WO2025072929A1 - Methods and compositions of treating age-related conditions - Google Patents

Abstract

Compositions and methods for treating age-related eye conditions and/or age-related brain conditions in a subject in need thereof The compositions comprise tetracospentaenoic add [24:5n-3], The method comprises the steps of identifying the subject presenting with the age-related eye condition, and administering tetracospentaenoic acid [24:5n-3j to the subject.

Description

Skowronska-Krawczyk and Gao September 24, 2024 METHODS AND COMPOSITIONS OF TREATING AGE-RELATED CONDITIONS CROSS-REFERENCE TO RELATED APPLICATIONS [0001]This application claims benefit of U.S. Provisional Application No. 63/586,251 filed September 28, 2023, the specification of which is incorporated herein in their entirety by reference. FIELD OF THE INVENTION [0002]The present invention provides methods and compositions for treating age-related eye conditions. Additionally, it offers methods and compositions for treating age-related brain conditions. BACKGROUND OF THE INVENTION [0003]The specific composition of lipids within membranes dictates their biophysical properties, such as diffusion, permeability, domain formation, and curvature generation. Age-related changes in membrane lipid composition have been postulated to be one of the hallmarks of aging. In the aged retina, polyunsaturated fatty acids (PUFAs), essential components of cellular membranes, show significantly decreased levels, and this decrease is further exacerbated in retinas affected by age-related macular degeneration (AMD). Retinal tissue is particularly enriched in long- and very long-chain PUFAs (LC-PUFA and VLC-PUFAs, respectively), which are integral components of photoreceptor disc membranes, and their depletion or reduced levels have been postulated to be one of the hallmarks of AMD. [0004]Strategies aimed at preserving or replenishing VLC-PUFAs in the aging retina, such as dietary interventions with n-3 PUFAs and LC-PUFAs, are being investigated as potential approaches to maintain retinal health and function in older individuals. While some studies indicate improvement of vision following supplementation with docosahexaenoic fatty acid (DHA) or both DHA and eicosapentaenoic fatty acid (EPA), others, like Age-Related Eye Disease Study 2 (AREDS2), report no such correlation, deeming DHA and EPA supplementation trials inconclusive. Interestingly, dietary and oral supplementation of animals with very high doses of VLC-PUFAs have shown promising results. Another study has shown some improvement in visual performance in a dietary supplementation experiment with fish oil enriched in n-3 C24-28 VLC-PUFAs. However, due to the high cost of synthesizing VLC-PUFAs and the limited accessibility Skowronska-Krawczyk and Gao September 24, 2024 to enriched fish oil, coupled with their relatively low efficacy, these methods currently lack practical applicability for preventing age-related decline in human vision. BRIEF SUMMARY OF THE INVENTION [0005] It is an objective of the present invention to provide methods and compositions that allow for treating age-related eye and brain conditions in a subject in need thereof, as specified in the independent claims. Embodiments of the invention are given in the dependent claims. Embodiments of the present invention can be freely combined with each other if they are not mutually exclusive. [0006]The exact mechanisms underlying the decrease 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 (Elongation of very long-chain fatty acids protein 2), which encodes an endoplasmic reticulum membrane-resident protein that produces precursors to DHA and VLC-PUFAs. Elovl2 is primarily expressed in tissues with high metabolic demands, such as the liver, retina, and brain. Importantly, increasing methylation of the ELOVL2 regulatory region has been shown to be one of the best biomarkers of chronological aging. [0007]Thus, the present invention identifies and describes critical molecular, structural, and functional changes in the aging retina and correlates many of these phenotypes with the lack of ELOVL2 activity and disturbed lipid composition. Intravitreal injection of the direct product of ELOVL2 elongation, 24:5n-3, improves visual function, reduces the severity of aging phenotypes, and decreases the accumulation of sub-RPE deposits in aged mice. Notably, no other PUFA demonstrated such a profound effect. The present invention underscores the importance of ELOVL2 activity in maintaining healthy vision and suggests a potential new therapy to reverse the symptoms of aging in the eye and prevent age-related eye diseases such as AMD. [0008] In some embodiments, the present invention features a method of treating age-related eye conditions (e.g., age-related macular degeneration and/or age-related vision loss/decline) in a subject in need thereof. In some embodiments, the method involves administering a composition of tetracospentaenoic acid [24:5n-3] to the subject, e.g., into the eye of the subject. In other embodiments, the method involves Skowronska-Krawczyk and Gao September 24, 2024 administering a composition of tetracospentaenoic acid [24:5n-3] and docosahexaenoic fatty acid (DHA) to the subject, e.g., into the eye of the subject. In some embodiments, the tetracospentaenoic acid [24:5n-3] and/or the DHA are administered as a free fatty acid, a fatty acid ethyl ester (FAEE), or a fatty acid methyl ester (FAME). The composition may be administered via an intravitreal injection, an intravenous injection, an intraperitoneal injection, or topically. The present invention may also feature a composition for use in treating age-related eye conditions in a subject in need thereof. In some embodiments, the composition comprises tetracospentaenoic acid [24:5n-3]. In other embodiments, the composition comprises tetracospentaenoic acid [24:5n-3] and DHA. [0009] In some embodiments, the present invention features a method of treating age-related liver conditions (e.g., nonalcoholic fatty liver disease, alcoholic liver disease, hepatitis C, fibrosis, or cirrhosis) in a subject in need thereof. In some embodiments, the method comprises administering a composition comprising tetracospentaenoic acid [24:5n-3] to the subject. In other embodiments, the method comprises administering a composition comprising tetracospentaenoic acid [24:5n-3] and DHA to the subject. In some embodiments, the tetracospentaenoic acid [24:5n-3] and/or the DHA are administered as a free fatty acid, a FAEE, or a FAME. The composition may be administered via an intravenous injection. The present invention may also feature a composition for use in treating age-related liver conditions in a subject in need thereof. In some embodiments, the composition comprises tetracospentaenoic acid [24:5n-3]. In other embodiments, the composition comprises