Methods and compositions for treating age-related diseases

Abstract

A composition and method for treating age-related eye disease and / or age-related brain disease in subjects requiring treatment for age-related eye disease and / or age-related brain disease. The composition comprises tetracosapentaenoic acid [24:5n-3]. The method comprises the steps of identifying a subject exhibiting age-related eye disease and administering tetracosapentaenoic acid [24:5n-3] to the subject.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the benefit of U.S. Provisional Application No. 63 / 586,251, filed on 28 September 2023, the specification of which is incorporated herein by reference in its entirety.

[0002] The present invention provides methods and compositions for treating age-related eye diseases. Furthermore, the present invention provides methods and compositions for treating age-related brain diseases. [Background technology]

[0003] The specific composition of lipids within a membrane determines biophysical properties such as diffusion, permeability, domain formation, and curvature generation. Age-related changes in membrane lipid composition are considered to be one of the characteristics of aging. In aged retinas, levels of polyunsaturated fatty acids (PUFAs), essential components of cell membranes, are significantly reduced, and this reduction is further exacerbated in retinas affected by age-related macular degeneration (AMD). Retinal tissue is particularly rich in long-chain and very-long-chain PUFAs (LC-PUFAs and VLC-PUFAs, respectively), essential components of photoreceptor disc membranes, and their depletion or reduction in levels is considered to be one of the characteristics of AMD.

[0004] Strategies aimed at maintaining or supplementing VLC-PUFAs in the aging retina, such as dietary therapy using n-3 polyunsaturated pufas (PUFAs) and LC-PUFAs, are being studied as promising approaches to maintain retinal health and function in older adults. While some studies have shown improved vision with docosahexaenoic acid (DHA) supplementation or supplementation with both DHA and eicosapentaenoic acid (EPA), others, such as the Age-Related Eye Disease Study 2 (AREDS2), have not reported such a correlation and have concluded that DHA and EPA supplementation trials are inconclusive. Interestingly, very high doses of VLC-PUFAs administered orally to animals have yielded promising results. Another study showed some improvement in visual ability in a dietary supplementation experiment using fish oil concentrated with n-3 C24-28 VLC-PUFAs. However, the high cost of synthesizing VLC-PUFAs, limited access to concentrated fish oil, and their relatively low efficacy mean that these methods currently lack practical applicability for preventing age-related vision loss in humans. [Overview of the project]

[0005] The object of the present invention is to provide a method and composition that enables the treatment of age-related eye and brain diseases in the subject requiring treatment, as specified in the independent claims. Embodiments of the present invention are described in the dependent claims. Embodiments of the present invention can be freely combined, as long as they are not mutually exclusive.

[0006] The precise mechanisms underlying the decline of VLC-PUFAs in aging and disease are not yet fully understood. The elongation and desaturation of essential fatty acids are tightly regulated and involve several enzymatic steps. The key enzyme involved in the elongation of LC- and VLC-PUFAs is ELOVL2 (Extra-Long Chain Fatty Acid Elongation Enzyme 2), whose gene encodes an endogenous endoplasmic reticulum membrane protein that produces precursors of DHA and VLC-PUFAs. ELOVL2 is primarily expressed in tissues with high metabolic demands, such as the liver, retina, and brain. Importantly, increased methylation of the ELOVL2 regulatory region has been shown to be one of the best biomarkers of aging by chronological age.

[0007] Therefore, this invention identifies and describes key molecular, structural, and functional changes in aging retinas, correlating many of these phenotypes with ELOVL2 activity deficiency and lipid composition disturbances. Intravitreal injection of 24:5n-3, a direct product of ELOVL2 elongation, improves visual function, reduces the severity of aging phenotypes, and decreases the accumulation of subepithelial retinal pigment deposits in aged mice. Notably, other PUFAs did not exhibit such remarkable effects. This invention highlights the importance of ELOVL2 activity in maintaining healthy vision and suggests the potential of novel therapeutic approaches to reverse aging symptoms of the eye and prevent age-related eye diseases such as AMD.

[0008] In some embodiments, the present invention features a method for treating age-related eye diseases (e.g., age-related macular degeneration and / or age-related vision loss / decline) in subjects requiring treatment. In some embodiments, the method includes administering a composition of tetracosapentaenoic acid [24:5n-3] to a subject, for example, to the subject's eye. In other embodiments, the method includes administering a composition of tetracosapentaenoic acid [24:5n-3] and docosahexaenoic acid (DHA) to a subject, for example, to the subject's eye. In some embodiments, tetracosapentaenoic acid [24:5n-3] and / or DHA are administered as free fatty acids, fatty acid ethyl esters (FAEEs), or fatty acid methyl esters (FAMEs). The compositions may be administered by intravitreal injection, intravenous injection, intraperitoneal injection, or topically. The present invention may also feature compositions for use in treating age-related eye diseases in subjects requiring treatment for age-related eye diseases. In some embodiments, the composition comprises tetracosapentaenoic acid [24:5n-3]. In other embodiments, the composition comprises tetracosapentaenoic acid [24:5n-3] and DHA.

[0009] In some embodiments, the present invention features a method for treating age-related liver disease (e.g., non-alcoholic fatty liver disease, alcoholic liver disease, hepatitis C, fibrosis, or cirrhosis) in subjects requiring treatment. In some embodiments, the method includes administering a composition comprising tetracosapentaenoic acid [24:5n-3] to the subject. In other embodiments, the method includes administering a composition comprising tetracosapentaenoic acid [24:5n-3] and DHA to the subject. In some embodiments, tetracosapentaenoic acid [24:5n-3] and / or DHA are administered as free fatty acids, FAEE, or FAME. The composition may be administered via intravenous injection. The present invention may also feature a composition for use in treating age-related liver disease in subjects requiring treatment of age-related liver disease. In some embodiments, the composition comprises tetracosapentaenoic acid [24:5n-3]. In other embodiments, the composition comprises tetracosapentaenoic acid [24:5n-3] and DHA.

[0010] In other embodiments, the present invention features a method of treating an age-related brain disease (e.g., Alzheimer's disease, ALS, age-related cognitive decline, dementia, and Parkinson's disease) in a subject in need of the treatment. In some embodiments, the method comprises administering to the subject, for example to the brain of the subject, a composition comprising tetracosapentaenoic acid [24:5n-3]. In other embodiments, the method comprises administering to the subject, for example to the brain of the subject, a composition comprising tetracosapentaenoic acid [24:5n-3] and DHA. In some embodiments, tetracosapentaenoic acid [24:5n-3] and / or DHA are administered as free fatty acids, FAEE or FAME. The composition may be administered by intravenous injection and / or stereotactic direct injection. The present invention may also feature a composition for use in treating an age-related brain disease in a subject in need of the treatment of the age-related brain disease. In some embodiments, the composition comprises tetracosapentaenoic acid [24:5n-3]. In other embodiments, the composition comprises tetracosapentaenoic acid [24:5n-3] and DHA.

[0011] In further embodiments, the present invention features a method of treating an age-related bone disease (e.g., multiple myeloma and other myelodysplastic syndromes (MDS), or anemia) in a subject in need of the treatment. In some embodiments, the method comprises administering to the subject a composition comprising tetracosapentaenoic acid [24:5n-3]. In other embodiments, the method comprises administering to the subject a composition comprising tetracosapentaenoic acid [24:5n-3] and DHA. In some embodiments, tetracosapentaenoic acid [24:5n-3] and / or DHA are administered as free fatty acids, FAEE or FAME. The composition may be administered by intravenous administration and / or intravenous injection. The present invention may also feature a composition for use in treating an age-related bone disease in a subject in need of the treatment of the age-related bone disease. In some embodiments, the composition comprises tetracosapentaenoic acid [24:5n-3]. In other embodiments, the composition comprises tetracosapentaenoic acid [24:5n-3] and DHA.

[0012] One of the unique and inventive technical features of the present invention is to directly administer tetracosapentaenoic acid [24:5n-3] to the eye. While not intending to limit the present invention to any theory or mechanism, it is believed that the technical feature of the present invention is advantageous for slowing the progression of, reversing the condition of, or curing ocular diseases. None of the currently known prior references or works possess the unique and inventive technical features of the present invention. For example, conventionally, oral administration of n-3 PUFAs has been regarded as the standard administration method. Therefore, attempting a direct intravitreal administration mode would not be considered obvious.

[0013] Furthermore, the inventive technical features of the present invention contributed to surprising results. For example, 24:5n-3 is found in very small amounts in the eye, and its level in diseased eyes does not change significantly compared with that in healthy eyes. Accordingly, a person skilled in the art would not expect 24:5n-3 to improve visual function. However, it has surprisingly been found that injection of 24:5n-3 can improve visual function and bring about the reversal or cure of eye-related diseases.

[0014] Any feature or combination of features described herein is included within the scope of the present invention, as is apparent from the context, the present specification, and the knowledge of those skilled in the art, provided that the features included in such combination are not mutually inconsistent. Further advantages and aspects of the present invention will be apparent in the following detailed description and claims.

[0015] The features and advantages of the present invention will become apparent from a consideration of the following detailed description presented in connection with the accompanying drawings. [Brief Description of the Drawings]

[0016] [Figure 1A] This is a diagram showing that the age-related decrease in VLC-PUFA levels in the retina is associated with decreased visual acuity. [Figure 1B] This is a diagram showing that the age-related decrease in VLC-PUFA levels in the retina is associated with decreased visual acuity. [Figure 1C] This figure shows that age-related decreases in VLC-PUFA levels in the retina are associated with decreased visual acuity. [Figure 1D] This figure shows that age-related decreases in VLC-PUFA levels in the retina are associated with decreased visual acuity. [Figure 1E] This figure shows that age-related decreases in VLC-PUFA levels in the retina are associated with decreased visual acuity. [Figure 1F] This figure shows that age-related decreases in VLC-PUFA levels in the retina are associated with decreased visual acuity. [Figure 1G] This figure shows that age-related decreases in VLC-PUFA levels in the retina are associated with decreased visual acuity. [Figure 1H] This figure shows that age-related decreases in VLC-PUFA levels in the retina are associated with decreased visual acuity. [Figure 1I] This figure shows that age-related decreases in VLC-PUFA levels in the retina are associated with decreased visual acuity. [Figure 1J]This figure shows that age-related decreases in VLC-PUFA levels in the retina are associated with decreased visual acuity. Figure 1A shows the age-dependent levels of 18:0, 20:4n-6, 22:6n-3, and VLC-PUFA in the retina (n=5, *=p<0.05, **=p<0.01). Figure 1B shows the levels of major phospholipid classes in the retina at 3 months and 18 months of age (n=5, *=p<0.05, **=p<0.01). Figure 1C shows the quantification of lipid classes in aged photoreceptor outer segments (POS) (n=10, *=p<0.05). Figure 1D shows that lipid ontology (LION) analysis of significantly altered lipids in aged POS indicates significant changes in the biophysical properties of the membrane. Figure 1E shows the downregulation of PC(48:12) and PC(50:12) in aged POS (n=10, *=p<0.05). Figure 1F shows the scotopic and photopic electroretinogram (ERG) responses in aged (18-month-old) mice compared to juvenile (3-month-old) mice (n=10, **=p<0.01, ***=p<0.001, ****=p<0.0001). Figure 1G shows the rate of recovery from rod-mediated dark adaptation in juvenile (3-month-old) and aged (17-month-old) mice. Figure 1H shows the oscillation potential amplitude of scotopic ERG under dark adaptation in aged (17-month-old) and juvenile (3.5-month-old) mice. Figure 1I shows the absolute levels of PDE6A and PDE6B in aged (18-month-old) retinas quantified using a stable isotope-labeled (SIL) peptide-based method (n=5, *=p<0.05). Figure 1J shows immunostaining of C3, APOE, and C5b-9, markers of AMD, in young (5-month-old) and aged (26-month-old) retinas. Scale bar is 25 μm.

