous, intramuscular, intrathecal, or buccal, intraperitoneal, or by inhalation.

[0055] Pharmaceutical dosage forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof, and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. In most cases, the form is sterile and, to the extent that easy syringability exists, liquid. Preparations must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, and / or vegetable oils. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size (in the case of dispersions), and by the use of surfactants. The action of microorganisms can be prevented by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it is preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be achieved by using in the composition an agent delaying absorption, for example, aluminum monostearate or gelatin.

[0056] For example, when administering an injectable aqueous solution, the solution may be suitably buffered, if necessary, and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. In this regard, sterile aqueous vehicles that can be employed are known to those skilled in the art. For example, one dosage can be dissolved in 1 mL of isotonic NaCl solution and added to 1000 mL of subcutaneous injection fluid or injected at the proposed infusion site (see, e.g., Remington's Pharmaceutical Sciences, 15th Edition, pp. 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the host. The person responsible for administration will, in any event, determine the appropriate dose for the individual host.

[0057] Sterile injection solution is prepared by incorporating the S6K1 inhibitor in the required amount with various other ingredients listed herein as needed into a suitable solvent, followed by filtration sterilization.Generally, dispersion is prepared by incorporating various sterilized active ingredients into a sterile vehicle that contains a basic dispersion medium and other necessary ingredients from those listed above.For sterile powder to prepare sterile injection solution, the preferred method of preparation is vacuum drying and freeze-drying, which produces a powder (from its previously sterile-filtered solution) consisting of active ingredient and any desired additional ingredients.

[0058] The S6K1 compositions disclosed herein can be formulated in neutral or salt forms. Pharmaceutically acceptable salts include acid addition salts (formed with the protein's free amino groups) and salts formed with inorganic acids, such as hydrochloric or phosphoric acids, or organic acids such as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with free carboxyl groups can also be derived from inorganic bases, such as sodium, potassium, ammonium, calcium, and the like, or ferric hydroxide, and organic bases such as isopropylamine, trimethylamine, histidine, procaine, and the like. Upon formulation, solutions are administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as injectable solutions, drug-release capsules, and the like.

[0059] Delivery vehicles such as liposomes, nanocapsules, microparticles, microspheres, lipid particles, vesicles, etc. can be used to introduce the compositions of the present disclosure into suitable host cells. In particular, S6K1 inhibitors can be formulated for delivery encapsulated in either lipid particles, liposomes, vesicles, nanospheres, nanoparticles, etc.

[0060] Such a formulation is considered to be preferable for introducing the pharmaceutically acceptable preparation of nucleic acid or S6K1 inhibitor disclosed herein.The formation and use of liposomes are generally known to those skilled in the art.Recently, liposomes with improved serum stability and circulation half-life have been developed (US Pat. No. 5,741,516).In addition, various methods have been described for liposomes and liposome-like preparations that can be used as drug carriers (US Pat. No. 5,567,434; US Pat. No. 5,552,157; US Pat. No. 5,565,213; US Pat. No. 5,738,868 and US Pat. No. 5,795,587).

[0061] Some liposomes have been used successfully in some cell types that are normally resistant to transfection by other procedures. In addition, liposomes are not subject to the DNA length constraints inherent in viral-based delivery systems. Liposomes have been effectively used to introduce genes, drugs, radiotherapeutic agents, viruses, transcription factors, and allosteric effectors into various cultured cell lines and animals. In addition, several clinical trials testing the effectiveness of liposome-mediated drug delivery have been successfully completed.

[0062] Liposomes are formed from phospholipids dispersed in an aqueous medium and spontaneously form multilamellar concentric bilayer vesicles (also called multilamellar vesicles (MLVs)). MLVs typically have diameters between 25 nm and 4 μm. Sonication of MLVs results in the formation of small unilamellar vesicles (SUVs) with diameters ranging from 200 to 500 Å, containing aqueous solution within their cores.

[0063] Alternatively, nanocapsule formulations of S6K1 inhibitors can be used. Nanocapsules can generally entrap substances in a stable and reproducible manner. To avoid side effects due to intracellular polymer overload, such ultrafine particles (approximately 0.1 μm in size) should be designed using polymers that can be degraded in vivo. Biodegradable polyalkylcyanoacrylate nanoparticles that meet these requirements are considered for use. Example [Example]

[0064] Activation of mTORC1 in human photoreceptors (PRs) is an adaptive response to the nutritional deficiencies that photoreceptors experience early in the disease process. Increased expression of aerobic glycolysis genes has been observed in photoreceptors from human AMD samples, suggesting enhanced mTORC1 activity in humans with AMD.

[0065] This example describes in vivo experiments performed on a mouse model of age-related macular degeneration (AMD). The mouse model of AMD was generated by genetically increasing the expression of aerobic glycolysis genes. Briefly, mammalian target of rapamycin 1 (mTORC1) activity was increased in mice by deleting the tuberous sclerosis complex (TSC1). The resulting mice ( 桿状体 TSC1 - / - AMD (referred to as "wet AMD") includes both early (e.g., "wet AMD") pathology, which involves accumulation of apolipoprotein E (ApoE) and complement factor H (CHF), and late (e.g., "dry AMD") pathology, which involves neovascularization of the RPE and lower photoreceptors and geographic atrophy (GA).

[0066] In addition, this mouse also shows a decrease in di-DHA lipids in phosphatidylethanolamine and phosphatidylcholine.At the same time, it has been found that DHA-rich food reduces the risk of disease progression.Data suggests that it is not the increase in aerobic glycolysis itself, but rather the gene expression changes that accompany the increase in mTORC1 activity that cause AMD.For example, in some embodiments, the decrease in di-DHA phospholipids is due to the decrease in the expression of the enzyme(s) involved in synthesis.