tetracospentaenoic acid [24:5n-3] and DHA. [0010] In other embodiments, the present invention features a method of treating age-related brain conditions (e.g., Alzheimer’s Disease, ALS, age-related cognitive decline, dementia, and Parkinson’s disease) in a subject in need thereof. In some embodiments, the method comprises administering a composition comprising tetracospentaenoic acid [24:5n-3] to the subject, e.g., into the subject's brain. In other embodiments, the method comprises administering a composition comprising tetracospentaenoic acid [24:5n-3] and DHA to the subject, e.g., into the subject's brain. In some embodiments, the tetracospentaenoic acid [24:5n-3] and/or the DHA are administered as a free fatty acid, a FAEE, or a FAME. The composition may be Skowronska-Krawczyk and Gao September 24, 2024 administered via an intravenous and/or stereotactic direct injection. The present invention may also feature a composition for use in treating age-related brain conditions in a subject in need thereof. In some embodiments, the composition comprises tetracospentaenoic acid [24:5n-3]. In other embodiments, the composition comprises tetracospentaenoic acid [24:5n-3] and DHA. [0011] In further embodiments, the present invention features a method of treating age-related bone conditions (e.g., multiple myeloma and other Myelodysplastic Syndromes (MDS), or anemia) in a subject in need thereof. In some embodiments, the method comprises administering a composition comprising tetracospentaenoic acid [24:5n-3] to the subject. In other embodiments, the method comprises administering a composition comprising tetracospentaenoic acid [24:5n-3] and DHA to the subject. In some embodiments, the tetracospentaenoic acid [24:5n-3] and/or the DHA are administered as a free fatty acid, a FAEE, or a FAME. The composition may be administered via an intravenous and/or intravenous injection. The present invention may also feature a composition for use in treating age-related bone conditions in a subject in need thereof. In some embodiments, the composition comprises tetracospentaenoic acid [24:5n-3]. In other embodiments, the composition comprises tetracospentaenoic acid [24:5n-3] and DHA. [0012]One of the unique and inventive technical features of the present invention is administering tetracospentaenoic acid [24:5n-3] directly to the eye. Without wishing to limit the invention to any theory or mechanism, it is believed that the technical feature of the present invention advantageously provides for a slowing down progression, reversal or healing of eye related conditions. None of the presently known prior references or works have the unique inventive technical feature of the present invention. For example, traditionally, oral administration of n-3 PUFAs was the gold standard mode of administration. Thus, it would not be obvious to try a direct intravitreal mode of administration. [0013]Moreover, the inventive technical features of the present invention contributed to a surprising result. For example, 24:5n-3 is found in very small amounts in the eye, and the levels in a diseased eye as compared to a healthy eye do not differ that much. As such, one of ordinary skill in the art would not expect 24:5n-3 to improve visual function. However, it was surprisingly found that when the 24:5n-3 was injected, 24:5n-3 was able Skowronska-Krawczyk and Gao September 24, 2024 to improve visual function, leading to a reversal or healing of eye related conditions. [0014]Any feature or combination of features described herein are included within the scope of the present invention provided that the features included in any such combination are not mutually inconsistent as will be apparent from the context, this specification, and the knowledge of one of ordinary skill in the art. Additional advantages and aspects of the present invention are apparent in the following detailed description and claims. BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S) [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 in which: [0016]FIG. 1A, 1B, 1C, 1D, 1E, 1F, 1G, 1H, 1I, and 1J shows age-related decreased VLC-PUFA levels in the retina are associated with vision decline. FIG.1A shows levels of 18:0, 20:4n-6, 22:6n-3, and VLC-PUFAs in retinas dependent on age (n=5, * = p<0.05, ** = p<0.01). FIG.1B shows levels of major phospholipid classes in 3-month-old and 18-month-old retinas (n=5, * = p<0.05, ** = p<0.01). FIG.1C shows quantification of lipid classes in aging photoreceptor outer segments (POS) (n=10, * = p<0.05). FIG.1D shows lipid ontology (LION) analysis of significantly changed lipids in aging POS showed severe alterations in membrane biophysical properties. FIG. 1E shows down-regulated PC (48:12) and PC (50:12) in aging POS (n=10, * = p<0.05). FIG.1F shows scotopic and photopic electroretinogram (ERG) responses in aged (18-month-old) mice compared to young (3-month-old) mice (n=10, ** = p<0.01, *** = p<0.001, **** = p<0.0001). FIG. 1G shows the rate of rod-mediated dark adaptation recovery in young (3-month-old) and aged (17-month-old) mice. FIG. 1H shows oscillatory potential amplitudes of dark-adapted scotopic ERG in old (17-month-old) and young (3.5-month-old) mice. FIG. 1I shows absolute levels of PDE6A and PDE6B in aged (18-month-old) retinas quantified using a stable isotope–labeled (SIL) peptides-based method (n=5, * = p<0.05). FIG.1J shows immunostaining of C3, APOE and C5b-9, markers of AMD in young (5-month-old) and in old (26-month-old) retinas; scale bar 25um [0017]FIG. 2A, 2B, 2C, 2D, 2E, 2F, 2G, 2H, and 2I shows that age-related decreased VLC-PUFA levels in the retina are associated with vision decline. FIG. 2A shows Skowronska-Krawczyk and Gao September 24, 2024 significantly changed lipids (FC>|1.5|, p<0.05) exhibited distinct clustering between young (3-month-old) and aged (18-month-old) retinas by principal component analysis (PCA). FIG. 2B shows a network of lipidomic changes derived from Bioinformatics Methodology for Pathway Analysis (BioPAN). The thin, light gray arrow indicates negative z-score. The dark gray arrow indicates positive z-score. Dark grey node color indicates that the node is involved in a pathway that is significantly active or suppressed. The highlighting of the dark green arrow indicates that the pathway is significantly altered. Black arrow indicates upregulation of DGAT2 in aged retina. FIG.2C shows that among the analyzed VLC-PEs, PE (48:12) was significantly downregulated in aging POS (n=10, *** = p<0.001). FIG. 2D shows decreased contrast sensitivity in aged (18-month-old) animals compared to young (3-month-old) animals (* = p<0.05, ** = p<0.01, *** = p<0.001). FIG.2E shows photopic ERG waveforms in response to UV and green flash in 3- 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 an average of n=5 animals in representative groups. FIG.2F (left) shows a decreased