[0017] [Figure 2A] This figure shows that age-related decreases in VLC-PUFA levels in the retina are associated with decreased visual acuity. [Figure 2B] This figure shows that age-related decreases in VLC-PUFA levels in the retina are associated with decreased visual acuity. [Figure 2C]This figure shows that age-related decreases in VLC-PUFA levels in the retina are associated with decreased visual acuity. [Figure 2D] This figure shows that age-related decreases in VLC-PUFA levels in the retina are associated with decreased visual acuity. [Figure 2E] This figure shows that age-related decreases in VLC-PUFA levels in the retina are associated with decreased visual acuity. [Figure 2F] This figure shows that age-related decreases in VLC-PUFA levels in the retina are associated with decreased visual acuity. [Figure 2G] This figure shows that age-related decreases in VLC-PUFA levels in the retina are associated with decreased visual acuity. [Figure 2H] This figure shows that age-related decreases in VLC-PUFA levels in the retina are associated with decreased visual acuity. [Figure 2I]This figure shows that age-related declines in VLC-PUFA levels in the retina are associated with decreased visual acuity. Figure 2A shows that principal component analysis (PCA) revealed clear clustering between young (3-month-old) and older (18-month-old) retinas for significantly altered lipids (FC>|1.5|, p<0.05). Figure 2B shows a network of lipidome changes derived from Bioinformatics Methodology for Pathway Analysis (BioPAN). Thin light gray arrows indicate negative z-scores. Dark gray arrows indicate positive z-scores. Dark gray nodes indicate involvement in pathways where the node is significantly activated or suppressed. Highlighting of dark green arrows indicates significant changes in that pathway. Black arrows indicate upregulation of DGAT2 in older retinas. Figure 2C shows that among the VLC-PE analyzed, PE(48:12) was significantly downregulated in aged POS (n=10, ***=p<0.001). Figure 2D shows that contrast sensitivity is reduced in older (18-month-old) animals compared to younger (3-month-old) animals (*=p<0.05, **=p<0.01, ***=p<0.001). Figure 2E shows photopic ERG waveforms in response to UV and green flash in 3-month-old and 18-month-old mice. Three light stimulus intensities are presented: 0.48log[cd s m-2], 1log[cd s m-2], and 1.48log[cd s m-2]. Each waveform is the mean value of n=5 animals in a representative group. Figure 2F (left) shows that the recovery of mean Amax was reduced in older mice (17 months old) compared to younger mice (3.5-month-old mice). Figure 2F (right) shows that the recovery of rod-driven ERGa wave sensitivity (Sf) after the same bleaching treatment was not impaired in older animals. Figure 2G shows that the relative amounts of Rho-normalized lighttransmission proteins remained unchanged in aging retinas. Figure 2H shows that immunofluorescence staining using rod bipolar cell-specific antibody (PKCa) revealed age-related morphological changes in the outer plexiform layer, including excessive extension of dendrites into the outer nucleus.Figure 2I shows immunofluorescence staining using glutamine synthase antibody (GS), illustrating that Müller cell processes associated with aging pass through the outer boundary membrane.

[0018] [Figure 3A] This figure shows that Elovl2 expression decreases with age. [Figure 3B] This figure shows that Elovl2 expression decreases with age. [Figure 3C] This figure shows that Elovl2 expression decreases with age. [Figure 3D] This figure shows that Elovl2 expression decreases with age. [Figure 3E] This figure shows that Elovl2 expression decreases with age. [Figure 3F] This figure shows that Elovl2 expression decreases with age. Figure 3A shows the VLC-PUFA elongation pathway. VLC-PUFAs are produced from the elongation of essential fatty acids by enzymes such as Elovl2, 4, and 5. Figure 3B shows that the Elovl2 promoter is methylated with age in the mouse retina. Figure 3C shows images of retinal sections from young (5-month-old) mice (top panel) and older (26-month-old) mice (bottom panel) stained with RNAscope probes designed for Elovl2, Elovl4, and Elovl5, and counterstained with Hoechst. Figure 3D shows that snRNA-seq demonstrated decreased Elovl2 expression in cone photoreceptors in older (18-month-old) mice compared to young (3-month-old) mice. Figure 3E shows GSEA analysis of the cone enrichment pathway in young (3-month-old) and older (18-month-old) mice. Figure 3F visualizes the intensity of interactions in the retinas of young and elderly individuals. Each plot represents an interaction between cell types. The thickness of the lines between cells indicates the intensity of the interaction, with thicker lines representing stronger interactions.

[0019] [Figure 4A] This figure shows the scRNA-seq analysis of genes involved in the VLC-PUFA biosynthesis pathway. [Figure 4B] This figure shows the scRNA-seq analysis of genes involved in the VLC-PUFA biosynthesis pathway. [Figure 4C] This figure shows the scRNA-seq analysis of genes involved in the VLC-PUFA biosynthesis pathway. Figure 4A shows the expression of genes involved in the VLC-PUFA biosynthesis pathway in various cell types of the eye, visualized using CZ CELLxGENE Discover. Figure 4B shows the visualization of the probability of ligand-receptor (LR) pair-mediated communication from specific cell populations to other cell populations in young (3-month-old) and elderly (18-month-old) retinas. The x-axis represents source-target cell population pairs, and the y-axis represents various LR pairs. The color intensity of the dots corresponds to the communication probability. The size of the dots represents the significance of the interaction in CellChat. Figure 4C shows the visualization of signaling pathways mediating interactions in young and elderly retinas. Each segment along the circle represents a cell population, and the arcs connecting them show the interactions between these cell populations. The color of the arcs corresponds to the cell population from which they originate. The thickness of the arcs indicates the strength of the interaction in the signaling pathway.

[0020] [Figure 5A] This figure shows that a disruption in the lipid composition of the retina of Elovl2C234W mice is correlated with vision loss. [Figure 5B] This figure shows that a disruption in the lipid composition of the retina of Elovl2C234W mice is correlated with vision loss. [Figure 5C] This figure shows that a disruption in the lipid composition of the retina of Elovl2C234W mice is correlated with vision loss. [Figure 5D] This figure shows that a disruption in the lipid composition of the retina of Elovl2C234W mice is correlated with vision loss. [Figure 5E] This figure shows that a disruption in the lipid composition of the retina of Elovl2C234W mice is correlated with vision loss. [Figure 5F]This figure shows that a disruption in the lipid composition of the retina of Elovl2C234W mice is correlated with vision loss. [Figure 5G] This figure shows that a disruption in the lipid composition of the retina of Elovl2C234W mice is correlated with vision loss. [Figure 5H] This figure shows that a disruption in the lipid composition of the retina of Elovl2C234W mice is correlated with vision loss. [Figure 5I] This figure shows that a disruption in the lipid composition of the retina of Elovl2C234W mice is correlated with vision loss. [Figure 5J]This figure shows that disturbances in the lipid composition of the Elovl2C234W mouse retina correlate with vision loss. Figure 5A shows that total fatty acid products, DHA (22:6) and VLC-PUFA (24:6, 32:6, 34:6, 36:6) were reduced in the retinas of Elovl2C234W mice compared to wild-type mice of the same age due to ELOVL2 elongation (n=4, *=p<0.05). Figure 5B shows the changes in major phospholipid classes in the Elovl2C234W retina compared to wild-type retinas of the same age, which are similar to the changes seen in aged retinas compared to young retinas (**=p<0.01). Figure 5C shows that lipid ontology (LION) analysis of lipids that were significantly altered in the Elovl2C234W retina revealed serious changes in the biophysical properties of the membrane. Figure 5D shows the VLC-PC species in the retina of Elovl2C234W mice (*=p<0.05). Figures 5E, 5F, and 5H show the decreased contrast sensitivity (Figure 5E), decreased scotopic electroretinogram (ERG) response (Figure 5F), and delayed rod-mediated dark adaptation (Figure 5H) in Elovl2C234W mice compared to wild-type mice of the same age (*=p<0.05, **=p<0.01, ***=p<0.001, ****=p<0.0001). Figure 5G shows that the maximum amplitude of the ERG a wave in the retina of 12-month-old Elovl2C234W mice is similar to the amplitude of the ERG a wave in the retina of 18-month-old wild-type mice. Figure 5I shows no difference in outer nuclear layer (ONL) thickness between wild-type and Elovl2C234W retinas measured by OCT at 12 months of age (n=5 / group), and between Elovl2C234W and wild-type retinas at 18 months of age (n=4 / group). Figure 5J shows a comparison of the GSEA normalized enrichment score (NES) of selected GO terms in Elovl2C234W and wild-type retinas at 12 months of age, and the NES of GO terms in retinas at 18 months and 12 months of age.

[0021] [Figure 6A] This figure shows that a disruption in the lipid composition of the retina of Elovl2C234W mice correlates with vision loss. [Figure 6B]This figure shows that a disruption in the lipid composition of the retina of Elovl2C234W mice correlates with vision loss. [Figure 6C] This figure shows that a disruption in the lipid composition of the retina of Elovl2C234W mice correlates with vision loss. [Figure 6D] This figure shows that a disruption in the lipid composition of the retina of Elovl2C234W mice correlates with vision loss. [Figure 6E] This figure shows that a disruption in the lipid composition of the retina of Elovl2C234W mice correlates with vision loss. [Figure 6F] This figure shows that a disruption in the lipid composition of the retina of Elovl2C234W mice correlates with vision loss. [Figure 6G] This figure shows that disturbances in the lipid composition of the retina of Elovl2C234W mice correlate with visual acuity loss. Figure 6A shows that the total level of VLC-PC in the retina of Elovl2C234W mice is reduced compared to the retina of wild-type mice of the same age (*=p<0.05). Figure 6B shows that free fatty acid analysis revealed a decrease in DHA and VLC-PUFA in the retina of Elovl2C234W mice (n=3, *=p<0.05). Figure 6C shows a decrease in the scotopic b-wave electroretinogram (ERG) response in 12-month and 18-month-old Elovl2C234W mice (*=p<0.05, **=p<0.01, ***=p<0.001). Figures 6D, 6E, and 6F show that in 12-month-old Elovl2C234W mice, recovery of photopic a-wave and b-wave (Figure 6D), scotopic c-wave ERG response (Figure 6E), and rod-driven Sf (Figure 6F) was unchanged compared to wild-type mice of the same age (n=6). Figure 6G shows that GSEA enrichment plots of the synapse formation (GO:0007416) pathway revealed similar enrichment in 12-month-old Elovl2C234W mice and wild-type retinas of the same age, as well as in 18-month-old and 12-month-old wild-type retinas.

[0022] [Figure 7A] This figure shows that intravitreal administration of 24:5n-3 in aged mice restores vision. [Figure 7B] This figure shows that intravitreal administration of 24:5n-3 in aged mice restores vision. [Figure 7C] This figure shows that intravitreal administration of 24:5n-3 in aged mice restores vision. [Figure 7D] This figure shows that intravitreal administration of 24:5n-3 restores vision in aged mice. Figure 7A shows a schematic diagram of intravitreal administration of 24:5n-3 in 18-month-old mice. Figures 7B, 7C, and 7D show improvements in electroretinogram (ERG) responses for scotopic and photopic waves a and b (Figure 7B), accelerated recovery of rod-mediated dark adaptation (Figure 7C), and improvement in visual evoked potentials (VEPs) (Figure 7D) 5 days after intravitreal administration of 0.36 nmol of 24:5n-3 (*=p<0.05, **=p<0.01; ***=p<0.005; ****=p<0.001). However, no improvement was shown with 0.36 nmol of 22:6n-3 (DHA).