[0067] Mice with activated mTORC1 in PR also exhibited other early disease features, such as delayed photoreceptor outer segment (POS) clearance, accumulation of lipofuscin in the retinal pigment epithelium (RPE) and lipoproteins in Bruch's membrane (BrM), and altered complement storage. POS are lipid-rich, and mTORC1 is known to regulate lipid synthesis. To clarify the cause of delayed POS clearance by the RPE, 桿状体 Tsc1 - / -Mouse retinal lipid composition was profiled. A ~3-fold decrease in diDHA (44:12)-containing phosphatidylethanolamine (PE) and phosphatidylcholine (PC) lipids was observed in whole retina preparations (Figure 4A) and POS preparations (Figure 4B). To test whether this decrease in diDHA-type PE and PC lipids contributes to the delay in POS clearance, 桿状体 Tsc1 - / - Mice were fed a diet enriched with 2% DHA. 桿状体 Tsc1 - / - Feeding mice a 2% DHA-enriched diet improved POS clearance at 2 months (Fig. 4C). To test whether the delayed POS clearance could be improved even after the delay had already occurred, we administered 2% DHA-enriched diets to mice at 6 months of age. 桿状体 Tsc1 - / - Mice were fed a DHA-enriched diet for 2 weeks. This had an even more pronounced effect at 6 months, as POS clearance was more strongly affected (Figure 4D). To clarify whether dietary DHA also affected overall RPE health, mice were fed a DHA diet from weaning until 6 months of age. This reduced the percentage of multinucleated RPE cells (Figure 4E), improved fundus pathology (Figure 4F), prevented the accumulation of ApoB, ApoE, and CFH, and restored C3 expression (Figure 4G). No significant differences in RPE hypertrophy were observed. None of the 12 DHA-fed mice (n = 12) developed any GA by 6 months, whereas one of six mice fed the control diet did. Re-profiling of retinal lipids after 10 weeks of DHA feeding suggested that the levels of di-DHA-containing PE and PC lipids were not restored. This suggests that DHA acted directly on the RPE to improve the overall health of the PRE (Figure 4H). Overall, the data suggest that activation of mTORC1 in rods affects retinal lipid composition, which in turn impacts the overall health of the RPE.

[0068] To investigate the effect of ribosomal protein S6 kinase beta-1 (S6K1, also known as p70S6 kinase) function on the development of AMD pathology, additional mouse models were generated, such as mice with activated mTORC1 and loss of S6K1, which did not develop advanced AMD pathology. Figure 1 shows the morphology of a mouse with loss of TSC1 in rods and two normal copies of S6K1 ( 桿状体 TSC1 - / - S6K1 + / + ), mice lacking TSC1 and S6K1 in rods ( 桿状体 TSC1 - / - S6K1 - / - ), mice lacking TSC1 in rods and one copy of S6K1 ( 桿状体 TSC1 - / - S6K1 - / + ), and mice with two normal copies of TSC1 and a complete loss of S6K1 ( 桿状体 TSC1 + / + S6K1 - / - ) shows the distribution of pathology in rods. Complete loss of S6K1 in the presence of loss of TSC1 prevents advanced AMD pathology. Figure 2 shows fundus images and retinal pigment epithelium flat preparations from mice with one copy of S6K1 and loss of TSC1 ( 桿状体 TSC1 - / - S6K1 - / + ) develop GA as seen in fundus pathology (left) and flat specimens. In contrast, mice lacking both TSC1 and S6K1 ( 桿状体 TSC1 - / - S6K1 - / - ), no pathology was observed. Figure 3 shows that S6K1 deficiency in the setting of TSC1 loss prevents the accumulation of ApoE and complement factor H (CHF), both hallmarks of early-stage AMD.

[0069] These data suggest that inhibiting S6K1 in the setting of increased mTORC1 activity prevents the development of both early and late AMD-associated pathology. [Example]

[0070] Human tissue samples The age and sex of the human postmortem ocular samples are shown in Figure 5A and Figures 11A-11B. All staining performed on human tissue samples used cryopreserved tissue sections.

[0071] animal Conditional Tsc1 and Raptor alleles, as well as rod iCre-75 and cone iCre, have all been described previously. All mice were genotyped for the absence of the rd8 mutation. Mice were maintained on a 12-hour light / 12-hour dark light cycle without dietary restriction. Equal numbers of male and female mice were used in all experiments. No sex-specific differences were observed. The DHA diet was created by replacing 2% soybean oil in the AIN-93G lab diet (obtained from Dyets, Inc.) with 2% DHASCO (obtained from DSM). The AIN-93G diet was used as the control diet in all DHA experiments. All animals remained on the control diet except for the DHA and DHA control experiments; the AIN-93G control diet and 5P75 * The facility diets differ in their soybean oil content, which is 7% and 5%, respectively.

[0072] Ophthalmoscopy and angiography Fundus examination was performed. The age and number of mice analyzed in a given experiment are indicated in the figure and / or legend. Immediately after fundus examination, angiography was performed by subcutaneous injection of 125 mg / kg sodium fluorescein solution behind the neck. Images were acquired using a Micron III from Phoenix Technology Group. The overall accuracy of fundus examination for GA diagnosis was confirmed on RPE flat specimens of 22 eyes, 7 of which were diagnosed with GA by fundus examination. On RPE flat specimens, 9 of the 22 eyes were confirmed to have GA.

[0073] Optical Coherence Tomography (OCT) OCT was performed using a Bioptigen system (Model 70-20000). The OCT in Figure 13 was acquired during the manuscript revision period using a new Micron IV system from Phoenix Technology Group. Mice were anesthetized with a mixture of ketamine / xylazine (100 mg / kg and 10 mg / kg). To dilate the pupils, one drop each of phenylephrine (2.5%) and tropicamide (1%) was applied 10 min before recording. After recording, mice were allowed to recover on a warming tray.

[0074] Electroretinogram (ERG) analysis ERGs were performed using a Celeris system for dark-adapted, light-adapted, and C-wave ERGs. The number of mice per group is indicated in the figure legends. Mice were not pre-screened for ocular pathology.

[0075] Lactate assay Lactate assays (L-lactate assay kit, Abcam, catalog no. ab65330) were performed on 2-month-old mice using four biological samples (each consisting of both retinas obtained from the same animal). Each biological measurement was performed in triplicate. Retinas were dissected in ice-cold PBS and processed according to the manufacturer's instructions.

[0076] NADPH assay NADPH assays (NADP / NADPH Assay Kit, Sigma, Cat. No. MAK312) were performed on 2-month-old mice using 7-8 biological samples (each consisting of one retina). Each biological measurement was performed in duplicate. Retinas were dissected in ice-cold PBS and processed according to the manufacturer's instructions.

[0077] Quantitative Western blot analysis All Western blot quantifications used three biological samples (each consisting of both retinas from the same mouse). Each sample was analyzed in triplicate. Protein was extracted as follows: After enucleation, the eyeballs were dissected in chilled PBS buffer. The dissected retinas were quickly transferred to RIPA buffer (Thermo Scientific, Cat. No. 89900) containing protease and phosphatase inhibitors (1:100 dilution; Cat. No. 1861281) and homogenized by sonication. After centrifugation at 13,000 RPM for 10 minutes at 4°C, the protein extract was transferred to a new tube, and protein concentration was quantified using Bio-Rad Protein Assay (Cat. Nos. 500-0113, 0114, and 0115). 5 μg and 10 μg of total protein were loaded to quantify PKM2 and p-S6 expression levels, respectively. The following primary antibodies were used, all from Cell Signaling Technology: rabbit anti-PKM2 antibody (1:4,000; Catalog No. 4053), rabbit anti-pS6 (Ser240 / 244) antibody (1:1,000; Catalog No. 5364), and mouse anti-β-actin antibody (1:1,000, Catalog No. 3700) for normalization. Protein detection was performed using fluorescently labeled secondary antibodies (1:10,000) from Licor in combination with the Odyssey system. Quantification was performed using Image Studio software.