recovery of the averaged Amax in aged mice (17-month-old) compared to young mice (3.5-month-old mice). FIG. 2F (right) shows the recovery of rod-driven ERG a-wave sensitivity (S_f) following the same bleach was not compromised in older animals. FIG. 2G shows relative amounts of proteins involved in phototransduction normalized to Rho remained unchanged in aging retina. FIG.2H shows immunofluorescence staining with rod bipolar-specific antibody (PKCa) showed age-related morphological changes in outer plexiform layer including overgrowth of dendrites into the outer nuclear layer. FIG. 2I shows immunofluorescence staining with glutamine synthetase antibody (GS) showed age-related Muller cell processes passing outer limiting membrane. [0018]FIG. 3A, 3B, 3C, 3D, 3E, and 3F shows age-related decrease in Elovl2 expression. FIG.3A shows VLC-PUFA elongation pathways. VLC-PUFAs are produced from essential FAs elongation by enzymes including Elovl2, 4, 5. FIG.3B shows Elovl2 promoter is increasingly methylated with age in mouse retina. FIG.3C shows images of mouse retina sections from young—5mo (top panels) and old—26mo (bottom panels) animals stained with RNAscope probes designed for Elovl2, Elovl4 and Elovl5, counterstained with Hoechst. FIG. 3D shows snRNA-seq demonstrated decreased expression of Elovl2 in cone photoreceptors in old (18-month-old) mice compared to young (3-month-old) mice. FIG. 3E shows GSEA analysis of pathways enriched in Skowronska-Krawczyk and Gao September 24, 2024 cones from young (3-month-old) and old (18-month-old) mice. FIG. 3F shows the visualization of interaction strength across young and old retinas. Each plot represents interactions among the cell types. The thickness of the lines between the cells indicates the interaction strength, with thicker lines representing stronger interactions. [0019]FIG. 4A, 4B, and 4C shows scRNA-seq analysis of genes involved in VLC-PUFAs biosynthesis pathway. FIG. 4A shows CZ CELLxGENE Discover visualization of expression of genes involved in VLC-PUFAs biosynthesis pathway in different cell types of the eye. FIG. 4B shows visualization of the communication probabilities mediated by ligand-receptor (L-R) pairs from specific cell groups to other cell groups in the young (3-month-old) and old (18-month-old) retinas. The x-axis represents the source-target cell group pairs, while the y-axis lists the different L-R pairs. The color intensity of the dots corresponds to the communication probability. The size of the dots represents the significance of the interactions CellChat interaction. FIG. 4C shows the visualization of the signaling pathways mediating interactions in young and old retinas. Each segment along the circle represents a cell group, and the connecting arcs indicate the interactions between these groups. The color of the arcs corresponds to the source cell group. The thickness of the arcs indicates the interaction strength of the signaling pathway. [0020]FIG. 5A, 5B, 5C, 5D, 5E, 5F, 5G, 5H, 5I, and 5J shows disturbed lipid composition in Elovl2^C234W mouse retinas is correlated with vision loss. FIG.5A shows total fatty acid products of ELOVL2 elongation, DHA (22:6) and VLC-PUFAs (24:6, 32:6, 34:6 and 36:6) were decreased in retinas of Elovl2^C234W mice compared to age-matched wildtype mice (n=4, * = p<0.05). FIG.5B shows changes in major phospholipid classes in Elovl2^C234W retinas compared to age-matched wildtype retinas, resembling changes seen in aged retinas compared to young retinas (** = p<0.01). FIG. 5C shows lipid ontology (LION) analysis of significantly changed lipids in Elovl2^C234W retinas showed severe alterations in membrane biophysical properties. FIG.5D shows VLC-PC species in Elovl2C234W retinas (* = p<0.05). FIG. 5E, 5F, 5H shows decreased contrast sensitivity (FIG.5E), scotopic electroretinogram (ERG) responses (FIG.5F) and delayed rod-mediated dark adaptation (FIG. 5H) of Elovl2^C234W mice compared to age-matched wildtype mice (* = p<0.05, ** = p<0.01, *** = p<0.001, **** = p<0.0001). FIG.5G shows maximum ERG a-wave amplitudes of 12-month-old Elovl^2C234W retinas were similar to those of 18-month-old wild-type retinas. FIG. 5I shows no difference in outer nuclear Skowronska-Krawczyk and Gao September 24, 2024 layer (ONL) thickness between 12-month-old wildtype and Elovl2^C234W retinas as measure by OCT (n=5/group), and 18-month-old Elovl2^C234W and wildtype retinas (n=4/group). FIG. 5J shows a comparison of GSEA Normalized Enrichment Scores (NES) of selected GO terms in 12-month-old Elovl2^C234W vs. 12-month-old wildtype retinas with the NES of GO terms in 18-month-old vs.12-month-old retinas. [0021]FIG. 6A, 6B, 6C, 6D, 6E, 6F, and 6G shows Disturbed lipid composition in Elovl2C234W mouse retinas is correlated with vision loss. FIG. 6A shows decreased total levels of VLC-PC in Elovl2^C234W retinas compared to age-matched wildtype retinas (* = p<0.05). FIG. 6B shows free fatty acids analysis showed decreased DHA and VLC-PUFAs in Elovl2^C234W retinas (n=3, * = p<0.05). FIG. 6C shows reduced scotopic b-wave electroretinogram (ERG) responses in 12- and 18-month-old Elovl2^C234W mice (* = p<0.05, ** = p<0.01, *** = p<0.001). FIG. 6D, 6E and 6F shows photopic a- and b-wave (FIG. 6D), scotopic c-wave ERG responses (FIG. 6E), and the recovery of rod-driven S_f (FIG.6F) remained unchanged in 12-month-old Elovl2^C234W mice compared to age-matched wildtype mice (n=6). FIG. 6G shows GSEA enrichment plots for synapse assembly (GO: 0007416) pathway showed similar enrichment in 12-month-old Elovl^2C234W vs. age-matched wildtype retinas and 18-month-old wildtype vs.12-month-old wildtype retinas. [0022]FIG. 7A, 7B, 7C, and 7D shows Intravitreal supplementation of 24:5n-3 in aged mice rescues visual function. FIG.7A shows a schematic of intravitreal supplementation of 24:5n-3 in 18-month-old mice. FIG.7B, 7C, and 7D shows improvement of scotopic and photopic a- and b-wave electroretinogram (ERG) responses (FIG. 7B), faster rod-mediated dark adaptation recovery (FIG. 7C), and improved visual-evoked potentials (VEP) (FIG. 7D) 5 days following intravitreal supplementation of 0.36 nmol 24:5n-3 (*=p<0.05, **=p<0.01; ***=p<0.005; ****=p<0.001), but not of 0.36nmol 22:6n-3 (DHA). [0023]FIG.8A, 8B, 8C, 8D, 8E, and 8F shows Lack of retinal toxicity or visual rescue in young mice following intravitreal supplementation of 24:5n-3 and optimal dosage of 24:5n-3, effect of different fatty acids on 18-month-old mice. FIG.8A shows a schematic of intravitreal supplementation of 24:5n-3 in 3-month-old mice. FIG. 8B, 8C, and 8D shows an electroretinogram (ERG) responses 2 days post-injection (FIG. 8B), 5 days post-injection (FIG. 8C) and rod-mediated dark adaptation recovery (FIG.8D) in young animals remained unchanged after supplementation. FIG. 8E and 8F shows photopic Skowronska-Krawczyk and Gao September 24, 2024 ERG responses 