[0023] [Figure 8A] This figure shows the absence of retinal toxicity or visual recovery in young mice after intravitreal administration of 24:5n-3 and optimal dose of 24:5n-3, as well as the optimal dose of 24:5n-3 and the effects of different fatty acids on 18-month-old mice. [Figure 8B] This figure shows the absence of retinal toxicity or visual recovery in young mice after intravitreal administration of 24:5n-3 and optimal dose of 24:5n-3, as well as the optimal dose of 24:5n-3 and the effects of different fatty acids on 18-month-old mice. [Figure 8C] This figure shows the absence of retinal toxicity or visual recovery in young mice after intravitreal administration of 24:5n-3 and optimal dose of 24:5n-3, as well as the optimal dose of 24:5n-3 and the effects of different fatty acids on 18-month-old mice. [Figure 8D] This figure shows the absence of retinal toxicity or visual recovery in young mice after intravitreal administration of 24:5n-3 and optimal dose of 24:5n-3, as well as the optimal dose of 24:5n-3 and the effects of different fatty acids on 18-month-old mice. [Figure 8E]This figure shows the absence of retinal toxicity or visual recovery in young mice after intravitreal administration of 24:5n-3 and optimal dose of 24:5n-3, as well as the optimal dose of 24:5n-3 and the effects of different fatty acids on 18-month-old mice. [Figure 8F] This figure shows the absence of retinal toxicity or visual recovery in young mice after intravitreal administration of 24:5n-3 and optimal dose of 24:5n-3, the optimal dose of 24:5n-3, and the effects of different fatty acids on 18-month-old mice. Figure 8A shows a schematic diagram of intravitreal administration of 24:5n-3 in 3-month-old mice. Figures 8B, 8C, and 8D show the electroretinogram (ERG) response and rod-mediated dark adaptation recovery (Figure 8D) in young animals 2 days after injection (Figure 8B) and 5 days after injection (Figure 8C), and no changes were observed after supplementation. Figures 8E and 8F show that the photopic ERG response in 18-month-old mice five days after injection remained unchanged after supplementation with 0.04 nmol, 0.72 nmol, and 2.5 nmol of 24:5n-3 (Figure 8E), and 0.36 nmol of 20:5n-3 and 32:6n-3 (Figure 8F).

[0024] [Figure 9A] This figure shows the reversal of the molecular aging phenotype in the eyes of 18-month-old animals supplemented with 24:5n-3. [Figure 9B] This figure shows the reversal of the molecular aging phenotype in the eyes of 18-month-old animals supplemented with 24:5n-3. [Figure 9C] This figure shows the reversal of the molecular aging phenotype in the eyes of 18-month-old animals supplemented with 24:5n-3. [Figure 9D] This figure shows the reversal of the molecular aging phenotype in the eyes of 18-month-old animals supplemented with 24:5n-3. [Figure 9E] This figure shows the reversal of the molecular aging phenotype in the eyes of 18-month-old animals supplemented with 24:5n-3. [Figure 9F]This figure shows the reversal of molecular aging phenotypes in 18-month-old eyes supplemented with 24:5n-3. Figure 9A shows that after intravitreal administration of 24:5n-3, the levels of a possible class of phospholipids incorporating VLC-PUFA increased in the isolated photoreceptor outer segment (POS) (*=p<0.05, **=p<0.01). Figure 9B shows that almost all differentially expressed genes (DEGs) (FC>|1.5|, pval<0.05) were downregulated in 18-month-old 24:5(n-3) supplemented retinas compared to vehicle-injected retinas (n=3). Figure 9C shows the gene set enrichment analysis (GSEA) enrichment plot, revealing downregulation of complement (NES=-1.77, FDR q=0.00) and oxidative stress and redox (NES=-1.70, FDR q=0.006) pathways after 24:5(n-3) supplementation (n=3). Figure 9D shows that Metascape analysis demonstrated downregulation of immune responses, inflammation, microglial phagocytosis, and cell migration pathways after 24:5n-3 supplementation (n=3). Figure 9E shows the STRING functional protein-protein interaction network of the top 20 transcription factors involved in gene regulation after 24:5n-3 supplementation. Figure 9F shows immunofluorescence staining showing decreased expression of complement C3, APOE, HTRA1, and C5b-9 proteins in the RPE cell layer of a retinal cross-section of an eye supplemented with 24:5n-3. Scale bar: 20 mm.

[0025] [Figure 10A] This figure shows the lipid analysis of the retina after lipid supplementation. [Figure 10B] This figure shows the lipid analysis of the retina after lipid supplementation. [Figure 10C] This figure shows the lipid analysis of the retina after lipid supplementation. [Figure 10D]This figure shows lipid analysis in the retina after lipid supplementation. Figure 10A shows that major phospholipid classes remain unchanged in vehicle-administered retinas and 24:5n-3 supplemented retinas. Figure 10B shows that PUFA levels in the retina after 22:6n-3, 24:5n-3, and 32:6n-3 injections remain unchanged compared to vehicle-administered injections. Figure 10C shows that DESeq2 normalized counts of Elovl2, Elovl4, and Elovl5 remain relatively unchanged after 24:5n-3 supplementation. Figure 10D shows the Metascape analysis of enriched clusters in the pathway after 24:5n-3 supplementation, color-coded by p-value. [Modes for carrying out the invention]

[0026] Any methods, apparatus, and materials similar or equivalent to those described herein may be used in carrying out the present invention. The following definitions are provided to facilitate the understanding of certain terms that are frequently used herein and are not intended to limit the scope of this disclosure. If there are multiple definitions for a term herein, the definition in this section shall prevail unless otherwise stated. The headings used herein are for structural purposes only and are not intended to limit the inventions described herein.

[0027] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art in the field to which the disclosed invention pertains. The singular terms “a,” “an,” and “the” encompass multiple referents unless the context clearly indicates otherwise. Similarly, the word “or” is intended to encompass “and” unless the context clearly indicates otherwise. The term “comprising” means that other elements may be present in addition to the predefined elements presented. The use of “comprising” indicates inclusion, not limitation. In other words, the term “comprising” means “primarily encompassing, but not necessarily only encompassing.” Furthermore, variations of “comprising,” such as “comprise” and “comprises,” have the same meaning. In some respects, the art described herein relates to the compositions, methods, and their respective components described herein as essential to the invention, but there is room to include unspecified elements, whether essential or not (“comprising”).

[0028] All publications, patent applications, patents, and other references referenced herein are incorporated in their entirety by reference for all purposes. In the event of any conflict, this specification, including the explanation of terms, shall prevail.

[0029] The disclosed technology can be carried out or tested using methods and materials similar or equivalent to those described herein, but suitable methods and materials are listed below. The materials, methods, and examples are illustrative and not intended to limit the scope of this invention.

[0030] As used herein, the terms “subject” and “patient” are interchangeable. As used herein, subject may be a mammal such as a non-primate (e.g., cattle, pigs, horses, cats, dogs, rats, etc.) or a primate (e.g., monkeys and humans). In certain embodiments, subject is human. This term does not indicate a specific age or sex. Therefore, it is intended to include adult and newborn subjects (whether male or female), as well as fetuses. In one embodiment, subject is a mammal (e.g., human) having the disease, disorder or condition described herein. In another embodiment, subject is a mammal (e.g., human) at risk of developing the disease, disorder or condition described herein. “Patient” is subject suffering from the disease or disorder.

[0031] As used herein, the terms “treat,” “treating,” or “treatment” refer to both therapeutic and preventive or protective measures aimed at preventing, reducing, delaying (mitigating), suppressing, or eliminating undesirable physiological changes, symptoms, diseases, or disorders. For example, a disease could be age-related macular degeneration (AMD). For the purposes of the present invention, beneficial or desirable clinical outcomes include, but are not limited to, symptom relief, reduction of the disease's scope, stabilization of the disease (i.e., no worsening), delay or slowing of disease progression, improvement or mitigation of the disease state, and (partial or complete) remission, whether detectable or undetectable. “Treatment” may also mean an extension of survival compared to the expected survival without treatment. Persons requiring treatment include those who already have a disease or disorder, as well as those who are susceptible to disease or disorder, or who need to prevent or delay the onset of disease or disorder. In some cases, the subject or patient may be identified (e.g., diagnosed) as having a disease or condition prior to administration of the composition of the present invention. Subjects at risk of disease can be identified, for example, by any or a combination thereof, of a suitable diagnostic assay or prognostic assay known in the art.

[0032] As used herein, “clinical improvement” may mean a significant reduction or cessation of the symptoms of the disorder.

[0033] The terms “to manage,” “to control,” and “manage” refer to preventing or slowing the progression, expansion, or worsening of a disease or disorder, or one or more of its symptoms. In some cases, the beneficial effects obtained from preventive or therapeutic drugs may not result in a cure for the disease or disorder.

[0034] Where used herein, unless otherwise specified, the term “therapeutic dose” of a composition herein means an amount sufficient to provide a therapeutic benefit in the treatment or management of a viral infection, or an amount sufficient to delay or minimize one or more symptoms associated with a viral infection. The therapeutic dose of a composition described herein means an amount of therapeutic agent, either alone or in combination with other therapies, that provides a therapeutic benefit in the treatment or management of a viral infection. The term “therapeutic dose” may include an amount that improves the overall treatment, reduces or avoids symptoms or causes, or enhances the therapeutic effect of another therapeutic agent.

[0035] The terms "administer" and "dosage" refer to a method of providing a pharmaceutical preparation to a subject. Such methods are well known to those skilled in the art and include, but are not limited to, intranasal administration, parenteral administration (e.g., intravenous and subcutaneous administration), intramuscular injection, intraperitoneal injection, intrathecal administration, transdermal administration, extracorporeal administration, and topical administration of the composition.

[0036] Referring here to Figures 1A to 10D, the present invention is characterized by a method and composition for treating age-related diseases (e.g., age-related eye diseases) in subjects requiring treatment for age-related diseases. Furthermore, the present invention provides a method and composition for treating age-related brain diseases in subjects requiring treatment for age-related brain diseases.

[0037] The present invention may feature a method for treating age-related eye disease in subjects requiring treatment for age-related eye disease. In some embodiments, the method includes administering a composition containing tetracosapentaenoic acid [24:5n-3] to a subject. In other embodiments, the method includes administering a composition containing tetracosapentaenoic acid [24:5n-3] and DHA to a subject. In some embodiments, the method may include identifying a subject exhibiting age-related eye disease and administering tetracosapentaenoic acid [24:5n-3] to the subject's eye.

[0038] In some embodiments, the method involves administering a composition containing tetracosapentaenoic acid [24:5n-3] to the eye of a subject. In other embodiments, the method involves administering a composition containing tetracosapentaenoic acid [24:5n-3] and DHA to the eye of a subject.

[0039] In some embodiments, tetracosapentaenoic acid [24:5n-3] is administered as a free fatty acid, fatty acid ethyl ester (FAEE), or fatty acid methyl ester (FAME). In some embodiments, DHA is administered as a free fatty acid, fatty acid ethyl ester (FAEE), or fatty acid methyl ester (FAME).

[0040] In some embodiments, compositions containing tetracosapentaenoic acid [24:5n-3] may be administered by intravitreous injection, intravenous injection, intraperitoneal injection, or topical administration.

[0041] In some embodiments, age-related eye disease is age-related macular degeneration and / or age-related vision loss / decline. In some embodiments, a therapeutically effective dose is administered for the disease.

[0042] The present invention may also feature methods for treating age-related liver disease in subjects requiring treatment for age-related liver disease. In some embodiments, the method includes administering tetracosapentaenoic acid [24:5n-3] to the subject. In other embodiments, the method includes administering a composition containing tetracosapentaenoic acid [24:5n-3] and DHA to the subject. In some embodiments, the method includes identifying a subject exhibiting age-related liver disease and administering tetracosapentaenoic acid [24:5n-3] to the subject. Non-limiting examples of age-related liver disease include, but are not limited to, non-alcoholic fatty liver disease, alcoholic liver disease, hepatitis C, and fibrosis and cirrhosis.

[0043] In some embodiments, tetracosapentaenoic acid [24:5n-3] is administered as a free fatty acid, fatty acid ethyl ester (FAEE), or fatty acid methyl ester (FAME). In some embodiments, DHA is administered as a free fatty acid, fatty acid ethyl ester (FAEE), or fatty acid methyl ester (FAME).

[0044] In some embodiments, the composition is administered by intravenous injection. In some embodiments, a therapeutically effective dose is administered for the disease.

[0045] The present invention may also feature a method for treating age-related brain disease in subjects requiring treatment for age-related brain disease. In some embodiments, the method includes administering tetracosapentaenoic acid [24:5n-3] to a subject. In other embodiments, the method includes administering a composition containing tetracosapentaenoic acid [24:5n-3] and DHA to a subject. In some embodiments, the method includes identifying a subject exhibiting age-related brain disease and administering tetracosapentaenoic acid [24:5n-3] to the subject's brain.