[0078] immunohistochemistry Immunohistochemistry (IHC) and immunofluorescence were performed on cryosections (10 μm thick) or RPE / retinal whole-mount specimens using the following primary antibodies: rabbit anti-PKM2 (1:1000; Cell Signaling Technology, catalog no. 4053), rabbit anti-ZO1 (1:100; Invitrogen, catalog no. 40-2200), and rabbit anti-Iba1 (1:300; Wako, catalog no. 019-19741), mouse anti-CRE-recombinase (1:500; Covance, catalog no. PRB-106P), and mouse anti-rhodopsin (1:100, originally obtained from the University of British Columbia, clone 1D4 (available from Abcam, catalog no. 5417)). All were diluted in PBS containing 0.3% Triton X-100 and 5% bovine serum albumin (BSA, Cell Signaling Technology). For rabbit anti-pS6 (Ser240 / 244) antibody (1:300; Cell Signaling Technology, catalog no. 5364), PBS was replaced with TBS. For rabbit anti-apolipoprotein B (ApoB) (1:800; Abcam, catalog no. 20737), goat anti-apolipoprotein E (ApoE) (1:1,000; Millipore, catalog no. 178479), rabbit anti-CFH (1:300; catalog no. ABIN3023097), and goat anti-mouse complement C3 (1:300; MP Biomedicals, catalog no. 55510), Triton X-100 was replaced with 0.2% saponin.The following reagents were pre-conjugated with chromophores: rhodamine phalloidin (1:1,000; Life Technologies, catalog no. R415), fluorescein peanut agglutinin lectin (PNA) (1:1,000; Vector Laboratories, catalog no. FL1071), and fluorescein Griffonia simplicifonia lectin I (GSL I) isolectin B4 (1:300; Vector Laboratories, catalog no. FL-1201). Nuclei were counterstained with 4',6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich, catalog no. 9542). All secondary antibodies (1:500, donkey) were purchased from Jackson Immuno Research and were purified F(ab)2 fragments that showed minimal cross-reactivity with other species. The exception was immunohistochemical staining, which used the ImmPACT VIP kit (Vector Laboratories, catalog number SK-4605). Expression changes in ApoB, ApoE, C3, and CFH were confirmed in at least three individual animals per genotype. All images were visualized using a Leica DM6 Thunder microscope equipped with a 16-bit monochrome camera.

[0079] Quantification of RPE multinucleation and cell size RPE whole specimens were collected and stained with anti-ZO1 antibody by immunofluorescence to highlight RPE cell boundaries. Quantification was performed within a 1.5 mm radius from the center, covering each 22,500 μm area. 2Ten images were selected. Because the distribution of affected areas may be random in control and experimental mice, the 10 most affected areas within one RPE flat were selected, avoiding the GA area in the experimental mice. Images for quantification were acquired at 20x magnification. IMARIS software was used to quantify the number of nuclei per RPE cell and cell area within a given image. Each image contained 30–50 RPE cells, meaning that 300–500 RPE cells were analyzed per RPE flat to calculate the average number of nuclei per RPE cell and average RPE cell size. Each experimental group consisted of 6–8 RPE flats. Age and number of RPE flats per group are listed in the corresponding figure legends.

[0080] Analysis of POS clearance by RPE POS clearance was quantified: 40,000 μm within a 1.5 mm radius from the center for each RPE flat specimen. 2 Ten areas were randomly selected to quantify the number of rhodopsin-positive dots per RPE cell. Images for quantification were acquired at 20x magnification. RPE cell boundaries were detected using an anti-ZO1 antibody. Quantification was performed by selecting dot diameters greater than 2 μm for dot counting and counting the number of RPE cells per imaged field using an IMARIS imaging processor. The average number of dots per RPE cell for a given RPE flat specimen was obtained by averaging the results from 10 fields. This number was then used to generate the average number of biological replicates, as displayed in individual figures for each genotype and time point. All POS clearance experiments were performed using 2-month-old mice (except for 6-month-old mice fed a DHA-enriched diet for 2 weeks).

[0081] Quantification of rod viability Quantification of rod viability was performed. Six retinas per group were used per quantification. Retinal sections were cut from the dorsal to the ventral direction. TUNEL assay. TUNEL assay (Roche, catalog number 12156792910) was performed according to the manufacturer's instructions. After the TUNEL reaction, tissues were processed for immunofluorescence staining as described above. Semi-thin and transmission electron microscopy (EM) were performed.

[0082] Lipid profiling Each biosample consisted of two retinas obtained from the same animal. The following number of biosamples were used: 桿状体 Tsc1 - / - = 9 cases; 桿状体 Tsc1 + / + = 6 cases; 錐体 Tsc1 - / - = 6 cases; 錐体&桿状体 Tsc1 - / -For the POS preparation, six retinas from three animals per genotype were pooled. Briefly, tissues were homogenized in 40% aqueous methanol and then diluted 1:40 with 2-propanol / methanol / chloroform (4:2:1 v / v / vol) containing 20 mM ammonium formate and 1.0 μM PC (14:0 / 14:0), 1.0 μM PE (14:0 / 14:0), and 0.33 μM PS (14:0 / 14:0) as an internal standard. Samples were introduced into a triple quadrupole mass spectrometer (TSQ Ultra, Thermo Scientific) using a chip-based nanoESI source (Advion NanoMate) operated in infusion mode. PC lipids were measured using precursor ion scanning at m / z 184, PE lipids were measured using neutral loss scanning at m / z 141, and PS lipids were measured using neutral loss scanning at m / z 185. All species detected per group are expressed as a relative percentage (%) of the total based on their response values. The abundances of lipid molecular species were calculated using Lipid Mass Spectrum Analysis (LIMSA) software (University of Helsinki, Helsinki, Finland).

[0083] statistical analysis Multiple t-tests were used for two-group comparisons, and two-way analysis of variance was used for comparisons of three or more groups. Both analysis types were two-tailed. Significance levels: * p<0.05; ** p<0.01; *** p<0.001; **** *p<0.0001. All bar graphs show the mean and error bars represent SEM. Fundus analysis bar graphs show the percentage of mice that developed the indicated retinal pathology, while error bars represent the margin of error calculated using a 90% confidence level.