5 days post-injection in 18-month-old mice remained unchanged after supplementation with 0.04 nmol, 0.72 nmol and 2.5 nmol 24:5n-3 (FIG. 8E), and 0.36 nmol 20:5n-3 and 32:6n-3 (FIG.8F). [0024]FIG.9A, 9B, 9C, 9D, 9E, and 9F shows Reversal of molecular aging phenotypes in 18-month-old 24:5n-3-supplemented eyes. FIG. 9A shows increased levels of possible classes of VLC-PUFA-incorporated phospholipids in isolated photoreceptor outer segments (POS) following intravitreal supplementation of 24:5n-3 (* = p<0.05, ** = p<0.01). FIG. 9B shows nearly all differentially expressed genes (DEGs) (FC > |1.5|, pval<0.05) in 18-month-old 24:5(n-3)-supplemented retinas were downregulated compared to vehicle-injected retinas (n=3). FIG. 9C shows Gene Set Enrichment Analysis (GSEA) enrichment plots revealed downregulation of complement (NES=-1.77, FDR qval=0.00) and oxidative stress and redox (NES=-1.70, FDR qval=0.006) pathways following 24:5(n-3) supplementation (n=3). FIG. 9D shows Metascape analysis demonstrated downregulation of immune response, inflammation, microglial phagocytosis and cell migration pathways following 24:5n-3 supplementation (n=3). FIG. 9E shows STRING functional protein-protein interaction network of top 20 transcription factors involved in gene regulation after 24:5n-3 supplementation. FIG. 9F shows immunofluorescence staining shows lower expression of complement C3, APOE, HTRA1 and C5b-9 proteins in RPE cell layer in retinal cross-sections from 24:5n-3 supplemented eyes. Scale Bars: 20mm. [0025]FIG. 10A, 10B, 10C, and 10D shows Lipid analysis in retina after lipid supplementation. FIG. 10A shows major phospholipid classes remained unchanged in retinas from vehicle and 24:5n-3 supplemented retina. FIG. 10B shows unchanged levels of PUFAs in retinas after 22:6n-3, 24:5n-3 and 32:6n-3 injection compared to vehicle injection. FIG. 10C shows DESeq2 normalized counts of Elovl2, Elovl4, and Elovl5 following 24:5n-3 supplementation remain relatively unchanged. FIG.10D shows metascape analysis of enriched clusters of pathways after 24:5n-3 supplementation colored by p-value. DETAILED DESCRIPTION OF THE INVENTION [0026]Any methods, devices, and materials similar or equivalent to those described herein can be used in the practice of this invention. The following definitions are provided to facilitate understanding of certain terms used frequently herein and are not meant to limit the scope of the present disclosure. In the event that there is a plurality of Skowronska-Krawczyk and Gao September 24, 2024 definitions for a term herein, those in this section prevail unless stated otherwise. Headings used herein are for organizational purposes only, and in no way limit, the invention described herein. [0027]Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which a disclosed invention belongs. The singular terms "a," "an," and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. The term "comprising" means that other elements can also be present in addition to the defined elements presented. The use of "comprising" indicates inclusion rather than limitation. Stated another way, the term "comprising" means "including principally, but not necessary solely". Furthermore, variation of the word "comprising", such as "comprise" and "comprises", have correspondingly the same meanings. In one respect, the technology described herein related to the herein described compositions, methods, and respective component(s) thereof, as essential to the invention, yet open to the inclusion of unspecified elements, essential or not ("comprising"). [0028]All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety for all purposes. In case of conflict, the present specification, including explanations of terms, will control. [0029]Although methods and materials similar or equivalent to those described herein can be used to practice or test the disclosed technology, suitable methods and materials are described below. The materials, methods, and examples are illustrative only and not intended to be limiting. [0030]As used herein, the terms “subject” and “patient” are used interchangeably. As used herein, a subject can be a mammal such as a non-primate (e.g., cows, pigs, horses, cats, dogs, rats, etc.) or a primate (e.g., monkey and human). In specific embodiments, the subject is a human. The term does not denote a particular age or sex. Thus, adult and newborn subjects, as well as fetuses, whether male or female, are intended to be included. In one embodiment, the subject is a mammal (e.g., a human) having a disease, disorder, or condition described herein. In another embodiment, the subject is a mammal (e.g., a human) at risk of developing a disease, disorder, or Skowronska-Krawczyk and Gao September 24, 2024 condition described herein. A “patient” is a subject afflicted with a disease or disorder. [0031]As used herein, the terms "treat," “treating,” or "treatment" refer to both therapeutic treatment and prophylactic or preventative measures, with the objective of preventing, reducing, slowing down (lessen), inhibiting, or eliminating an undesired physiological change, symptom, disease, or disorder. For example, the disease may be Age-Related Macular Degeneration (AMD). For purposes of this invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already with the condition or disorder as well as those prone to have the condition or disorder or those in which the condition or disorder is to be prevented or onset delayed. Optionally, the subject or patient may be identified (e.g., diagnosed) as one suffering from the disease or condition prior to administration of the compositions of the invention. Subjects at risk for the disease can be identified by, for example, any or a combination of appropriate diagnostic or prognostic assays known in the art. [0032]As used herein, “clinical improvement” may refer to a noticeable reduction in the symptoms of a disorder, or cessation thereof. [0033]The terms “manage,” “managing,” and “management” refer to preventing or slowing the progression, spread, or worsening of a disease or disorder, or of one or more symptoms thereof. In certain cases, the beneficial effects that a subject derives from a prophylactic or therapeutic agent do not result in a cure of the disease or disorder. [0034]As used herein, and unless otherwise specified, the term “therapeutically effective amount” of a composition herein is an amount sufficient to provide a therapeutic benefit in the treatment or management of viral infection or to delay or minimize one or more symptoms associated with the viral infection. A therapeutically effective amount of a composition described herein means an amount of therapeutic agent, alone or in combination with other therapies, which provides a therapeutic benefit Skowronska-Krawczyk and Gao September 24, 2024 in the treatment or management of a viral