[0046] In some embodiments, the method involves administering tetracosapentaenoic acid [24:5n-3] to the target brain. In other embodiments, the method involves administering a composition containing tetracosapentaenoic acid [24:5n-3] and DHA to the target brain.

[0047] In some embodiments, tetracosapentaenoic acid [24:5n-3] is administered as a free fatty acid, fatty acid ethyl ester (FAEE), or fatty acid methyl ester (FAME). In some embodiments, DHA is administered as a free fatty acid, fatty acid ethyl ester (FAEE), or fatty acid methyl ester (FAME).

[0048] In some embodiments, the composition is administered by intravenous injection and / or stereotactic direct injection.

[0049] In some embodiments, age-related brain diseases are selected from diseases comprising Alzheimer's disease, amyotrophic lateral sclerosis (ALS), age-related cognitive decline, dementia, Parkinson's disease, parkinsonism, and other Parkinson's-like disorders. In some embodiments, a therapeutically effective dose is administered for the disease.

[0050] The present invention may also feature a method for treating age-related bone disease (e.g., myeloma) in subjects requiring treatment for age-related bone disease. In some embodiments, the method includes administering tetracosapentaenoic acid [24:5n-3] to the subject. In other embodiments, the method includes administering a composition containing tetracosapentaenoic acid [24:5n-3] and DHA to the subject. In some embodiments, the method includes identifying a subject exhibiting age-related bone disease and administering tetracosapentaenoic acid [24:5n-3] to that subject. Non-limiting examples of age-related bone disease include, but are not limited to, multiple myeloma and other myelodysplastic syndromes (MDS), anemia, etc.

[0051] In some embodiments, the composition is administered by intravenous administration and / or intravenous injection.

[0052] The present invention may also feature compositions for use in the treatment of age-related eye diseases in subjects requiring treatment for age-related eye diseases. In some embodiments, the composition comprises tetracosapentaenoic acid [24:5n-3]. In other embodiments, the composition comprises tetracosapentaenoic acid [24:5n-3] and docosahexaenoic acid (DHA). In some embodiments, the composition is administered to the eye of the subject. In some embodiments, the composition is administered by intravitreous injection, intravenous injection, intraperitoneal injection, or topical administration.

[0053] The present invention may also feature compositions for use in the treatment of age-related brain diseases in subjects requiring treatment for age-related brain diseases. In some embodiments, the composition comprises tetracosapentaenoic acid [24:5n-3]. In other embodiments, the composition comprises tetracosapentaenoic acid [24:5n-3] and docosahexaenoic acid (DHA). In some embodiments, the composition is administered to the brain of the subject. In some embodiments, the composition is administered by intravenous injection and / or stereotactic direct injection.

[0054] The present invention may also feature compositions for use in the treatment of age-related liver disease in subjects requiring treatment for age-related liver disease. In some embodiments, the composition comprises tetracosapentaenoic acid [24:5n-3]. In other embodiments, the composition comprises tetracosapentaenoic acid [24:5n-3] and docosahexaenoic acid (DHA). In some embodiments, the composition is administered by intravenous injection.

[0055] The present invention may also feature compositions for use in the treatment of age-related bone disease in subjects requiring treatment of age-related bone disease. In some embodiments, the composition comprises tetracosapentaenoic acid [24:5n-3]. In other embodiments, the composition comprises tetracosapentaenoic acid [24:5n-3] and docosahexaenoic acid (DHA). In some embodiments, the composition is administered by intravenous administration and / or intravenous injection.

[0056] example

[0057] The following are non-limiting examples of the present invention. It should be understood that these examples are not intended to limit the present invention in any way. Equivalents or substitutions are within the scope of the present invention.

[0058] Age-related vision loss is associated with a decrease in VLC-PUFA levels in the retina: To investigate changes in the lipid composition of aging retinas, a series of lipidomics analyses were performed on dissected mouse tissues at 3, 6, 12, 18, and 23 months of age. For complete lipidomics analysis, lipids were extracted using the Bligh-Dyer method. For comprehensive fatty acid (FA) analysis, FAs were released from lipids using acid hydrolysis and extracted using hexane. Complex lipids and FAs were analyzed by liquid chromatography-mass spectrometry (LC-MS) as described herein. In FA analysis, the abundance of individual polyunsaturated fatty acids (PUFAs) was normalized to an internal standard (FA 21:5) and tissue weight. This data revealed that, compared to the retinas of 3-month-old mice, DHA levels (34% in 18-month-old mice (p=0.0178), 33% in 23-month-old mice (p=0.0165)) and VLC-PUFA, specifically 32:6 (31% in 18-month-old mice, p=0.0128), 34:6 (52% in 18-month-old animals (p=0.0042), 39% in 23-month-old animals (p=0.0290)), and 36:6 ​​(55% in 18-month-old mice (p=0.0046), 59% in 23-month-old mice (p=0.0029)), gradually decreased in aged retinas (Figure 1A).

[0059] After performing FA analysis, changes in lipid composition in the retinas of aging mice were examined. The levels of extracted complex lipids were analyzed using data-dependent acquisition (DDA) LC-MS, and lipid species were identified using LipidSearch software 4.2.21 (Thermo). Of the 528 lipids quantified in the retina, the levels of 15 lipids significantly decreased and the levels of 53 lipids increased in aged retinas (multiplier > |1.5|, p < 0.05). Principal component analysis revealed clear clustering based on differences in lipid composition between 3-month-old and 18-month-old retinas (Figure 2A). In the 18-month-old retinas, two major membrane lipid classes, phosphatidylcholine (PC) and phosphatidylethanolamine (PE), changed in opposite directions. Specifically, in aged retinas, PC significantly decreased (p = 0.0039, n = 5) and PE increased (p = 0.0329, n = 5) (Figure 1B). Next, photoreceptor outer segments (POSs) were isolated from the retinas of 3-month-old and 18-month-old mice. During lipid extraction, PCs decreased significantly (5%), while phosphatidylglycerol (PG), sphingomyelin (SM), and triglycerides (TG) increased (18%, 26%, and 25%, respectively) (Figure 1C). Based on the significant changes in lipids in the POS membrane, Bioinformatics for Pathway Analysis (BioPAN) was performed on the lipidomics dataset.

[0060] BioPAN revealed that in 18-month-old POS (Positive Erythematosus) cells, TG synthesis from diglycerides (DG) was highly activated (Z-score = 2.957), while DG formation from TG was inhibited (Z-score = -3.165) (Figure 2B). Analysis of previous transcriptome data from young and aged retinas revealed that while the expression levels of triacylglycerol lipase and transacylase enzymes (PNPLA4-5) remained unchanged, the level of DGAT2, the enzyme that catalyzes the final reaction of TG synthesis, was elevated in aged retinas (Figure 2B). Next, to further analyze the dataset, lipid ontology (LION) enrichment analysis was performed, revealing highly enriched changes in membrane components, as well as a decrease in the thickness of the membrane bilayer and high lateral diffusion within the membrane (Figure 1D). Since PC and PE are mainly present in the cell membrane, further analysis was performed to evaluate the effects of aging on membrane composition. In particular, the levels of VLC-PUFA incorporated into complex lipids and their effects on the membrane of aging retinas were focused on. PC-VLC-PUFA levels were analyzed using Lipid Data Analyzer with a customized database. This data revealed a decrease in the levels of these lipids. Specifically, in POS, PC (50:12) and PE (48:12) decreased by 30% and 42%, respectively (Figures 1E and 2E).

[0061] To correlate age-related lipid changes with visual function, a series of visual tests were performed on 3-month-old and 18-month-old mice. First, quantitative optomotor response (OMR) analysis was performed to measure contrast sensitivity under scotopic conditions, mimicking nocturnal light levels. Unsurprisingly, 18-month-old mice had significantly lower contrast sensitivity under scotopic conditions compared to 3-month-old mice (Figure 2D). Using full-field electroretinography (ERG), the amplitudes of both scotopic wave a and b were significantly reduced (Figure 1F). Maximum scotopic wave a was approximately 34% lower in 18-month-old mice (166±5μV, n=14) compared to 3-month-old mice (250±12μV, n=14, ***P<0.001). Photopic green / UV wave a and b were also reduced in 18-month-old mice compared to younger controls (Figures 1F and 2E). Next, we investigated whether dark adaptation of rod photoreceptors was suppressed in aged mice. After bleaching approximately 90% of the visual pigment, the rods in both groups gradually recovered their light response in the dark over the following 60 minutes. max The recovery could be explained by a single exponential function with a time constant of 18.9 ± 0.7 mins, and the level 60 minutes after bleaching was approximately 80 ± 6% of the pre-bleaching value (Figure 2F, left). Rods from aged mice also showed a robust recovery of the maximum response after bleaching, although their rate (24.3 ± 2.7 mins) was slightly lower compared to juvenile mouse rods (approximately 1.3 times, p<0.001). However, the maximum response amplitude 60 minutes after bleaching reached the same relative level as juvenile control rods (approximately 81 ± 4%) (Figure 1G). Oscillatory potentials were also compared between juvenile and aged mice, and a consistent and significant decrease was observed in all four oscillatory potential peaks in both weak and strong flash scotopic responses (Figures 2E and 1H). However, the same post-bleaching rod-driven ERGa wave sensitivity (S f The recovery of ) was not impaired in older animals (Figure 2F, right), which suggests that the visual cycle is functioning normally in older animals. In summary, these results indicate that aging affects visual function through a decline in visual contrast sensitivity and photoreceptor function.

[0062] To investigate whether visual function decline in aging animals may be due to changes in the light transmission cascade, levels of proteins involved in signal transduction in POS, including Rho, PDE6A, PDE6B, GNAT1, and GBB1, were quantified using an absolute quantification method based on stable isotope-labeled (SIL) peptides. To compare levels of PDE6 and transducin proteins in the retinas of 3-month-old and 18-month-old animals, an optimal amount of SIL peptide was added to protein samples, which were trypsinized and analyzed by LC-MS / MS. The amount of each protein was calculated using the peak area ratio of endogenous peptide to SIL peptide, and the quantification results were normalized to Rho and expressed as mean ± standard error of the mean. Absolute quantification of these proteins showed a slight decrease in PDE6A and PDE6B in the retinas of 18-month-old animals (Figure 1I). However, the relative amounts of all measured proteins (PDE6A, PDE6B, GNAT1, and GBB1) remained unchanged (Figure 2G). This suggests that the ratio of proteins involved in light transmission signaling pathways is not affected in aged retinas.

[0063] Finally, to assess age-related changes in protein levels associated with retinal lesions, immunofluorescence staining was performed on retinal sections of adult (5-month-old) and aged (26-month-old) mice. Antibodies targeting complement factor 3 (C3), apolipoprotein E (ApoE), and subunits of the membrane attack complex (MAC) in the complement pathway C5b-9, which are associated with an increased risk of age-related macular degeneration (AMD), were used. The data demonstrated higher levels of these proteins in the retinal pigment epithelium (RPE) cell layer of 26-month-old animals compared to retinal sections of 5-month-old animals (Figure 1J). Furthermore, immunofluorescence staining with rod bipolar cell-specific and Müller cell-specific antibodies (protein kinase C alpha, PKCa, and glutamine synthetase, GS, respectively) revealed age-related morphological changes in the outer plexiform layer, including dendritic overgrowth into the outer nucleus (Figure 2H-2I).

[0064] Age-related changes in Elovl2 expression: ELOVL2 encodes a key enzyme in the biosynthesis of long-chain fatty acids (Figure 3A). Specifically, it elongates 22:5n-3 to 24:5n-3, which is then elongated to VLC-PUFA by other elongation enzymes, including ELOVL4, or further processed in peroxisomes to produce 22:6n-3,DHA. The Elovl2 regulatory region has high methylation levels in aged retinas. Here, we analyzed methylation levels at each CpG site of the Elovl2 promoter, which has been shown to undergo age-related methylation changes in human blood cells (Figure 3B). To achieve this, DNA was isolated from the retinas of middle-aged (<12 months) and aged (24 months) mice and treated with sodium bisulfite to distinguish between methylated and unmethylated cytosine. Next, both converted and unconverted DNA were used to amplify the target region using methylation-insensitive primers. Next, the PCR products were sequenced using Sanger sequencing, and the relative levels of methylated and unmethylated cytosine were calculated by comparing the area under the sequencing peaks of both DNA samples. This data revealed a group of five CpGs with significantly higher methylation levels in aged retinas, while the other four closest to the transcription start site remained unchanged. Notably, the CpG sites that showed higher methylation in mice had direct homology to methylated CpGs in aged human blood.