[0084] Expression of HK2 and PKM2 is increased in PR of AMD patients To clarify whether PR metabolism differs in individuals with AMD, we investigated the expression of these two key metabolic genes in human donor eyes with and without AMD. On retinal sections, we observed increased expression of PKM2 and HK2 in PR from AMD patients (n = 3), with the greatest increase found in cones (Figure 5A and Figures 11A-11B). Regarding PKM2 expression in non-diseased retina, its expression was low, as these sections required up to 5x longer exposure to histochemical reagents to reveal a strong signal (Figure 5A). To allow for more linear comparisons between samples, we repeated the experiment using immunofluorescence (Figure 11A). A 2-fold increase in the signal between non-diseased and diseased tissue was sufficient to reveal the PR signal in non-diseased tissue without overexposing the signal in diseased retina. Expression of both genes in mice was observed to decline with age (Figure 11C). The data show that levels of HK2 and PKM2 are increased in PR of individuals with AMD, suggesting that glucose utilization is reduced in diseased individuals.

[0085] 桿状体 Tsc1 - / - Mice develop advanced AMD pathology To clarify the effects of metabolic changes on retinal and RPE health in wild-type mice, we used the Cre-lox system to knock out the Tsc1 gene (hereafter referred to as 桿状体 Tsc1 - / - mTORC1 was constitutively activated in rods by a phospho-p-S6 (referred to as mTORC1). mTORC1 activity was confirmed by immunofluorescence and Western blot analysis for phosphorylated ribosomal protein S6 (p-S6) (Figure 5B-C). Similarly, PR metabolic changes were confirmed by quantifying retinal PKM2, lactate, and NADPH levels (Figure 5C-E).

[0086] 桿状体 Tsc1 - / -To determine whether mice would develop advanced AMD-like pathology, mice were followed for 18 months (18M) by fundus examination and fluorescein angiography (Fig. 6 and Fig. 12). At 2 months, migration and accumulation of microglia into the subretinal space was observed, and at 4 months, the formation of retinal folds, some of which were filled with microglia, was observed (Fig. 13). Flat mount and section analysis revealed highly autofluorescent RPE cells on the side opposite the folds (Fig. 7A-B), suggesting acute RPE cell damage or loss in the mice.

[0087] Geographic atrophy was observed in 5% of mice at 6 months and in 25% of mice at 18 months (Figure 7C). GA also overlapped with areas of retinal folds, but the presence of these folds is not required for the development of GA. In general, pathology worsened with age in the same animals (Figure 12). To confirm that the area of GA correlates with regional PR atrophy and that RPE atrophy precedes PR atrophy, we compared the RPE and corresponding retina by flat-mount analysis (Figures 8A-C), identifying intermediate RPE pathology (Figure 8D), and also performed semithin sections through the area of GA, which were identified by optical coherence tomography (OCT) (Figures 8E-F).

[0088] Neovascular pathology was observed to reach a frequency of 7% (lower than GA) by 18 months (Figure 7C), most of which coincided with areas of GA. Retinal neovascular pathology was routinely detected in semithin sections (Figure 8F), but choroidal neovascular pathology was not evident on RPE flat specimens. Except for subretinal microglial accumulation, heterozygous neovascular pathology was not observed. 桿状体 Tsc1 + / - None of the mice were Cre- - Littermate control mice ( 桿状体 Tsc1 + / + ) did not develop advanced pathology (Figures 7B-7C). Consistent with this, neither activation of mTORC1 nor increased expression levels of PKM2 桿状体 Tsc1 + / -It was minimal in mice (Fig. 5C).

[0089] To clarify whether RPE stress and atrophy also occurred in regions outside of GA, we determined the percentage of multinucleated RPE cells and measured changes in RPE cell size in non-GA areas. At 18 months, we found a significant increase in multinucleated, denucleated, and hypertrophic RPE cells (Figure 8G). The data suggest that loss of Tsc1 within rods contributes to widespread RPE pathology that causes localized GA in some animals. We next investigated whether changes occurred in overall PR viability and function. Consistent with widespread RPE pathology, a slight decrease in PR layer thickness was observed at 18 months (Figure 14A). Rod a-wave amplitude was significantly increased. 桿状体 Tsc1 - / - The amplitude was higher in HK2 mice at early time points, but declined to that of littermate controls by 18 months (Fig. 14B). The higher initial amplitude is consistent with the observation that loss of HK2 reduces the dark adaptation response and reduces retinal lactate and NADPH levels. Therefore, the higher initial amplitude may reflect higher energy availability. Alternatively, increased transcription or translation of phototransduction genes due to increased PKM2 expression or increased mTORC1 activity, respectively, may be responsible for the increased dark adaptation response and reduced retinal lactate and NADPH levels. 桿状体 Tsc1 - / - This may also explain the higher a-wave amplitude in mice. C-wave amplitude, which in part reflects RPE health, 桿状体 Tsc1 - / - There was no difference between mice and controls (Figure 14D). Overall, the data suggest that loss of Tsc1 within rods slows disease progression, except in areas where advanced pathology has been established.

[0090] To confirm that GA was not caused by aberrant CRE recombinase expression in the RPE, RPE flat specimens were stained for p-S6. 桿状体 Tsc1 - / -In both mice and controls, CRE recombinase expression was observed in p-S6-positive cells at 2 months (Figure 15A), but not in p-S6-positive cells (Figure 15B). Furthermore, the number of p-S6-positive cells dramatically increased with age (Figures 15A and 15C). This increase is likely due to the fact that increased mTORC1 activity in the RPE is associated with RPE dysfunction, senescence, and cell loss. 桿状体 Tsc1 - / - This may reflect an increase in the number of diseased RPE cells in the mice.

[0091] 桿状体 Tsc1 - / - Mice also show early disease features It has been proposed that the metabolic demands of the PR contribute to lipoprotein accumulation and drusen formation. To clarify whether metabolic changes induced in the PR also contribute to lipoprotein accumulation, we investigated the distribution of ApoB and ApoE in the BrM. We observed accumulation of both lipoproteins in the basal layer of the RPE and the BrM, independent of any advanced pathology (Figure 16A). Electron microscopy (EM) analysis revealed neutral lipid and basal layer deposits within the BrM, as well as thickened BrM in areas of GA (Figure 16B). However, drusen-like deposits were not observed; rather, basal prominences were quite common (Figure 16C). Increased autofluorescence was also observed. 桿状体 Tsc1 - / - This was observed in the mouse RPE, suggesting increased lipofuscin accumulation (Figure 16D).

[0092] 桿状体 Tsc1 - / - In mice, we observed a uniform downregulation of C3 and a uniform upregulation of CFH in the BrM (Fig. 16A). The data suggest that this early disease feature, induced by mTORC1 activation in rods, occurs uniformly throughout the tissue, regardless of the presence of any advanced pathology.