infection. The term “therapeutically effective amount” can encompass an amount that improves overall therapy, reduces, or avoids symptoms or causes, or enhances the therapeutic efficacy of another therapeutic agent. [0035]The terms “administering,” and “administration” refer to methods 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, administering the compositions intranasally, parenterally (e.g., intravenously and subcutaneously), by intramuscular injection, by intraperitoneal injection, intrathecally, transdermally, extracorporeally, topically or the like. [0036]Referring now to FIGs. 1A–10D, the present invention features methods and compositions for treating age-related conditions (e.g., age-related eye conditions) in a subject in need thereof. In addition, the present invention provides methods and compositions for treating age-related brain conditions in a subject in need thereof. [0037]The present invention may feature a method of treating age-related eye conditions in a subject in need thereof. In some embodiments, the method comprising administering a composition comprising tetracospentaenoic acid [24:5n-3] to the subject. In other embodiments, the method comprises administering a composition comprising tetracospentaenoic acid [24:5n-3] and DHA to the subject. In some embodiments, the method may comprise identifying the subject presenting with the age-related eye condition and administering tetracospentaenoic acid [24:5n-3] into the eye of the subject. [0038] In some embodiments, the method comprises administering a composition comprising tetracospentaenoic acid [24:5n-3] into an eye of the subject. In other embodiments, the method comprises administering a composition comprising tetracospentaenoic acid [24:5n-3] and DHA into an eye of the subject. [0039] In some embodiments, the tetracospentaenoic acid [24:5n-3] is administered as a free fatty acid, a fatty acid ethyl ester (FAEE), or a fatty acid methyl ester (FAME). In some embodiments, the DHA is administered as a free fatty acid, a fatty acid ethyl ester (FAEE), or a fatty acid methyl ester (FAME). [0040] In some embodiments, the composition comprising tetracospentaenoic acid Skowronska-Krawczyk and Gao September 24, 2024 [24:5n-3] may be administered via intravitreal injection, intravenous injection, intraperitoneal injection, or topically. [0041] In some embodiments, the age-related eye condition 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 a method of treating age-related liver conditions in a subject in need thereof. In some embodiments, the method comprises administering tetracospentaenoic acid [24:5n-3] to the subject. In other embodiments, the method comprises administering a composition comprising tetracospentaenoic acid [24:5n-3] and DHA to the subject. In some embodiments, the method comprises identifying the subject presenting with the age-related liver condition and administering tetracospentaenoic acid [24:5n-3] to the subject. Non-limiting examples of age-related liver conditions include but are not limited to nonalcoholic fatty liver disease, alcoholic liver disease, hepatitis C, and fibrosis and cirrhosis. [0043] In some embodiments, the tetracospentaenoic acid [24:5n-3] is administered as a free fatty acid, a fatty acid ethyl ester (FAEE), or a fatty acid methyl ester (FAME). In some embodiments, the DHA is administered as a free fatty acid, a fatty acid ethyl ester (FAEE), or a fatty acid methyl ester (FAME). [0044] In some embodiments, the composition is administered an intravenous injection. In some embodiments, a therapeutically effective dose is administered for the disease. [0045]The present invention may also feature a method of treating age-related brain conditions in a subject in need thereof. In some embodiments, the method comprises administering tetracospentaenoic acid [24:5n-3] to the subject. In other embodiments, the method comprises administering a composition comprising tetracospentaenoic acid [24:5n-3] and DHA to the subject. In some embodiments, the method comprises identifying the subject presenting with the age-related brain condition and administering tetracospentaenoic acid [24:5n-3] into a brain of the subject. [0046] In some embodiments, the method comprises administering tetracospentaenoic acid [24:5n-3] into the brain of the subject. In other embodiments, the method comprises administering a composition comprising tetracospentaenoic acid [24:5n-3] and DHA into Skowronska-Krawczyk and Gao September 24, 2024 the brain of the subject. [0047] In some embodiments, the tetracospentaenoic acid [24:5n-3] is administered as a free fatty acid, a fatty acid ethyl ester (FAEE), or a fatty acid methyl ester (FAME). In some embodiments, the DHA is administered as a free fatty acid, a fatty acid ethyl ester (FAEE), or a fatty acid methyl ester (FAME). [0048] In some embodiments, the composition is administered via an intravenous and/or stereotactic direct injection. [0049] In some embodiments, the age-related brain condition is selected from the diseases consisting of Alzheimer’s Disease, Amyotrophic Lateral Sclerosis (ALS), age-related cognitive decline, dementia, Parkinson’s disease, Parkinsonism and other diseases that resemble Parkinson. In some embodiments, a therapeutically effective dose is administered for the disease. [0050]The present invention may also feature a method of treating age-related bone conditions (e.g., myeloma) in a subject in need thereof. In some embodiments, the method comprises administering tetracospentaenoic acid [24:5n-3] to the subject. In other embodiments, the method comprises administering a composition comprising tetracospentaenoic acid [24:5n-3] and DHA to the subject. In some embodiments, the method comprises identifying the subject presenting with the age-related bone condition and administering tetracospentaenoic acid [24:5n-3] to the subject. Non-limiting examples of age-related bone conditions include but are not limited to multiple myeloma and other Myelodysplastic Syndromes (MDS), anemia and the like. [0051] In some embodiments, the composition is administered via an intravenous and/or intravenous injection. [0052]The present invention may also feature a composition for use in treating age-related eye conditions in a subject in need thereof. In some embodiments, the composition comprises tetracospentaenoic acid [24:5n-3]. In other embodiments, the composition comprises tetracospentaenoic acid [24:5n-3] and docosahexaenoic fatty acid (DHA). In some embodiments, the composition is administered to the eye of the subject. In some embodiments, the composition is administered via an intravitreal injection, an intravenous injection, intraperitoneal injection, or topically. Skowronska-Krawczyk and Gao September 24, 2024 [0053]The present invention may also feature a composition for use in treating age-related brain conditions in a