[0065] To visualize the cell type and age-specific expression of key enzymes in the PUFA elongation pathway, RNA in situ hybridization was performed on retinal sections of adult (5-month-old) and aged (26-month-old) mice using RNAscope probes targeting Elovl2, Elovl4, and Elovl5 (Figure 3C). Elovl2 expression was mainly observed in the photoreceptor layer, while Elovl4 was expressed in all layers of the retina, and Elovl5 was expressed in the inner granular layer and ganglion cell layer. Expression levels of Elovl4 and Elovl5 did not change relatively with age, but Elovl2 expression was decreased in aged (26-month-old) mice compared to 5-month-old animals.

[0066] To identify specific cell types expressing Elovl2 and other PUFA elongation enzymes, single-nuclear RNA sequencing (snRNAseq) was performed on retinas isolated from young (3-month-old) and aged (18-month-old) mice. After normalizing the mean expression levels to TBP, Elovl2 expression was significantly reduced in aged retinas (0.080 compared to 0.100 in young retinas, p-value = 3.59E-6). Elovl2 expression was highest in cones and barely detectable in other cell types (Figure 3D). This data is consistent with data obtained from mouse and human retinas using the CZI CELL×GENE Discover program (Figure 3D), confirming the enrichment of Elovl2 in cones. Furthermore, when cell type-specific expression of other enzymes from the PUFA elongation pathway was examined, only Elovl2 showed cone-specific expression, while other enzymes were not significantly restricted (Figures 3D and 4D). Subsequently, the pathways of cone photoreceptor changes associated with aging were investigated. Differential expression genes (DEGs) (log factor change > 0.1, padj < 0.05) were identified in the cones of young (3 months old) and older (18 months old) mice and subsequently used for GO function enrichment analysis (Figure 3E). Biological pathways enriched in young cones were associated with visual and light stimulus detection. Pathways enriched in older cones were involved in synaptic transmission, ion channels, and membrane assembly.

[0067] To gain deeper insights into changes in intercellular interactions in aging retinas, we used the computational package CellChat, focusing on interactions between cones, rods, and Müller glia (Figures 3F, 4B, 4C). Specifically, CellChat infers intercellular communication from snRNA-seq data by calculating the expression levels of signaling ligands and receptors, including soluble agonists, antagonists, and stimulative or inhibitory membrane-bound coreceptors. First, we assessed the number and intensity of inferred interactions within and between cell types. In young retinas, the strongest interactions were detected between Müller glia and cones. These interactions were further increased in aged tissues. Similarly, signaling between rods and Müller glia increased in aged retinas. Interestingly, cone-rod interactions were not observed in young retinas, but several new and significant interactions were detected in 18-month-old tissues. Analysis of specific ligand-receptor pairs within these signaling interactions revealed the emergence of binding between the neural adhesion molecule (Negr1-Negr1) and the metabotropic glutamate receptor (Glu-Grm8) between rod and cone cells, particularly in aged retinas. Furthermore, several interactions between metabotropic glutamate receptors (Glu-Grm8, Glu-Grm7) and ion channel glutamate receptors (Glu-Gria4) were revealed within Müller glia in aged retinas (Figures 4B and 4C). In addition, this analysis revealed a novel interaction between cyclosporine A and CD147 (Ppia-Bsg) between Müller cells and photoreceptor cells. These are recognized as infection sensors and inflammation initiation pathways (Figure 4B).

[0068] Lack of ELOVL2 activity accelerates age-related phenotypic changes in the retina: Elovl2 mutant mice with loss of ELOVL2 enzyme activity due to substrate binding disorder (Elovl2 C234W ) was created. Elovl2, 18 months old C234WFor the mouse retina, a detailed analysis of the lipid composition including total fatty acids and a global lipidomics analysis were performed. Quantification revealed that the levels of PUFAs synthesized from ELOVL2 products, including 22:6, 24:6, 32:6, 34:6 and 36:6, in 18-month-old Elovl2 C234W retinas were significantly lower than in age-matched wild-type retinas (Figure 5A). Untargeted lipidomics analysis identified 595 lipid species, of which 28 showed significantly lower levels compared to wild-type tissues and 84 showed significantly higher levels in Elovl2 C234W retinas (fold change > |1.5|, p-value < 0.05). Furthermore, Elovl2 C234W tissues exhibited decreased PC levels and increased PE levels, similar to the changes detected in aged retinas (Figure 5B) (Figure 1B). Lipid Ontology (LION) enrichment analysis based on significantly altered lipids suggested highly enriched changes in cell membrane components, as well as decreased bilayer thickness and high lateral diffusion, which were also similar to the changes in aged POS (Figure 5C). Therefore, further analysis of VLC-PC revealed decreases in PC(46:12), PC(48:12), PC(50:12), PC(54:12), and PC(56:12) (Figure 5D). This contributed to the reduction in total VLC-PC levels in 18-month-old Elovl2C234W retinas (Figure 6A). Analysis of free fatty acids (FFA) showed that 22:6, 24:6, 26:6, 32:6, 34:6 and 36:6 were decreased by 45% (p=0.048), 70% (p=0.002), 92% (p=0.002), 62% (p=0.008), 57% (p=0.008) and 32% (p=0.128), respectively, in 18-month-old Elovl2 C234W retinas compared to age-matched wild-type samples (Figure 6B).

[0069] Next, the visual function of 12-month-old and 18-month-old Elovl2 C234W mice was compared with that of age-matched wild-type mice. At both ages, Elovl2 C234WThe animals' scotopic contrast sensitivity was lower compared to wild-type mice of the same age (Figure 5E). Scotopic wave a and b ERG responses were also reduced at both ages compared to wild-type controls (Figures 5F and 6C). Furthermore, in wild-type mice, scotopic wave a and b ERG responses decreased dramatically between 12 and 18 months of age (Figures 5F and 6C). In contrast, Elovl2 C234W In mice, the change in ERG amplitude between these ages is minimal (Figures 5F and 6C). Notably, in 12-month-old Elovl2 C234W The maximum ERG a-wave amplitude (120 μV) in mice decreased to a level comparable to that of 18-month-old control animals (150 μV, p=0.103) (Figure 5G). C234W There were no differences between mice and mice in the amplitudes of photopic ERG a and b waves, or in the RPE-generated ERG c wave (Figures 6D and 6E).

[0070] Next, after nearly complete rhodopsin bleaching with green light, rod dark adaptation was evaluated in ELOVL2-deficient mice. In control mice, the mean scotopic ERG a-wave maximal response A max It recovered with a time constant of 28.6 ± 2.6 minutes, and its level 60 minutes after bleaching was approximately 90 ± 7% of the pre-bleaching value (Figure 5H). In contrast, Elovl2 C234W Recovery of the scotopic α-wave response in mice was significantly suppressed compared to controls. Consistent with this, Elovl2 C234W Rod A in mice max The average recovery rate (42.7 ± 7.3 minutes) was approximately 1.5 times slower than that of control animals (p = 8E-6), and by the end of the 60-minute recording, it had only reached about 69 ± 5% of its pre-bleaching level (Figure 5H). Dark adaptation wave a photosensitivity (S f ) However, this was also lower in the same group of mice lacking ELOVL2 (approximately 16%) (compared to 1.79 ± 0.08 m in the control group). 2 cd -1 s -1 1.50±0.05 m 2 cd -1 s -1 (*p<0.05). However, the same bleached rod-driven S fRecovery was not suppressed in ELOVL2-deficient animals (Figure 6F). This suggests that visual chromophores are recycled normally in mutant mice.

[0071] Elovl2 C234W To determine whether a decrease in ERG amplitude in mice could be due to cell loss or retinal degeneration, the thickness of the outer nuclear layer (photoreceptors) was measured using optical coherence tomography (OCT). The study compared 12-month-old wild-type and Elovl2 mice. C234W (n=5), and wild-type and Elovl2 at 18 months of age. C234W In the group of four (n=4) eyes, no statistically significant difference in ONL thickness was observed using two-way ANOVA (Figure 5I). Therefore, in the group of 12-month-old Elovl2 C234W In animals, decreased rod function preceded the loss of detectable photoreceptor cells or retinal degeneration.

[0072] Elovl2 C234W Considering that mice showed accelerated phenotypic changes associated with aging in the retina, we investigated whether these also involved transcriptome changes that mimic those seen in aging retinas. First, bulk RNA sequencing was performed on wild-type retinas at 12 and 18 months of age. Next, this data was collected from 12-month-old wild-type and Elovl2 mice. C234W We identified potential transcriptome similarities in retinal changes observed between ELOVL2 enzyme deficiency and aging, compared to the retinal transcriptome. Gene set enrichment analysis (GSEA) was performed on differentially expressed genes (change multiplier >|1|, padj < 0.05) using gene ontology, biological processes, cellular components, and molecular functional gene sets. Downregulated pathways in both aging and ELOVL2-deficient retinas included telomere maintenance, RNA polymerase I-mediated transcription, negative regulation of gene expression (epigenetic), and gene silencing (Figure 5J). Conversely, pathways such as synapse formation, transmembrane transporter activity, TORC1 signaling, DNA damage response-signaling-mediated transcription, and ceramide metabolic processes were downregulated in 12-month-old Elovl2-deficient retinas compared to wild-type retinas.C234W Upward control was observed in both the retina and the 18-month-old wild-type retina (Figures 5J and 6G).

[0073] Restoration of Vision in Aged Animals by Intravitreous Fatty Acid Injection: While we do not wish to limit the present invention to any theory or mechanism, a deficiency of 24:5n-3, an ELOVL2 product in aging retinas, is considered one of the main causes of age-related vision loss, and supplementation with this fatty acid may improve vision in aged animals. First, the strategy of intravitreous supplementation was adopted. This allowed for precise control of the amount of lipid administered to the eye, in contrast to forced oral administration. To evaluate the retinal toxicity of 24:5n-3, the compound was administered intravitreously to unilateral mice at 3 months of age, and ERG responses were measured on days 2 and 5 post-injection (Figure 8A). Compared to the vehicle-injected eye, the eye injected with 24:5n-3 showed no significant change in the a-wave or b-wave amplitude of the scotopic ERG on day 2 (Figure 8B). Furthermore, no significant changes were observed in either the scotopic or photopic response on day 5 (Figure 8C). In summary, intravitreal injection of 24:5n-3 did not cause retinotoxicity in 3-month-old mice and did not affect rod or cone-driven retinal responses.

[0074] To investigate the effects of 24:5n-3 supplementation on visual function in aged mice, the compound was intravitreously administered to one eye of each 18-month-old animal, while the other eye was injected with a control vehicle. After 5 days, the effects on visual function were tested using a multimodal approach, and then the retinas were collected and the fatty acid and lipid composition was analyzed to investigate molecular changes (Figure 7A). First, various doses of 24:5n-3 were tested to determine the optimal level that could improve visual acuity in 18-month-old mice. Interestingly, eyes treated with 24:5n-3 showed improvement in both the a-wave and b-wave responses of the scotopic and photopic ERGs at an injectable dose of 0.36 nmol (Figure 7B, right), while no changes were observed with other doses (Figure 8E). Next, the procedure was repeated using the optimal dose (0.36 nmol) to test whether PUFAs upstream or downstream of 24:5n-3, such as 20:5n-3, 22:6n-3 (DHA), and 32:6n-3, had similar effects. Intravitreal injections of 20:5n-3, 22:6n-3, and 32:6n-3 were administered, followed by functional analysis (Figure 7B left and Figure 8F). Surprisingly, only the eyes supplemented with 24:5n-3 intravitreously showed statistically significant improvement in both the a-wave and b-wave responses of scotopic and photopic ERGs when evaluated 5 days after injection. In contrast, injections of 22:6n-3 and 20:5n-3 did not result in functional improvement (Figure 7B and Figure 8F left). In eyes treated with 32:6n-3, a slight improvement was observed in the b-wave response of the ERG generated by green and blue light in photopic vision, but no improvement was observed in the scotopic vision response (Figure 8F, right).