[0093] AMD-like pathology depends on the dose of activated mTORC1 To test whether mTORC1 is required for the observed pathology, we used mice simultaneously deficient in Tsc1 and the mTORC1 adaptor protein Raptor ( 桿状体 Tsc1 - / -桿状体 Raptor - / - Heterozygous Raptor mice (referred to as rhabdomyosarcoma mice) were acquired. Fundus imaging revealed no pathology except for microglial accumulation (observed in 76% of 12- to 18-month-old mice) (Figures 8A and 8B). 桿状体 Tsc1 - / -桿状体 Raptor - / + ) did not develop any GA or neovascular pathology by 12 months (Figure 8B). However, retinal folds were present, albeit at a low frequency. The absence of any severe pathology was consistent with quantification of multinucleated RPE cells and RPE cell size, revealing no substantial differences between these strains at 12 months (Figure 8C). Western blot analysis for p-S6 and PKM2 confirmed reduced mTORC1 activity (Figure 8E). 桿状体 Tsc1 - / -桿状体 Raptor + / - The p-S6 level in 桿状体 Tsc1 - / - PKM2 levels showed a dose-dependent decline when compared with mice. 桿状体 Tsc1 - / - PKM2 levels remained similar to those in heterozygotes (compare Figure 8D with Figure 5C). 桿状体 Tsc1 - / -桿状体 Raptor + / - In mice, lactate and NADPH levels increased with Cre expression. - The accumulation of ApoB, ApoE, C3, and CFH was analyzed to clarify the extent to which this affected early pathology. The accumulation of these markers was 桿状体 Tsc1 - / -桿状体 Raptor - / - In mice, the gene was restored to normal, but heterozygous 桿状体 Tsc1 - / -桿状体 Raptor + / -Mice showed a more intermediate phenotype (Fig. 8G). Little accumulation of ApoB was observed, whereas ApoE accumulation was 桿状体 Tsc1 - / - The accumulation was similar to that observed in mice. Similarly, CFH showed very little accumulation, and C3 was substantially reduced. The data suggest that the development of early and late pathology is promoted in a dose-dependent manner by increased mTORC1 activity.

[0094] RPE phagocytosis 桿状体 Tsc1 - / - Variable in mice Impaired RPE lysosomal activity is associated with AMD. The nature of RPE cell stress is uniform, resulting in impaired POS clearance. 桿状体 Tsc1 - / - We investigated whether rod POS clearance varies in mice. Because rod POS outflow is diurnal, clearance can be monitored over time on RPE flat preparations stained for rhodopsin protein. Rod POS clearance is 桿状体 Tsc1 - / - In mice, a significant slowing was observed at 2 months, and 桿状体 Tsc1 - / -桿状体 Raptor - / - The effect was rescued in mice, suggesting that this effect was due to increased mTORC1 activity in rods (Figures 9A-C).

[0095] POS are rich in lipids, and mTORC1 is known to regulate lipid synthesis. To clarify the cause of delayed POS clearance by the RPE, 桿状体 Tsc1 - / - The retinal lipid composition of mice was profiled. A roughly three-fold decrease in diDHA (44:12)-containing phosphatidylethanolamine (PE) and phosphatidylcholine (PC) lipids was observed in whole retina preparations (Figure 9D) and POS preparations (Figure 9E). To test whether this decrease in diDHA-type PE and PC lipids contributes to the delay in POS clearance, 桿状体 Tsc1- / - Mice were fed a diet enriched with 2% DHA. 桿状体 Tsc1 - / - Feeding mice a 2% DHA-enriched diet improved POS clearance at 2 months (Fig. 9F). To test whether delayed POS clearance could be improved even after it had already occurred, we administered DHA to mice at 6 months of age. 桿状体 Tsc1 - / - Mice were fed a DHA-enriched diet for 2 weeks, which had an even more pronounced effect, as POS clearance was more affected at 6 months (Figure 9G).

[0096] To clarify whether dietary DHA also affects the overall health of the RPE, mice were fed a DHA diet from weaning until 6 months of age. This reduced the percentage of multinucleated RPE cells (Figure 9H), improved fundus pathology (Figure 9I), prevented the accumulation of ApoB, ApoE, and CFH, and restored C3 expression (Figure 9J). Differences in RPE hypertrophy were not evident in younger mice, likely because hypertrophy was less pronounced. None of the 12 DHA-fed mice (n = 12) developed any GA by 6 months, whereas one of six mice fed the control diet did. Re-profiling of retinal lipids after 10 weeks of DHA feeding revealed that the levels of di-DHA-containing PE and PC lipids were not restored. This suggests that DHA may be acting directly on the RPE to improve overall RPE health (Figure 9K). Overall, the data suggest that activation of mTORC1 in rods affects retinal lipid composition and impacts the overall health of the RPE.

[0097] Contribution of cones to disease differs from rods A cell line with cone-specific Tsc1 deletion ( 錐体 Tsc1 - / - ), and cell lines with defects in rods and cones ( 錐体&桿状体 Tsc1 - / - ) was obtained by fundus examination and angiography. 錐体Tsc1 - / - Mice were found to develop similar pathology without the formation of retinal folds (Figure 10A). The combined metabolic changes in rods and cones did not increase the overall frequency of advanced pathology by 12 months. However, advanced pathology had already begun to develop at 4 months (Figure 10A). 錐体 Tsc1 - / - Choroidal neovascular pathology in mice 桿状体 Tsc1 - / - It was easier to identify on RPE flat preparations when compared to mice (FIG. 10B). 錐体 Tsc1 - / - and 錐体&桿状体 Tsc1 - / - The mice also developed large drusen-like deposits that were positive for ApoE (Fig. 10C and 10D). 桿状体 Tsc1 - / - EM analysis revealed that loss of Tsc1 in cones was sufficient to cause the accumulation of small lipoprotein vesicles reminiscent of basal linear deposits within the BrM (Fig. 10E), which may explain the difference in deposit size. Finally, the GA area 桿状体 Tsc1 - / - or 錐体 Tsc1 - / - When compared to a mouse, 錐体&桿状体 Tsc1 - / - The RPE was generally larger in the 3-lineage mice (Figure 10F). This allowed visualization of the ongoing RPE atrophy by TUNEL (Figure 10F). All other pathology, such as uniform accumulation of lipoproteins and altered C3 and CFH expression, was similar in all 3 lines, but 錐体 Tsc1 - / - Mice showed significant but minimal changes (Figures 17A-17B). Rod POS clearance was 錐体 Tsc1 - / - This is also affected in mice, and loss of Tsc1 in cones affects rod POS clearance (Figure 17C). 錐体Tsc1 - / - The di-DHA-type PE lipids were also significantly reduced in mice (Figure 17D), suggesting that reduction of both lipids may affect RPE health. Overall, the data suggest that there are distinct mechanisms contributing to advanced AMD pathology between rods and cones (consistent with observations in humans). [Example]

[0098] Age-related macular degeneration (AMD) is one of the leading causes of visual impairment in the elderly. The disease is multifactorial, involving both genetic and non-genetic risk factors. Diets rich in omega-3 fatty acids, particularly docosahexaenoic acid (DHA), have been shown to reduce disease risk (e.g., Souied, E. et al., "Omega-3 Fatty Acids and Age-Related Macular Degeneration," Ophthalmic Res 55, 62-69, (2015)). Similarly, high DHA plasma levels correlate with reduced disease risk (e.g., Merle, BM et al., "High concentrations of plasma n3 fatty acids are associated with decreased risk for late age-related macular degeneration," J Nutr 143, 505-511, (2013)). Additionally, retinal DHA levels are reduced by 30% in individuals with AMD. Despite these findings, along with the identification of over 30 risk alleles, animal models generated to date do not accurately recapitulate the complex disease progression of AMD, nor is the role of DHA in pathogenesis fully understood.