subject in need thereof. In some embodiments, the composition comprises tetracospentaenoic acid [24:5n-3]. In other embodiments, the composition comprises tetracospentaenoic acid [24:5n-3] and docosahexaenoic fatty acid (DHA). In some embodiments, the composition is administered to the brain of the subject. In some embodiments, the composition is administered via an intravenous and/or stereotactic direct injection. [0054]The present invention may also feature a composition for use in treating age-related liver conditions in a subject in need thereof. In some embodiments, the composition comprises tetracospentaenoic acid [24:5n-3]. In other embodiments, the composition comprises tetracospentaenoic acid [24:5n-3] and docosahexaenoic fatty acid (DHA). In some embodiments, the composition is administered via an intravenous injection. [0055]The present invention may also feature a composition for use in treating age-related bone conditions in a subject in need thereof. In some embodiments, the composition comprises tetracospentaenoic acid [24:5n-3]. In other embodiments, the composition comprises tetracospentaenoic acid [24:5n-3] and docosahexaenoic fatty acid (DHA). In some embodiments, the composition is administered via an intravenous and/or intravenous injection. [0056]EXAMPLE [0057]The following is a non-limiting example of the present invention. It is to be understood that said example is not intended to limit the present invention in any way. Equivalents or substitutes are within the scope of the present invention. [0058]Age-related vision decline is associated with decreased VLC-PUFA levels in the retina: To investigate changes in the lipid composition of aging retinas a series of lipidomic analyses were performed on 3-, 6-, 12-, 18-, and 23-month-old dissected mouse tissues. For a complete lipidomic analysis, lipids were extracted using the Bligh-Dyer method. For comprehensive fatty acids (FAs) analyses, FAs were released from lipids using the acid hydrolysis method and extracted using hexanes. Complex lipids and FAs were analyzed by Liquid Chromatography Mass Spectrometry (LC-MS), as described herein. For FA analysis, the abundance of individual polyunsaturated fatty Skowronska-Krawczyk and Gao September 24, 2024 acids (PUFAs) was normalized to internal standard (FA 21:5) and tissue weight. This data revealed a progressive decline in levels of DHA (34% (p=0.0178) and 33% (p=0.0165) in 18-month-old and 23-month-old mice, respectively) and VLC-PUFAs, specifically 32:6 (31% in 18-month-old, p=0.0128), 34:6 (52% (p=0.0042) and 39% (p=0.0290) in 18-month-old and 23-month-old animals, respectively) and 36:6 (55% (p=0.0046) and 59% (p=0.0029) in 18-month-old and 23-month-old mice, respectively), in aged retinas compared to 3-month-old retinas (FIG.1A). [0059]After conducting FA analysis, the lipidomic changes in the aging mouse retina were explored. The levels of extracted complex lipids were analyzed using a data-dependent acquisition (DDA) LC-MS method, and lipid species were identified using LipidSearch software 4.2.21 (Thermo). Among the 528 lipids quantified in retinas, levels of 15 lipids were significantly reduced, and levels of 53 lipids were increased in aged retinas (fold change > |1.5|, p-value <0.05). Principal component analysis revealed clear clustering based on differences in lipid composition between 3- and 18-month-old retinas (FIG.2A). The two major membrane lipid classes, phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were altered in opposite directions in 18-month-old retinas. Specifically, PC was significantly decreased (p=0.0039, n=5), and PE was increased (p=0.0329, n=5) in aged retinas (FIG. 1B). Photoreceptor outer segments (POS) were next isolated from 3- and 18-month-old mouse retinas. Upon lipid extraction, a significant (5%) decrease in PC, and an increase in phosphatidylglycerol (PG), sphingomyelin (SM), and triglycerides (TG) (18%, 26%, and 25%, respectively) (FIG.1C). Based on the significantly changed lipids in POS membranes, Bioinformatics Methodology for Pathway Analysis (BioPAN) was performed on the lipidomics dataset. [0060]BioPAN revealed that the synthesis of TG from diglycerides (DG) was highly activated (Z-score=2.957) in 18-month-old POS, whereas the formation of DG from TG was inhibited (Z-score=−3.165) (FIG.2B). Analysis of previous transcriptomic data from young and old retinas revealed that, although the expression levels of enzymes with triacylglycerol lipase and transacylase activities (PNPLA4-5) remain unchanged, the level of DGAT2— the enzyme responsible for catalyzing the final reaction in TG synthesis— is elevated in aged retina (FIG. 2B). Then, to analyze the dataset further, lipid ontology (LION) enrichment analysis was performed, which revealed highly enriched changes in membrane components, as well as decreased bilayer thickness Skowronska-Krawczyk and Gao September 24, 2024 and high lateral diffusion in the membranes (FIG. 1D). Since PC and PE are primarily present in plasma membranes, further analysis was conducted to assess the effects of aging on membrane composition. In particular, the focus was on the levels of VLC-PUFAs incorporated in complex lipids and their impact on membranes in the aging retina. PC-VLC-PUFA levels were analyzed using a Lipid Data Analyzer with a customized database. This data revealed lower levels of these lipids; specifically, in POS, where PC (50:12) and PE (48:12) decreased by 30% and 42%, respectively (FIG. 1E and FIG.2E). [0061]A series of visual tests were performed on 3- and 18-month-old mice to correlate age-related lipid changes with visual functions. First, quantitative optomotor response (OMR) analysis was performed to measure contrast sensitivity in scotopic conditions to mimic nighttime light levels. Unsurprisingly, 18-month-old mice had significantly lower contrast sensitivity in scotopic conditions compared to 3-month-old mice (FIG. 2D). Using full-field electroretinography (ERG), a significant decrease in both scotopic a- and b-wave amplitudes (FIG. 1F). Maximal scotopic a-wave was reduced by ~ 34% in 18-month-old mice (166 ± 5 μV, n = 14) as compared to that in 3-month-old animals (250 ± 12 μV, n = 14, ***P < 0.001). Photopic green/UV a- and b-waves were also reduced in 18-month-old mice compared to young controls (FIG.1F and FIG.2E). Then, whether the dark adaptation of rod photoreceptors is suppressed in aged mice was investigated. After bleaching ~90% of their visual pigment, rods in both groups gradually recovered their photoresponses over the following 60-min period in the dark. The recovery of the averaged A_max in young rods could be described by a single exponential function with a time constant of 18.9 ± 0.7 min, and its level by 60 min after the bleach was ~ 80 ± 6% of the pre-bleach value (FIG.2F, left). Although rods in aged mice also demonstrated robust