[0075] Next, the effect of intraocular 24:5n-3 supplementation on rod dark adaptation was evaluated by in vivo ERG. In 17-month-old mice, this treatment improved the final percentage of recovered rod Amax response after bleaching by approximately 30% compared to the contralateral eye injected with the vehicle (Figure 7C, left), but did not affect the recovery of sensitivity (Figure 7C, right). In contrast, similar administration of 24:5n-3 did not affect the recovery of either of the two parameters in 3.5-month-old animals (Figure 8D). Therefore, the therapeutic effect of this lipid was shown to be specific to aged mice.

[0076] Following the observation of improved ERG responses, which measure photoreceptor and medial retinal function, in the supplemented retina, we next investigated whether this improvement was related to increased signaling from the retina to the brain by recording visual evoked potentials (VEPs). Recordings in the superior colliculus showed a significant increase in VEP peak amplitude in 18-month-old animals supplemented with 24:5n-3 compared to eyes administered with the vehicle (Figure 7D). Specifically, VEP amplitude increased from 44.15±5.95 μV in 18-month-old animals injected with the vehicle (n=4 animals; n=14 recording sites) to 86.67±8.61 μV in the supplemented eyes (n=4; n=16; p=0.00082). Thus, these results demonstrate that the improvement in photoreceptor visual function after intravitreous supplementation with 24:5n-3 is reliably transmitted to the brain.

[0077] Molecular rejuvenation of the retina after 24:5n-3 supplementation: To gain insight into the potential mechanisms underlying the improvement in visual acuity in mice treated with 24:5n-3, we first investigated whether lipid supplementation could cause any changes in the lipid composition of the retina. Retinal tissue was collected 5 days after lipid or vehicle injection, and lipids were extracted using the Bligh and Dyer method. Levels of all possible VLC-PUFA-containing phospholipids were analyzed from isolated POS, and all were elevated in the 24:5n-3 injection group. Among these, VLC-PUFA-containing PE, PC, LPC, PS, and plasmalogen PE increased by 25% (p=0.035), 26% (p=0.070), 20% (p=0.017), 22% (p=0.025), and 40% (p=0.007), respectively, in the 24:5n-3 supplementation group (Figure 9A). Overall, levels of phospholipids containing VLC-PUFAs increased by approximately 25% in POS-received fatty acid supplementation (p=0.033). However, analysis of total FA levels did not show a significant increase in total VLC-PUFA levels in the retina after supplementation, suggesting that either other forms of VLC-PUFA, such as free VLC-PUFA, or levels of VLC-PUFA in retinal compartments other than POS, could compensate for the observed changes (Figures 10A and 10B).

[0078] To understand the molecular changes occurring in the retinas of 18-month-old mice after fatty acid supplementation, bulk RNA sequencing analysis was performed on 24:5n-3-injected and vehicle-injected retinas isolated 5 days after intravitreal injection. Transcriptome changes were identified using the Bioconductor package DESeq229. First, the expression of Elovl2, Elovl4, and Elovl5, key enzymes involved in PUFA elongation, remained unchanged after supplementation (Figure 10C). 234 significantly altered genes (plicity change >|1.5|, p-adj <0.05) were identified. Interestingly, almost all differentially expressed genes (DEGs) were downregulated in eyes supplemented with 24:5n-3 (Figure 9B). Gene set enrichment analysis (GSEA) showed significant downregulation of complement (NES=-1.77, FDR q=0.00) and oxidative stress and redox pathways (NES=-1.70, FDR q=0.006) in retina supplemented with 24:5n-3 (Figure 9C). Visualization of enriched terms as a network using Metascape demonstrated downregulation of pathways involved in immune response, inflammation, microglial phagocytosis, and cell migration (Figures 9D and 10D).

[0079] To further identify transcription factors (TFs) involved in the gene expression changes observed after 24:5n-3 supplementation, we used ChIP-X Enrichment Analysis 3 (ChEA3), a tool for predicting DEG-related TFs based on a curated library of TF target gene sets from human, mouse, and rat. For downstream analysis, we constructed a global TF co-expression network using average rank output, which averages the ranks integrated across libraries. RNA sequencing data demonstrated downregulation of Sp100, Lyl1, Sp140, Irf5, Stat6, Fli1, Ikzf1, Spi1, Relb, Elf4, and Sp110, and upregulation of Trafd1. To visualize protein-protein interactions between these related TFs, we used STRING (v12.0) to create a functional interaction network of the top 20 TFs (Figure 9E). We removed low-expression TFs defined by a normalized read count (DESeq2 baseMean) < 10 and calculated the difference in mean Z-scores after 24:5n-3 supplementation for the remaining 16 TFs. This data demonstrated significant downregulation of almost all major TFs, including several interferon regulators (IRFs) involved in activating the immune response: Irf1, Irf2, Irf5, Irf7, and Irf8.

[0080] Further analysis revealed that, following 24:5n-3 supplementation, the “aging cerebellar” gene set was significantly downregulated in the retina of 18-month-old mice compared to controls (NES=-1.82, FDR=5.83E-4). This included significant downregulation of apolipoprotein E (Apoe) transcripts (log2FC=-0.79, p-adj=0.001). To support RNA sequencing data showing decreased expression of Apoe and complement cascade-related genes after supplementation, immunohistochemical analysis of retinal sections was performed (Figure 9F). First, a significant decrease in complement component C3, a major inflammatory protein activated by AMD35, was observed in the RPE of eyes supplemented with 24:5n-3. A decrease in the accumulation of APOE, a major component of aging retinal pigment epithelial drusenoid deposits, was also observed. Furthermore, levels of HTRA1, a serine protease secreted by RPE (Rapid Physiognomycete), which is increased in AMD, decreased after treatment with 24:5n-3. Finally, a decrease in levels of C5b-9, a subunit of the membrane attack complex (MEF), the terminal complex of the complement cascade, was observed, indicating reduced complement activation in eyes supplemented with 24:5n-3.

[0081] Animals: All animal experimentation procedures were approved by the Animal Science Committee (IACUC) of the University of California, Irvine. Elovl2 C234W The mice were previously generated using CRISPR / Cas9 technology. C57BL / 6J mice were purchased from Jax Laboratory for lipid supplementation studies. All mice were housed in the University of California, Irvine's housing facility under a standard 12-hour / 12-hour light (<150 lux) / dark cycle and fed ad libitum on a standard rodent diet (Envigo Teklad 2020X) without soy protein. Fatty acid levels in the diet are included in Table 1 below. [Table 1]

[0082] Electroretinography (ERG): Animals were dark-adapted overnight and anesthetized by IP injection of ketamine (100 mg / kg) and xylazine (4 mg / kg). Body temperature was maintained at 37-38°C with a heating pad during recording. Pupils were dilated using 1% atropine sulfate (rod dark-adaptation experiment) or 1% tropicamide and 2.5% phenylephrine (normal scotopic and photopic recording). The eyes were lubricated with corneal gel. A reference electrode needle was inserted subcutaneously into the skull. Responses to 11 stimuli of gradually increasing intensity were recorded and averaged. Wave a and wave b responses were plotted from the averaged ERG waveform. Scotopic and photopic ERG recordings were performed using a commercially available platform (Celeris, Diagnostics LLC). For photopic recording, mice were placed in a lit enclosure for 10 minutes prior to the experiment. Specific functions of M and S cones were measured. Stimulation was performed by alternating UV and green light irradiation using flashes of gradually increasing intensity. Green light stimulation was performed at -0.5, 0.5, 1.5, and 2.5 log cd·s / m 2 The intensity was increased through stages. UV light stimulation was -1, 0, 1, and 2 log cd·s / m 2 The response exhibited increasing intensity. The responses of the treated and untreated eyes were grouped separately and averaged together. Wave a and wave b traces represent the mean ERG waveform. Data were analyzed using Espion v.6 software (Diagnosys). GraphPad Prism was used for graph generation and statistical analysis.

[0083] Dark adaptation measurement: In rod dark adaptation experiments, full-field ERG was recorded using a UTAS BigShot device (LKC Technologies). The scotopic response to calibrated green (530nm) LED light was recorded for both eyes. Measurements from multiple tests were averaged, and the intervals between tests were adjusted to ensure that the response amplitude did not decrease across the series of tests in each step.

[0084] The a-wave flash sensitivity (Sf) of the rod ERG was calculated from the linear portion of the intensity-response curve as follows:

number

[0085] Optomortic Response (OMR): Optomortic responses (OMR) were recorded using a commercially available OMR device (PhenoSys GmbH, Berlin, Germany). This software automatically tracks the animal's head movement (optomotor reflex) in the direction of a rotating grid stimulus and calculates correct / incorrect tracking behavior presented as an OMR (optomotor) index. Mice were placed on a raised platform in the OMR arena. The grid stimulus (rotating at 12° / sec) was presented for approximately 12 minutes per test. The spatial frequency of the grid was set to 0.2 cycles per viewing angle. The stimulus was presented with varying contrasts of light and dark stripes: 3, 6, 10, 12.5, 15, 17.5, 20, 25, 37.5, 50, 75, and 100 contrasts. The stimulus at each contrast level lasted for 60 seconds. For the scotopic portion of the experiment (night light levels), the animals were dark-adapted for 12 hours before the experiment. The OMR arena was dimmed (to approximately 0.03 lux) using four neutral density filters in front of the stimulus display. Results with 100% contrast were excluded for adjustment (adjustment to the mouse system). OMR results that resulted in a correct / incorrect ratio of less than 0.8 were excluded. For each mouse, the results of multiple tests were averaged for analysis. Each data point represents the average of all mice tested. GraphPad Prism was used for statistical analysis and curve creation.

[0086] Neurophysiology (Visual Evoked Potentials - VEP): Animals were first anesthetized with 2% isoflurane (70% / 30%) in a mixture of N2O / O2, then placed in a stereotactic device, followed by local subcutaneous injection of analgesia (flunixine meglumine, 2.5 mg / kg SC every 8 hours) and lidocaine (0.5%). A small, custom-made plastic chamber was fixed to the exposed skull using dental acrylic. After 1 day of recovery, the re-anesthetized animals were placed in a custom-made hammock and maintained under isoflurane anesthesia (1-2% in N2O / O2). A craniotomy was performed, and multiple single tungsten electrodes were inserted into layers V1 II-VI. After electrode placement, the chamber was filled with sterile agar and sealed with sterile bone wax. Next, the animals were sedated with chlorprothixene hydrochloride (1 mg / kg; intramuscular administration) and maintained under mild isoflurane anesthesia (0.2-0.4% in 30% O2) throughout the recording procedure. EEG and EKG were monitored throughout, and body temperature was maintained with a heating pad (Harvard Apparatus, Holliston, Massachusetts).

[0087] Data was acquired using a multi-channel Scout recording system (Ripple, Utah, USA). Local electric field potentials (LFPs) obtained from multiple locations at the same cortical depth were processed with a 0.1Hz–250Hz band-pass filter and stored with spike data at a sampling rate of 1kHz. To calculate visually evoked potentials (VEPs), LFP signals were tuned to stimulus timestamps and averaged across tests at each recording depth. Spike signals from single neurons were processed with a 500Hz–7kHz band-pass filter and stored at a sampling frequency of 30kHz. Spikes were screened online using Trellis (Ripple, Utah, USA) while visual stimuli were applied. Visual stimuli were generated in Matlab (Mathworks, USA) using Psychophysics Toolbox and displayed on a gamma-corrected LCD monitor (55-inch, 60Hz; 1920×1080 pixels; average brightness 52cd / m2). A proprietary photodiode system was used to compensate for monitor delay during stimulus onset. Visual responses were evaluated according to previously published methods. To record visually evoked responses, animals were exposed to a 500 ms strong flash (10⁵ cd / m²). 2 The test was repeated 100 times.