[0099] AMD is considered a retinal pigment epithelium (RPE) disease. In the early stages of the disease, deposits called drusen form between the RPE and the underlying basement membrane, known as Bruch's membrane (BrM). Over time, these deposits increase in number and size, affecting the health of the RPE. Eventually, affected individuals progress to one of two advanced disease forms: geographic atrophy (GA) or choroidal neovascularization (CNV). In GA, widespread loss of confluent RPE leads to secondary photoreceptor (PR) death, as the RPE is responsible for transporting nutrients from the adjacent choroidal vasculature to the PR. In CNV, the choroidal vasculature breaks through Bruch's membrane, and the RPE develops retinal edema, resulting in PR loss. While CNV can be treated with vascular endothelial growth factor (VEGF) inhibitors, which prevent excessive edema formation, no treatments exist for GA or for preventing progression from the early drusen stage to the advanced stage. This is due to a lack of understanding of the causes and progression of the disease. As 85% of patients with advanced AMD develop GA, there is an unmet need to develop treatments that prevent the progression of the disease from the drusen stage to the advanced stage or prevent further progression of GA.

[0100] Although PR metabolism has been associated with both early and late disease stages, photoreceptors have long been considered bystanders in pathogenesis. Studies of the distribution of lipoprotein-rich drusen deposits (a marker of early disease stages) have revealed that two major types of pathological drusen are found in patients with AMD, whose locations reflect the density distribution of cone and rod PRs. Macular translocation procedures (used to treat late disease stages of GA) suggest that PRs may also cause this condition. Patients who underwent retinal rotation to move macular cones away from areas of dying RPE and toward healthy RPE experienced recurrence of GA in the areas where the cones had been relocated. In both cases, the high and differential metabolic demands of cones and rods have been proposed to underlie their pathogenesis. Therefore, we investigated whether the metabolic demands of PRs differ in patients with AMD. We found increased expression of two key metabolic PR genes, suggesting that PRs adapt to nutrient deprivation. Because mTORC1 controls cellular metabolism under nutrient stress, we constitutively activated mammalian target of rapamycin complex 1 (mTORC1)16 in mouse PRs to clarify the long-term effects of such metabolic adaptations. This was achieved by deleting the tuberous sclerosis complex 1 protein (TSC1). We found that the onset of pathology was age- and mTORC1-dependent (reminiscent of pathologies observed in humans, including drusen, GA, and CNV). The mouse model described in this disclosure is therefore the first animal model to develop all cardinal features of early and late disease stages. Importantly, disease progression in our mouse model depends on dietary DHA levels, and, similar to humans, our mice exhibit a reduction in certain di-DHA-containing retinal phospholipids. Our mice therefore offer an opportunity to identify new disease-causing mechanisms that lie downstream of mTORC1 and contribute to disease progression, as well as to test the efficacy of potential therapeutic candidates in delaying disease progression.

[0101] Since mTORC1 controls cellular metabolism under nutrient stress, we constitutively activated mTORC1 in mice to mimic the adaptive changes suggestive of nutrient deprivation observed in PR of AMD patients. Metabolic processes controlled by mTORC1 include glycolysis, fatty acid synthesis, protein translation, autophagy, and the activity of the second mTOR complex, mTORC2 (which also controls AKT activity). It has previously been confirmed that the pathology observed when TSC1 is lost in rods requires the activity of mTORC1. Furthermore, to confirm that the pathology was not related to the unknown function of the TSC1 protein, we selectively removed the second TSC complex protein, TSC2, in rods ( 桿状体 Tsc2 - / - ), disrupted the TSC complex. This caused global pathology and disease progression similar to that of loss of TSC1 in rods (Figures 19A-10F). The mice also showed delayed photoreceptor outer segment (POS) digestion, reduced diDHA-containing phosphatidylethanolamine (PE) and phosphatidylcholine (PC) lipids, and increased dark-adapted electroretinogram (ERG) recordings (Figures 20A-20D). Increased mTORC1 activity and aerobic glycolysis resulted in increased phosphorylated ribosomal protein S6 (p-S6) and pyruvate kinase muscle isoenzyme M2 (PKM2), both of which are involved in the activation of the TSC complex. 桿状体 Tsc2 - / - ) showed higher levels in mice (Figure 19A).

[0102] Next, to clarify which aspects downstream of mTORC1 are required for the development of early and late pathology, we investigated the mechanism by which the TSC complex was disrupted by also inhibiting the activity of hexokinase-2 (HK2). 桿状体 Tsc2 - / -桿状体 HK2 - / -) tested the contribution of glycolysis. This reduced the increase in lactate levels (Fig. 21A) and the level of the dark-adapted ERG response (Fig. 21G) caused by disruption of the TSC complex, thereby reversing some of the changes in glycolysis induced by mTORC1 activation. However, the data show that loss of HK2 in the context of mTORC1 hyperactivation still results in pathology (Fig. 21A-F) similar to that observed upon loss of TSC1 in rods or loss of TSC2 in rods (Fig. 19A-D).

[0103] Similarly, to test the contribution of the mTORC2 complex and AKT together, we generated mice simultaneously deficient in TSC1 and the mTORC2 adaptor protein Rictor ( 桿状体 Tsc1 - / -桿状体 Rictor - / - ) was generated. 桿状体 Tsc2 - / -桿状体 HK2 - / - Like the mouse, 桿状体 Tsc1 - / -桿状体 Rictor - / - The mice still developed advanced AMD pathology (FIGS. 22A-B), suggesting that alterations in glycolysis, AKT signaling, or mTORC2 activity do not contribute to advanced AMD.