recovery of their maximal responses after the bleach, its rate (24.3 ± 2.7 min) was slightly decreased (by ~ 1.3 times, p<0.001) as compared to that in young mouse rods. Yet, the maximal response amplitude 60 min post-bleach reached the same relative level (~ 81 ± 4%) as in young control rods (FIG.1G). The oscillatory potentials were also compared in young and old mice, and a consistent significant reduction was found in all four oscillatory potential peaks in both dim flash and bright flash scotopic responses (FIG. 2E and FIG. 1H). However, the recovery of rod-driven ERG a-wave sensitivity (Sf) following the same bleach was not compromised in older animals (FIG. 2F, right), suggesting the normal operation of their visual cycle. Taken Skowronska-Krawczyk and Gao September 24, 2024 together, these results show that aging affects visual function through decreased visual contrast sensitivity and photoreceptor function. [0062]To address whether the decrease in visual function in aged animals could be attributed to alterations in the phototransduction cascade, levels of proteins involved in signal transduction in POS, including Rho, PDE6A, PDE6B, GNAT1, and GBB1, were quantified using a stable isotope-labeled (SIL) peptides-based absolute quantification method. To compare the levels of PDE6 and transducin proteins in 3- and 18-month-old retinas, the optimal amount of SIL peptides was spiked into the protein samples, trypsinized, and analyzed by LC-MS/MS. The amount of each protein was calculated using the endogenous/SIL peptides peak area ratio, and the quantification results were normalized to Rho and displayed as the mean ± standard error of the mean. Absolute quantification of these proteins showed slight decreases in PDE6A and PDE6B in 18-month-old retinas (FIG. 1I); however, relative amounts of all measured proteins (PDE6A, PDE6B, GNAT1, and GBB1) remained unchanged (FIG. 2G). This suggests that the ratio of proteins involved in the phototransduction signaling pathway was unaffected in aged retina. [0063]Finally, to evaluate age-related changes in levels of proteins associated with retinal pathologies, immunofluorescence staining was performed on retinal cross-sections from adult (5-month-old) and advanced-aged (26-month-old) mice. Antibodies targeting Complement factor 3 (C3), Apolipoprotein E (ApoE), and subunit of the membrane attack complex (MAC) in the complement pathway C5b-9, all of which are correlated with increased risk of AMD, were used. This data demonstrated higher levels of these proteins in the retinal pigment epithelial (RPE) cell layer of 26-month-animals when compared to 5-month-old retina sections (FIG.1J). Additionally, immunofluorescence staining with rod bipolar- and Müller cell-specific antibodies (protein kinase C alpha, PKCa, and glutamine synthetase, GS, respectively) showed age-related morphological changes in the outer plexiform layer, including overgrowth of dendrites into the outer nuclear layer (FIGs.2H-2I). [0064]Age-related changes in Elovl2 expression: ELOVL2 encodes a key enzyme in the biosynthesis of long-chain fatty acids (FIG.3A). Specifically, it elongates 22:5n-3 to 24:5n-3, which is then elongated to VLC-PUFAs by other elongases including ELOVL4, or further processed in peroxisomes to produce 22:6n-3, DHA. The Elovl2 regulatory Skowronska-Krawczyk and Gao September 24, 2024 region has higher methylation levels in aged retina. Here, methylation levels were analyzed at each CpG site in the Elovl2 promoter that has been shown to undergo age-related methylation changes in human blood cells (FIG.3B). To achieve this, DNA from middle-aged (<12-month-old) and old (24-month-old) mouse retinas were isolated and subjected it to treatment with sodium bisulfite to distinguish methylated from unmethylated cytosines. Then, using both converted and non-converted DNA, regions of interest were amplified with primers insensitive for methylation. PCR products were then sequenced using Sanger sequencing, and relative levels of methylated versus non-methylated cytosines were calculated by comparing the area under the sequencing peak in both DNA samples. This data revealed a group of five CpGs carrying significantly higher methylation levels in older retinas, while the other four, the closest to the transcription start site, were not changed. Notably, the CpG sites that exhibited higher methylation in mice are direct homologs of CpGs methylated in aging human blood. [0065]To visualize cell type- and age-specific expression of key enzymes in the PUFA elongation pathway, RNA in situ hybridization was performed on retinal cross-sections from adult (5-month-old) and advanced-aged (26-month-old) mice using RNAscope probes targeting Elovl2, Elovl4, and Elovl5 (FIG. 3C). Elovl2 expression was predominantly observed in the photoreceptor layer, whereas Elovl4 was expressed throughout all retinal layers, and Elovl5 was expressed in the inner nuclear and ganglion cell layers. While Elovl4 and Elovl5 expression levels were relatively unchanged with age, Elovl2 expression 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-nucleus RNA sequencing (snRNAseq) was performed on retinas isolated from young (3-month-old) and old (18-month-old) mice. After normalizing mean expression levels to TBP, Elovl2 expression was significantly decreased in aged retinas (0.080 vs.0.100 in young retinas, p-value = 3.59E-6). Expression of Elovl2 was highest in cones and barely detectable in other cell types (FIG.3D). This data is in agreement with those obtained by the CZI CELL×GENE Discover program from mouse and human retinas (FIG. 3D), confirming the enrichment of Elovl2 in cones. In addition, the cell type-specific expression of other enzymes from the PUFA elongation pathway was Skowronska-Krawczyk and Gao September 24, 2024 investigated, and only Elovl2 exhibited cone-specific expression, while other enzymes were less restricted (FIG. 3D and FIG. 4D). Subsequently, the pathways that undergo alterations in cone photoreceptors during aging were investigated. Differentially expressed genes (DEGs) (log fold change > 0.1, padj < 0.05) were identified in young (3-month-old) and aged (18-month-old) cones, which were then used in GO functional enrichment analysis (FIG. 3E). Biological pathways that were enriched in young cones were related to visual perception and light stimulus detection. Pathways that were enriched in aged cones were involved in synaptic transmission, ion channels, and membrane assembly. [0067]To gain a deeper insight into the changes in intercellular interactions in the aging retina, the computational package CellChat was employed, focusing on interactions between cones, rods, and Müller glia (FIGs. 3F, 4B, and 4C). Specifically, CellChat infers cell-cell communication from snRNAseq data by calculating the expression levels of signaling ligands and receptors, including