[0088] Optical coherence tomography (OCT): Mice were anesthetized by intraperitoneal injection of ketamine / xylazine (10 mg / kg), followed by pupil dilation with 1% tropicamide and 2.5% phenylephrine. Optical coherence tomography (OCT) was performed using a Bioptigen spectral domain OCT system (Leica Microsystems, Buffalo Grove, Illinois), and four frames of OCT b-scan images were acquired from a series of 1200 a-scans. The thickness of the outer nuclear layer (ONL) was measured at a position 500 μm from the optic nerve head (ONH) in the nasal, temporal, superior, and inferior quadrants of each eye, and the mean ONL thickness was determined by averaging these values.

[0089] POS Separation: Photoreceptor outer segments (POSs) were separated using a slightly modified version of the previously described OptiPrep® density gradient centrifugation method. Briefly, one retina was placed in Ringer's solution (10 mM HEPES (pH 7.4), 130 mM NaCl, 3.6 mM KCl, 12 mM MgCl2, 1.2 mM CaCl2, and 0.02 mM EDTA) containing 8% OptiPrep® and vortexed for 1 minute. The sample was centrifuged at 200 × g for 1 minute at 4°C, and the supernatant containing ROS was gently collected. The vortexing and precipitation steps were repeated six times. The supernatants were combined for lipid extraction.

[0090] Intravitreal injection: All animals were anesthetized with isoflurane. Their eyes were anesthetized with propalacaine (0.5%, Bausch-Lomb), followed by pupil dilation with one drop of tropicamide (1%, Alcon Laboratories) and phenylephrine (2.5%, Akorn Pharmaceuticals, Lake Forest, Illinois). All drops were applied by pipette. Injections were performed under a surgical microscope (Zeiss). First, one drop of Gonak (Akorn Pharmaceuticals, Lake Forest, Illinois) was applied to the corneal surface to improve vision and prevent reflux from the eye. Next, a tunnel was created to reach the vitreous cavity using a 33-gauge subcutaneous needle. A 33-gauge blunt needle (World Precision Instruments) was connected to an RPE-KIT (World Precision Instruments) via a SilFlex tube (World Precision Instruments; SILFLEX-2) and advanced through the tunnel. Each mouse was injected with 1 μl of the compound per eye.

[0091] Lipid preparation: Fatty acids were mixed with 5% bovine serum albumin (BSA) filtered through a 0.22 μm membrane in physiological saline at a concentration of 36 mmol / L as a stock solution, and then incubated on a 40°C shaker for 1 hour to enable binding of fatty acids to BSA. The stock solution was diluted with physiological saline to obtain the required concentration. The vehicle was physiological saline containing a matching amount of BSA.

[0092] Lipid Extraction: Lipid extraction was performed according to the methodology of Bligh and Dyer. Briefly, the tissue was homogenized in 200 μL of water, transferred to a glass vial, and 750 μL of 1:2 (v / v) CHCl3:MeOH was added and vortexed. Next, 250 μL of CHCl3 was added and vortexed. Finally, 250 μL of ddH2O was added and vortexed. The sample was centrifuged at 3000 RPM for 5 minutes at 4°C. The lower phase was transferred to a new glass vial, dried under nitrogen, and stored at -20°C until subsequent lipid analysis.

[0093] LC-MS / MS: Lipid separation was performed using an Accucore C30 column (2.6 μm, 2.1 mm × 150 mm, Thermo Scientific). Q Exactive MS was operated in full MS scan mode (m / z 200, resolution 70,000), followed by ddMS2 (resolution 17,500) operation in both positive and negative modes. The AGC target values ​​were set to 1E6 and 1E5 for MS and MS / MS scan, respectively. The maximum injection time was 200 ms for MS and 50 ms for MS / MS. HCD was performed with stepwise collision energies of 30 ± 10% in negative ion mode and 25% and 30% in positive ion mode, using a separation window of 1.5 Da.

[0094] Data Analysis and Post-Processing: Data were analyzed using LipidSearch 4.2.21 software. Only peaks with molecular identification grade A or B were accepted (A: lipid class and fatty acid fully identified, or B: lipid class and some fatty acids identified). The relative abundance of each lipid species was obtained by normalizing to the total lipid intensity. VLC-PUFAs embedded in the PC were analyzed using Lipid Data Analyzer with a customized database. Lipid pathway analysis was performed using MetaboAnalyst, but BioPAN software (www.lipidmaps.org / biopan) was also used. Pathways with p-value < 0.05 and multiplier change > 1.5 were considered significant. The most active and most suppressed lipid response was considered significant (corresponding to p-value < 0.05 and Z-score > 1.645)21. Lipid species that showed significant changes (FC > 1.5, P < 0.05) were submitted to Lipid Ontology (LION) for lipid ontology analysis. Data visualization was performed using Prism 7 software (GraphPad Software, Inc.).

[0095] FA Analysis: Separation of VLC-PUFAs was achieved using an Acquity UPLC® BEH C18 column (1.7 μm, 2.1 mm × 100 mm, Waters Corporation). A Q Exactive MS was operated in negative mode full MS scan mode (resolution 70,000 at m / z 200). For the target compound, a scan range of m / z 250–800 was selected. Fatty acid identification was based on retention time and chemical formula.

[0096] RNA sequencing

[0097] Sample collection and preparation: Fresh retinal tissue was dissected from each mouse eye. Total RNA was extracted from the retina using the RNeasy Plus Mini Kit (Qiagen) according to the manufacturer's instructions. The quantity and quality of RNA were evaluated using the Qubit RNA HS assay kit (Thermo Fisher Scientific) and the Agilent Bioanalyzer RNA 6000 Pico kit (Agilent Technologies), respectively.

[0098] Library Construction and Sequencing: RNA sequencing libraries were prepared using the Illumina TruSeq RNA Library Prep kit. Paired-end sequencing was performed using a NovaSeq 6000 system with flow cell type S4. This generated paired-end reads of 100 base pairs in length, yielding approximately 30 million reads per sample.

[0099] Bioinformatics analysis: Raw sequencing reads were preprocessed using Trimmomatic to remove adapter sequences and low-quality bases. Washed reads were mapped to the mm10 assembled mouse reference genome using the aligner tool HISAT2. Gene-level expression counts were quantified using featureCounts. Differential expression analysis was performed using DESeq2, and genes with adjusted p-values ​​<0.05 and |multiplicity change| >1.5 were considered differentially expressed genes.

[0100] Single-nuclear RNA sequencing (snRNA-seq)

[0101] Sample collection and preparation: Retinals (n=3 per age group) were dissected and rapidly frozen in liquid nitrogen. Deep-frozen samples were sent to Active Motif for further processing. Active Motif is a 10x Genomics certified service provider for Single-Cell Multiomes for measuring whole-genome gene expression and open chromatin. Samples were processed according to Illumina's "Isolation of Nuclei for Single Cell RNA Sequencing & Tissues for Single Cell RNA Sequencing" procedure and subjected to snRNAseq according to Illumina's protocol.

[0102] Quality control, integration, and clustering: 91bp sequencing reads were generated by Illumina sequencing. The reads were mapped to a reference genome, and feature counts were identified using Cell Ranger for Single Cell Gene Expression software with default parameters (mkfastq and count function). Quality control was performed, and cell barcodes were retained that met the following three criteria: number of unique molecular identifiers (UMIs) within three median absolute deviations (MADs) of the population median, number of expressed genes within three MADs of the population median, and percentage of reads mapped to mitochondrial genes less than 15%. Approximately 1000 gene expressions per nucleus were detected in each sample. Downstream analysis was performed using the Seurat (v4.3.0) R package. Data were normalized using Seurat's LogNormalize function, and the optimal cluster resolution was determined using a clustering tree method.

[0103] Uniform Manifold Projection (UMAP) was used to visualize cells in a two-dimensional space. Cell identity was assigned using known markers established in previous studies and markers identified in this study. Differential gene expression analysis between clusters was performed using the Wilcoxon rank-sum test. Genes with a p-value less than 0.05 and a log-multiple change greater than 1 were considered to have altered expression. Furthermore, differential gene expression analysis between young and old conditions within each cell type was performed using the Wilcoxon rank-sum test. Genes with a p-value less than 0.05 and a log-multiple change greater than 0.1 were considered to have altered expression. Feature plots of Elovl2 expression levels were normalized to TBP.

[0104] Intercellular communication analysis: Intercellular communication analysis was performed using the CellChat package (v.2.0.0). Only pyramidal, rod, and Müller glial cells were used to infer intercellular communication. CellChat was first applied separately to young (3-month-old) and elderly (18-month-old) datasets to infer intercellular communication, and then a comparative analysis was performed by merging CellChat objects obtained from the young and elderly datasets. In short, following the official workflow, normalized counts were loaded into CellChat, and the preprocessing functions "identifyOverExpressedGenes", "identifyOverExpressedInteractions", and "projectData" were applied with standard parameters. For the main analysis, the core functions "computeCommunProb", "computeCommunProbPathway", and "aggregateNet" were applied using standard parameters.

[0105] Gene Set Enrichment Analysis (GSEA): Gene set enrichment analysis (GSEA) was performed using GSEA software (v4.2.3)76. Differential expression genes identified by DESeq2 were ranked using the signal-to-noise ratio rank index. Enrichment analysis was performed using predefined gene sets from databases such as Gene Ontology (GO), the Molecular Signatures Database Hallmark gene set, Reactome, and Wikpathways. The statistical significance of enrichment was determined using a weighted (p=1) scoring calculation scheme with 1000 substitutions. Gene sets with an mp size greater than 500 and less than 5 were excluded from the analysis.

[0106] Metascape gene enrichment and functional analysis: Differential expression genes (DEGs) (adjusted p-value < 0.05, |multiplicative change| > 1.5) were input into the Metascape platform, and pathway and process enrichment analysis was performed using selected ontology sources including GO Biological Processes, KEGG pathways, Reactome Gene Set, and WikiPathways. Terms with p-value < 0.01, minimum count of 3, and enrichment coefficient > 1.5 (the enrichment coefficient is the ratio of observed counts to the expected count by chance) were collected and grouped into clusters based on their membership similarity. Next, to further understand the relationships between terms, a network diagram was created for a subset of enriched terms. In this diagram, terms with similarity > 0.3 are connected by edges. This network was visualized using Cytoscape (v3.10.0), where each node represents an enriched term, proportional to the number of input genes for each term, and color-coded by its cluster ID or p-value.

[0107] Transcription Factor Enrichment Analysis: Transcription factor (TF) enrichment analysis was performed on differentially expressed genes (DEGs) using the ChEA3 (ChIP-X Enrichment Analysis 3) web tool (adjusted p-value < 0.05, |multiplicity change| > 1.5). TFs were ranked using a mean rank index, which averages the integrated rank across the entire gene set library. A global transcriptional co-expression network was constructed for the top 20 TFs from GTEx expression data and clustered using GO enrichment terms. Protein-protein interaction analysis was performed on the top 20 relevant TFs using the Search Tool for the Retrieval of Interaction Genes / Protein (STRING) database search tool. After excluding TFs with low expression levels (normalized read count DESeq2 baseMean < 10), protein-protein interactions were visualized as nodes and edges using Cytoscape (v3.10.0), and each node was color-coded by the difference in mean Z-scores.

[0108] Bisulfite sequencing for DNA methylation analysis: DNA methylation profiles were determined using PCR amplification followed by DNA Sanger sequencing. Methylated DNA was converted using the EZ DNA Methylation-Direct® Kit (Zymo Research, USA) according to the manufacturer's instructions. The converted DNA was amplified using primers that did not overlap with CpGs. The amplified products were sequenced to detect the methylation status of individual cytosines within the amplified region. The methylation rate at each CpG site was calculated as the ratio of methylated cytosines to the total signal level of cytosines.

[0109] Retinal cryosections and immunohistochemistry: Mouse eyes were excised after euthanasia and fixed in 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS, pH 7.4) at 4°C for 2 hours. The cornea, lens, and vitreous humor were removed, and the optic cup was fixed overnight in 4% PFA at 4°C. After fixation, the optic cup was cryoprotected by immersion in a sucrose density gradient (1 hour in 10% and 20% sucrose solutions at room temperature, and overnight in 30% sucrose solution at 4°C). The optic cup was embedded in Tissue-Tek OCT (Sakura, Torrance, California) and frozen on a conductive metal block placed on dry ice. Cryosections were prepared using a cryostat, and the retinal sections were cut into 10 μm thick serial sections.