[0104] The remaining processes controlled by mTORC1 are lipid synthesis, protein synthesis, and autophagy. Since autophagy and the overall increase in protein synthesis are directly controlled by mTORC1, most lipid synthesis pathways are controlled by mTORC1 in an S6K1-dependent manner. To test this theory, we generated mice lacking TSC1 and S6K1 ( 桿状体 Tsc1 - / - S6K1 - / - ) was generated. It was found that depletion of S6K1 in the absence of TSC1 completely inhibited the development of all pathologies (Figures 23A-23B). 桿状体 Tsc1 - / - S6K1 - / -At the Bruch's membrane (BrM) and RPE interface in the mouse eye, early disease stage markers, such as apolipoprotein E (ApoE) and complement factor H (CFH) accumulation, or reduced complement factor 3 (C3) expression, were all restored to their age-matched wild-type levels (Figure 24). To clarify whether a dose-dependent effect exists, heterozygous mice were treated with CFH. 桿状体 Tsc1 - / - S6K1 + / - We also tested mice with S6K1. Loss of one allele of S6K1 still prevented neovascular pathology from developing and dramatically reduced the frequency of GA (Figures 23A-B). Consistent with this data, the observed expression changes for early disease markers were variable (Figure 24). ApoE showed accumulation similar to that observed in diseased mice with both S6K1 wild-type alleles. In contrast, CFH and C3 levels were intermediate, and CHF accumulation was lower and C3 expression was higher when compared to diseased mice with both S6K1 wild-type alleles (Figures 21A-G). Collectively, the data suggest that alterations in lipid synthesis (but not autophagy or global protein synthesis) underlie the development and progression of AMD. Importantly, reducing S6K1 expression can attenuate or prevent AMD pathology in a dose-dependent manner. This suggests that either inhibiting S6K1 function or expression may be beneficial in slowing disease progression. Thus, complete inhibition of S6K1 function is not required for a successful therapeutic approach.

[0105] To test whether loss of S6K1 actually affects lipid synthesis, we profiled retinal phospholipids. We observed a significant decrease in di-DHA-containing phosphatidylethanolamine (PE) and phosphatidylcholine (PC) lipids in mice lacking TSC1. Similarly, in mice lacking TSC2 in rods, we found a strong decrease in di-DHA-containing PE and PC lipids, although baseline levels differed between the two strains (Figures 20A–20D). This may suggest background differences between the strains rather than differences due to loss of TSC1 or TSC2. Interestingly, loss of S6K1 resulted in a dose-dependent increase in these two di-DHA-containing phospholipids, regardless of mTORC1 hyperactivation (Figure 25). Complete loss of S6K1 resulted in an approximately 30% increase, similar to the decrease in retinal DHA levels (approximately 30%) observed in patients with AMD. Because feeding mice a DHA-enriched diet prevented disease progression, the data suggest that part of the protective effect mediated by the loss of S6K1 may be due to increased retinal DHA levels. The protective effect of dietary DHA is further highlighted by more than 15 epidemiological studies and studies linking high blood omega-3 fatty acid levels with reduced disease risk. Finally, a small-scale human study using omega-3 fatty acid levels five times higher than those in the NIH-funded AREDS2 study revealed the protective effect of dietary omega-3 fatty acids such as DHA in reducing the risk of disease progression. Importantly, in our mice lacking TSC1, although pathology was significantly alleviated, retinal di-DHA levels of PE and PC lipids did not increase after feeding DHA (Figure 26). DHA may directly act on the RPE to improve its overall health. This approach requires high levels of DHA supplementation. In contrast to control wild-type mice, feeding DHA increased retinal di-DHA levels in PE and PC lipids to a similar level as observed when S6K1 expression was lost. Genetic approaches that reduce S6K1 expression levels or activity therefore allow for increased DHA levels in the retina without the need for excessive dietary supplementation.Because the RPE phagocytose DHA-rich POS, increasing retinal DHA levels by reducing or inhibiting S6K1 is more beneficial than increasing DHA levels in the RPE through high-dose dietary DHA supplementation. Furthermore, because reduced retinal DHA levels caused by excessive S6K1 activity are unlikely to be the sole cause of AMD development and progression, therapeutically reducing S6K1 by knocking down or inhibiting its function is a better therapeutic approach.

[0106] Finally, to verify that S6K1 activity is indeed increased in patients with AMD, we performed immunohistochemistry for p-S6 on retinal sections from healthy individuals and patients with AMD. p-S6 is one of the canonical targets of S6K1 and is therefore a bona fide readout of S6K1 activity. Similarly, S6K1 is a bona fide target of mTORC1. Therefore, increased levels of p-S6 imply the presence of increased mTORC1 and increased S6K1 activity. The results showed that p-S6 levels were significantly increased in PRs from AMD patients (Figure 27), suggesting that the proposed mechanism of action is indeed correct. Increased activation of mTORC1 in PRs from AMD patients contributes to advanced pathology through increased activation of S6K1, one of the canonical targets of mTORC1 activation.

[0107] equivalent While several embodiments of the present invention have been described and illustrated herein, those skilled in the art will readily conceive of various other means and / or structures for performing the functions and / or obtaining one or more of the results and / or advantages described herein, and each such variation and / or modification is deemed to be within the scope of the present invention. More generally, those skilled in the art will readily recognize that all parameters, dimensions, materials, and configurations described herein are intended to be exemplary, and that the actual parameters, dimensions, materials, and / or configurations will depend on the particular application(s) in which the teachings of the present invention are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. It is therefore to be understood that the above-described embodiments are presented by way of example only, and that within the scope of the appended claims and their equivalents, the invention may be practiced otherwise than as specifically described and claimed. The present invention relates to each individual feature, system, article, material, and / or method described herein. Additionally, any combination of two or more such features, systems, articles, materials, and / or methods is within the scope of the present invention, where such features, systems, articles, materials, and / or methods are not inconsistent with one another.

[0108] The indefinite articles "a" and "an," as used in the specification and claims, unless clearly indicated to the contrary, should be understood to mean "at least one."

[0109] The phrase "and / or," as used in the specification and claims, should be understood to mean "either or both" of the elements so connected, i.e., elements present conjunctively in some cases and non-conjunctively in other cases. Other elements, whether related or unrelated to the elements specifically identified, may optionally be present, unless evidence to the contrary is clearly present. Thus, as a non-limiting example, "A and / or B," when used in conjunction with open-ended language such as "comprising," may refer, in one embodiment, to A without B (optionally including elements other than B); in another embodiment, to B without A (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements), etc.

[0110] As used in this specification and the claims, "or" should be understood to have the same meaning as "and / or" as defined above. For example, when separating items in a list, "or" or "and / or" should be interpreted as inclusive, i.e., including at least one, but also including two or more of several or a series of elements, and optionally additional items not listed. Only when the term clearly indicates the contrary, such as "only one of" or "exactly one of," or when used in the claims, "consisting of" refers to the inclusion of exactly one element of several or a series of elements. In general, the term "or," as used herein, when preceded by terms of exclusivity, such as "either," "one of," "only one of," or "exactly one of," should be interpreted as indicating exclusive alternatives (i.e., "one or the other, but not both"). "Consisting essentially of," when used in the claims, shall have its ordinary meaning as used in the field of patent law.