[0110] For immunohistochemistry, sections were blocked with 0.3% TritonX-100 in 5% BSA at room temperature for 1 hour to minimize nonspecific binding. Next, sections were incubated overnight at 4°C with primary antibody diluted in 0.1% TritonX-100 in 5% BSA (see Table 1). After three washes with PBS, sections were incubated with fluorescently labeled secondary antibody diluted in 0.1% TritonX-100 in 5% BSA (see Table 1) at room temperature for 1 hour. After three washes with PBS, the nuclei were counterstained with Hoescht 33342 (Thermo), and sections were mounted using ProLong Gold Antifade (Thermo). Immunostained sections were imaged at 40x and 100x magnification using a Keyence all-in-one fluorescence microscope (BZ-X810, Keyence, Itasca, Illinois).

[0111] In situ hybridization was performed using RNAscope® Multiplex Fluorescent Assay v2 (ACD Diagnostics). The probes used were manufacturer-designed. Briefly, fresh frozen tissue sections of mouse eyes were pre-treated with a target retrieval reagent containing hydrogen peroxide and protease IV according to the manual. Next, the probes were hybridized according to the protocol and then detected using fluorescein, cyanine 3, and cyanine 5 of TSA Plus® Fluorophores. Sections were mounted on ProLong Gold Antifade (Thermo Fisher) and imaged (Keyence BZ-X810).

[0112] Proteome analysis: Mouse retinas were harvested and suspended in urea buffer containing 8M urea, 0.1M Tris-HCl (pH 8.5), and a protease inhibitor cocktail (cOmplete® Protease Inhibitor Cocktail, Roche). The suspension was sonicated on ice for 4 minutes, followed by centrifugation at 12,000 g for 10 minutes at 4°C. The supernatant was collected and digested by filter-assisted sample preparation (FASP) method 83. Briefly, the supernatant was transferred to a spin-filter column (30 kDa cutoff). The proteins were reduced with 10 mM DTT at 56°C for 1 hour and alkylated with 20 mM iodoacetic acid for 30 minutes at room temperature in the dark. The buffer was then replaced with 50 mM NH4HCO3 by washing the membrane three times.

[0113] For the quantification of Rho, PED6A, PDE6B, GNAT1, and GBB1, stable isotope-labeled (SIL) peptides were added to the protein sample, and then free trypsin was added to the protein solution to achieve a trypsin-to-protein ratio of 1:50. The solution was incubated overnight at 37°C. The trypsin digest was centrifuged, washed further with water, and collected in the supernatant. The combined supernatant was vacuum-dried and then dissolved in 20 μL of 0.1% FA aqueous solution.

[0114] As used herein, the term “approximately” refers to ±10% of the referenced number.

[0115] While preferred embodiments of the present invention have been shown and described, it will be readily apparent to those skilled in the art that modifications may be made therein, without exceeding the scope of the appended claims. Therefore, the scope of the present invention is limited only by the appended claims. In some embodiments, the drawings presented in this patent application are drawn to scale, including angles and dimensional ratios. In some embodiments, the drawings are representative only, and the claims are not limited by the dimensions in the drawings. In some embodiments, descriptions of the present invention using the phrase "including" in this specification encompass embodiments that may be described as "essentially consisting of" or "consisting of." Therefore, the requirements for claiming one or more embodiments of the present invention using the phrase "essentially consisting of" or "consisting of" are met.

Claims

1. A method for treating age-related eye diseases in subjects requiring treatment for age-related eye diseases, comprising administering a composition containing tetracosapentaenoic acid [24:5n-3] to the subjects.

2. The method according to claim 1, wherein the composition is administered to the eye of the subject.

3. A method for treating age-related eye diseases in subjects requiring treatment for age-related eye diseases, comprising administering a composition containing tetracosapentaenoic acid [24:5n-3] to the eyes of the subjects.

4. The method according to any one of claims 1 to 3, wherein tetracosapentaenoic acid [24:5n-3] is administered as a free fatty acid, a fatty acid ethyl ester (FAEE), or a fatty acid methyl ester (FAME).

5. The method according to any one of claims 1 to 4, wherein the composition further comprises docosahexaenoic acid (DHA).

6. The method according to claim 5, wherein DHA is administered as a free fatty acid, a fatty acid ethyl ester (FAEE), or a fatty acid methyl ester (FAME).

7. The method according to any one of claims 1 to 6, wherein the composition is administered by intravitreous injection, intravenous injection, intraperitoneal injection, or local administration.

8. The method according to any one of claims 1 to 7, wherein the age-related eye disease is age-related macular degeneration and / or age-related loss / decline in visual acuity.

9. The method according to any one of claims 1 to 8, wherein a therapeutically effective dose is administered to the disease.

10. A method for treating age-related liver disease in a subject requiring treatment for age-related liver disease, comprising administering a composition containing tetracosapentaenoic acid [24:5n-3] to the subject.

11. The method according to claim 10, wherein tetracosapentaenoic acid [24:5n-3] is administered as a free fatty acid, a fatty acid ethyl ester (FAEE), or a fatty acid methyl ester (FAME).

12. The method according to claim 10 or claim 11, wherein the composition further comprises docosahexaenoic acid (DHA).

13. The method according to claim 12, wherein DHA is administered as a free fatty acid, a fatty acid ethyl ester (FAEE), or a fatty acid methyl ester (FAME).

14. The method according to any one of claims 10 to 13, wherein the composition is administered by intravenous injection.

15. The method according to any one of claims 10 to 14, wherein the age-related liver disease is non-alcoholic fatty liver disease, alcoholic liver disease, hepatitis C, fibrosis, or cirrhosis.

16. The method according to any one of claims 10 to 15, wherein a therapeutically effective dose is administered to the disease.

17. A method for treating age-related brain disease in a subject requiring treatment for age-related brain disease, comprising administering a composition containing tetracosapentaenoic acid [24:5n-3] to the subject.

18. The method according to claim 17, wherein the composition is administered to the target brain.

19. A method for treating age-related brain disease in a subject requiring treatment for age-related brain disease, comprising administering a composition containing tetracosapentaenoic acid [24:5n-3] to the brain of the subject.

20. The method according to any one of claims 17 to 19, wherein tetracosapentaenoic acid [24:5n-3] is administered as a free fatty acid, a fatty acid ethyl ester (FAEE), or a fatty acid methyl ester (FAME).

21. The method according to any one of claims 17 to 20, wherein the composition further comprises docosahexaenoic acid (DHA).

22. The method according to claim 21, wherein DHA is administered as a free fatty acid, a fatty acid ethyl ester (FAEE), or a fatty acid methyl ester (FAME).

23. The method according to any one of claims 17 to 22, wherein the composition is administered by intravenous injection and / or stereotactic direct injection.

24. The method according to any one of claims 17 to 23, wherein the age-related brain disease is selected from diseases comprising Alzheimer's disease, amyotrophic lateral sclerosis (ALS), age-related cognitive decline, dementia, and Parkinson's disease.

25. The method according to any one of claims 17 to 24, wherein a therapeutically effective dose is administered to the disease.

26. A method for treating age-related bone disease in a subject requiring treatment for age-related bone disease, comprising administering a composition containing tetracosapentaenoic acid [24:5n-3] to the subject.

27. The method according to claim 26, wherein tetracosapentaenoic acid [24:5n-3] is administered as a free fatty acid, a fatty acid ethyl ester (FAEE), or a fatty acid methyl ester (FAME).

28. The method according to claim 26 or claim 27, wherein the composition further comprises docosahexaenoic acid (DHA).

29. The method according to claim 28, wherein DHA is administered as a free fatty acid, a fatty acid ethyl ester (FAEE), or a fatty acid methyl ester (FAME).

30. The method according to any one of claims 26 to 29, wherein the composition is administered by intravenous administration and / or intravenous injection.

31. The method according to any one of claims 26 to 30, wherein the age-related bone disease is multiple myeloma and other myelodysplastic syndromes (MDS) or anemia.

32. The method according to any one of claims 26 to 31, wherein a therapeutically effective dose is administered to the disease.

33. A composition for use in treating age-related eye diseases in subjects requiring treatment of age-related eye diseases, wherein the composition comprises tetracosapentaenoic acid [24:5n-3].

34. The composition according to claim 33, wherein tetracosapentaenoic acid [24:5n-3] is administered as a free fatty acid, a fatty acid ethyl ester (FAEE), or a fatty acid methyl ester (FAME).

35. The composition according to claim 33 or claim 34, wherein the composition further comprises docosahexaenoic acid (DHA).

36. The method according to claim 35, wherein DHA is administered as a free fatty acid, a fatty acid ethyl ester (FAEE), or a fatty acid methyl ester (FAME).

37. The composition according to any one of claims 33 to 36, wherein the composition is administered to the eye of the subject.

38. The composition according to any one of claims 33 to 37, wherein the composition is administered by intravitreal injection, intravenous injection, intraperitoneal injection, or local administration.

39. The composition according to any one of claims 33 to 38, wherein the age-related eye disease is age-related macular degeneration and / or age-related loss / decline in vision.

40. A composition for use in the treatment of age-related brain disease in subjects requiring treatment for age-related brain disease, wherein the composition comprises tetracosapentaenoic acid [24:5n-3].

41. The composition according to claim 40, wherein tetracosapentaenoic acid [24:5n-3] is administered as a free fatty acid, a fatty acid ethyl ester (FAEE), or a fatty acid methyl ester (FAME).

42. The composition according to claim 40 or claim 41, further comprising docosahexaenoic acid (DHA).

43. The method according to claim 42, wherein DHA is administered as a free fatty acid, a fatty acid ethyl ester (FAEE), or a fatty acid methyl ester (FAME).

44. The composition according to any one of claims 40 to 43, wherein the composition is administered by intravenous injection and / or stereotactic direct injection.

45. The composition according to any one of claims 40 to 44, wherein the composition is administered to the brain of the subject.

46. The composition according to any one of claims 40 to 45, wherein the age-related brain disease is selected from diseases comprising Alzheimer's disease, amyotrophic lateral sclerosis (ALS), age-related cognitive decline, dementia, and Parkinson's disease.

47. A composition for use in the treatment of age-related liver disease in subjects requiring treatment for age-related liver disease, wherein the composition comprises tetracosapentaenoic acid [24:5n-3].

48. The composition according to claim 47, wherein tetracosapentaenoic acid [24:5n-3] is administered as a free fatty acid, a fatty acid ethyl ester (FAEE), or a fatty acid methyl ester (FAME).

49. The composition according to claim 47 or claim 48, wherein the composition further comprises docosahexaenoic acid (DHA).

50. The method according to claim 49, wherein DHA is administered as a free fatty acid, a fatty acid ethyl ester (FAEE), or a fatty acid methyl ester (FAME).

51. The composition according to any one of claims 47 to 50, wherein the composition is administered by intravenous injection.

52. The composition according to any one of claims 47 to 51, wherein the age-related liver disease is non-alcoholic fatty liver disease, alcoholic liver disease, hepatitis C, fibrosis, or cirrhosis.

53. A composition for use in the treatment of age-related bone disease in subjects requiring treatment for age-related bone disease, wherein the composition comprises tetracosapentaenoic acid [24:5n-3].

54. The composition according to claim 53, wherein tetracosapentaenoic acid [24:5n-3] is administered as a free fatty acid, a fatty acid ethyl ester (FAEE), or a fatty acid methyl ester (FAME).

55. The composition according to claim 53 or claim 54, wherein the composition further comprises docosahexaenoic acid (DHA).

56. The method according to claim 55, wherein DHA is administered as a free fatty acid, a fatty acid ethyl ester (FAEE), or a fatty acid methyl ester (FAME).

57. The composition according to any one of claims 53 to 56, wherein the composition is administered by intravenous administration and / or intravenous injection.

58. The composition according to any one of claims 53 to 57, wherein the age-related bone disease is multiple myeloma and other myelodysplastic syndromes (MDS) or anemia.

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