[0111] As used herein and in the claims, the phrase "at least one," in connection with a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements included in the list of elements, but not necessarily including at least one of every element specifically listed in the list of elements, and not excluding any combination of elements included in the list of elements. This definition also allows for the optional presence of elements other than those specifically identified in the list of elements to which the phrase "at least one" refers, whether related or not to the elements specifically identified. Thus, as a non-limiting example, "at least one of A and B" (or, equivalently, "at least one of A or B," or, equivalently, "at least one of A and / or B") could refer in one embodiment to at least one, optionally including more than one, i.e., A, with no B present (and optionally including elements other than B); in another embodiment to at least one, optionally including more than one, i.e., B, with no A present (and optionally including elements other than A); in yet another embodiment to at least one, optionally including more than one, i.e., A, and at least one, optionally including more than one, i.e., B (optionally including other elements), etc.

[0112] In the claims, as well as in the above specification, all transitional phrases, such as "comprising," "including," "carrying," "having," "containing," "involving," "holding," and the like, are understood to be open-ended, i.e., to mean "including, but not limited to." Only the transitional phrases "consisting of" and "consisting essentially of" shall be closed or semi-closed transitional phrases, respectively, as defined in Section 2111.03 of the United States Patent Office Manual of Patent Examining Procedure.

[0113] The use of ordinal terms in the claims, e.g., "first," "second," "third," etc., to modify claim elements does not, by itself, imply any priority, precedence, or ranking of one claim element over another, or the chronological order in which the actions of a method are performed, but is merely used as a marker to distinguish one claim element having a particular name from another element having the same name (but excluding the use of ordinal terms) to distinguish the claim elements.

[0114] array All NCBI gene and accession number sequences are incorporated herein by reference.

Claims

1. A composition administered to a subject at the drusen stage to inhibit drusen formation in the ocular tissue of the subject, the composition comprising one or more inhibitors of ribosomal protein S6 kinase beta-1 (S6K1), the one or more inhibitors of S6K1 comprising an inhibitory nucleic acid that binds to a nucleic acid encoding the S6K1 protein and reduces or prevents expression of the S6K1 protein.

2. The composition of claim 1 , wherein the ocular tissue comprises Bruch's membrane tissue, retinal pigment epithelium (RPE) tissue, macular tissue, or a combination thereof.

3. 3. The composition of claim 1 or 2, wherein the ocular tissue comprises photoreceptor cells, retinal pigment epithelial cells (RPE), ganglion cells, or a combination thereof.

4. 4. The composition of claim 1, wherein the composition is formulated to be administered by topical administration, intravitreal administration, subconjunctival injection, intrachoroidal injection, systemic injection, or any combination thereof.

5. The one or more S6K1 inhibitors further comprise a small molecule, peptide, protein, or antibody; the small molecule is PF-4708671, rosmarinic acid methyl ester (RAME), A77 1726, or a salt, solvate, or analog thereof, which directly binds to S6K1 and inhibits its activity; the peptide is a peptide that targets S6K1, the protein is a dominant negative S6K1 protein that directly binds to S6K1 and inhibits the activity of S6K1; The composition of claim 1 , wherein the antibody is an S6K1 antibody.

6. The composition of claim 1, wherein the inhibitory nucleic acid is a dsRNA, siRNA, shRNA, miRNA, ami-RNA, antisense oligonucleotide (ASO), or aptamer.

7. The composition of claim 5 , wherein the small molecule is a selective inhibitor of S6K1.

8. 8. The composition of claim 1, wherein the S6K1 inhibitor does not bind to or inhibit the expression or activity of mammalian target of rapamycin 1 (mTORC1).

9. 9. The composition of any one of claims 1 to 8, wherein the composition is formulated to, upon administration, reduce drusen formation in the ocular tissue by about 2-fold, 3-fold, 5-fold, 10-fold, 50-fold, 100-fold, or more than 100-fold compared to ocular tissue to which the composition has not been administered.

10. 10. The composition of any one of claims 1 to 9, wherein the ocular tissue is in vivo, and optionally the ocular tissue is present in the eye of a subject.

11. A composition for treating age-related macular degeneration (AMD) in an ocular tissue of a subject, the composition comprising one or more inhibitors of ribosomal protein S6 kinase beta-1 (S6K1), the composition being configured to be administered to the subject, the one or more inhibitors of S6K1 comprising an inhibitory nucleic acid that binds to a nucleic acid encoding an S6K1 protein and reduces or prevents expression of the S6K1 protein.

12. The composition of claim 11 , wherein the ocular tissue comprises Bruch's membrane tissue, retinal pigment epithelium (RPE) tissue, macular tissue, or a combination thereof.

13. 13. The composition of claim 11 or 12, wherein the ocular tissue comprises photoreceptor cells, retinal pigment epithelial cells (RPE), ganglion cells, or a combination thereof.

14. 14. The composition of any one of claims 11 to 13, wherein the composition is formulated to be administered by topical administration, intravitreal administration, subconjunctival injection, intrachoroidal injection, systemic injection, or any combination thereof.

15. The one or more S6K1 inhibitors further comprise a small molecule, peptide, protein, or antibody; the small molecule is PF-4708671, rosmarinic acid methyl ester (RAME), A77 1726, or a salt, solvate, or analog thereof, which directly binds to S6K1 and inhibits its activity; the peptide is a peptide that targets S6K1, the protein is a dominant negative S6K1 protein that directly binds to S6K1 and inhibits the activity of S6K1; The composition of any one of claims 11 to 14, wherein the antibody is an S6K1 antibody.

16. The composition of claim 11, wherein the inhibitory nucleic acid is a dsRNA, siRNA, shRNA, miRNA, ami-RNA, antisense oligonucleotide (ASO), or aptamer.

17. The composition of claim 15, wherein the small molecule is a selective inhibitor of S6K1.

18. 18. The composition of any one of claims 11 to 17, wherein the S6K1 inhibitor does not bind to or inhibit the expression or activity of mammalian target of rapamycin 1 (mTORC1).

19. 19. The composition of any one of claims 11 to 18, wherein the composition is formulated to, upon administration, reduce drusen formation in the ocular tissue by about 2-fold, 3-fold, 5-fold, 10-fold, 50-fold, 100-fold, or more than 100-fold compared to ocular tissue that has not been administered the one or more S6K1 inhibitors.

20. 20. The composition of any one of claims 11 to 19, wherein the ocular tissue is in vivo, and optionally the ocular tissue is present in the eye of a subject.

21. 21. The composition of any one of claims 11 to 20, wherein the composition is formulated to be administered to the subject in conjunction with an effective amount of di-docosahexaenoic acid (DHA).

22. 22. The composition of claim 21, wherein the DHA is a dietary supplement.

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