soluble agonists, antagonists, and stimulatory or inhibitory membrane-bound co-receptors. First, the overall inferred number and strength of interactions were evaluated within and between the cell types. In the young retinas, the strongest interactions were detected between Müller glia and cones. These interactions were further increased in aged tissue. Similarly, signaling between rods and Müller glia was increased in aged retina. Interestingly, cone-rod interactions were not found in young retinas, while several new significant interactions were detected in 18-month-old tissues. The analysis of specific ligand-receptor pairs within these signaling interactions revealed the emergence of links between neuronal adhesion molecules (Negr1 - Negr1) and metabotropic glutamate receptors (Glu - Grm8) between rods and cones, specifically in aged retinas. In addition, several interactions between metabotropic glutamate receptors (Glu - Grm8, Glu - Grm7) and ionotropic glutamate receptors (Glu - Gria4) emerged within Müller glia in aged retinas (FIGs. 4B and 4C). In addition, this analysis showed new Cyclosporin A and CD147 (Ppia - Bsg) interactions between Müller and photoreceptor cells, which are recognized as an infection sensor and an inflammation initiation system (FIG.4B). [0068] Lack of ELOVL2 activity accelerates age-related phenotypic changes in the retina: Elovl2-mutant mice (Elovl2^C234W) were generated with the loss of ELOVL2 enzymatic activity due to impaired substrate binding. Detailed analysis was performed of Skowronska-Krawczyk and Gao September 24, 2024 lipid composition, including total fatty acids and global lipidomic analysis on retinas from 18-month-old Elovl2^C234W mice. The quantification revealed that levels of PUFAs synthesized from ELOVL2 products, including 22:6, 24:6, 32:6, 34:6 and 36:6, were significantly lower in 18-month-old Elovl2C234W retinas than in age-matched wildtype retinas (FIG. 5A). Untargeted lipidomic analysis identified 595 lipids, of which 28 exhibited significantly lower levels, and 84 showed significantly higher levels in Elovl2^C234W retinas compared to wild-type tissues (fold change > |1.5|, p-value < 0.05). In addition, Elovl2^C234W tissue displayed a decrease in PC and increase in PE levels (FIG. 5B), similar to the changes detected in aging retinas (FIG. 1B). Lipid ontology (LION) enrichment analysis based on the significantly changed lipids suggested highly enriched changes in plasma membrane components, as well as decreased bilayer thickness and high lateral diffusion, which were also similar to the changes in aging POS (FIG.5C). Therefore, VLC-PCs were further analyzed and a decreased PC (46:12), PC (48:12), PC (50:12), PC (54:12), PC (56:12) (FIG.5D) was found, which contributed to a reduced total level of VLC-PCs in 18-month-old Elovl2C234W retina (FIG.6A). Analysis of free fatty acids (FFA) showed 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) decrease of 22:6, 24:6, 26:6, 32:6, 34:6 and 36:6 in 18-month-old Elovl2^C234W retinas, respectively, as compared to age-matched wildtype samples (FIG.6B). [0069]Next, the visual function in 12- and 18-month-old Elovl2^C234W mice were compared to age-matched wildtype mice. Scotopic contrast sensitivity in both ages was lower in Elovl2^C234W animals compared to age-matched wildtype mice (FIG.5E). Scotopic a- and b-wave ERG responses were also reduced at both ages compared to wildtype controls (FIG. 5F and FIG. 6C). Furthermore, in wildtype mice, scotopic a- and b-wave ERG responses decrease dramatically from 12 to 18 months (FIG. 5F and FIG. 6C). In contrast, Elovl2^C234Wmice show minimal change in ERG amplitudes between these ages (FIG.5F and FIG.6C). Notably, the maximum ERG a-wave amplitudes in 12-month-old Elovl2^C234W (120 μV) mice were decreased to levels comparable to those in 18-month-old control (150 μV, p = 0.103) animals (FIG.5G). There were no differences in photopic ERG a- and b-wave amplitudes as well as RPE-generated ERG c-waves between wildtype and Elovl2^C234W mice (FIG.6D and 6E). [0070]The rod dark adaptation in ELOVL2-deficient mice was then evaluated after a Skowronska-Krawczyk and Gao September 24, 2024 nearly complete rhodopsin bleach with green light. In control mice, the averaged scotopic ERG a-wave maximal response, A_max, recovered with the time constant of 28.6 ± 2.6 min, and its level by 60 min after the bleach was ~ 90 ± 7% of the pre-bleach value (FIG.5H). In contrast, the recovery of the scotopic a-wave response in Elovl2^C234W mice was significantly suppressed compared to controls. Consistent with this, the average rate of rod A_max recovery in Elovl2^C234W mice (42.7 ± 7.3 min) was ~ 1.5 times slower than in control animals (p=8E-6), and reached only ~ 69 ± 5% of its prebleached level by the end of 60-min recordings (FIG.5H). The dark-adapted a-wave photosensitivities (S_f) were also lower (by ~ 16%) in the same group of mice lacking ELOVL2 (1.50 ± 0.05 m² cd^-1 s^-1 vs. 1.79 ± 0.08 m² cd^-1 s^-1 in controls, *p < 0.05). However, the recovery of rod-driven S_f following the same bleach was not suppressed in ELOVL2-deficient animals (FIG. 6F), suggesting the normal recycling of the visual chromophore in the mutant mice. [0071]To determine whether the reduction in ERG amplitude in Elovl2^C234W mice could be due to cell loss or retinal degeneration, the outer nuclear layer (photoreceptor) thickness was measured by optical coherence tomography (OCT). No statistically significant difference by 2-way ANOVA in the ONL thickness between 12-month-old wildtype and Elovl2^C234W (n=5), and 18-month-old wildtype and Elovl2^C234W (n=4) eyes (FIG. 5I). Therefore, the reduction in rod function in 12-month-old Elovl2^C234W animals preceded any detectable photoreceptor cell loss or retinal degeneration. [0072]Given that Elovl2^C234W mice exhibited accelerated age-related phenotypic changes in the retina, whether they also underwent transcriptomic alterations that mimic those seen in the aging retina was investigated. First, bulk RNA sequencing was performed on 12-month-old and 18-month-old wildtype retinas. This data was then compared to the transcriptomes of 12-month-old wildtype and Elovl2^C234W retinas to identify potential transcriptomic parallels in retinal changes associated with the absence of the ELOVL2 enzyme and in aging. Gene set enrichment analysis (GSEA) was performed for differentially expressed genes (fold change > |1|, padj < 0.05) using Gene Ontology Biological Processes, Cellular Component, and Molecular Function gene sets. Pathways that were downregulated in both aging retinas and retinas lacking ELOVL2 included telomere maintenance, transcription by RNA polymerase I, negative regulation of gene expression (epigenetic), and regulation of gene silencing (FIG.5J). On the other Skowronska-Krawczyk and Gao September 24, 2024 hand, pathways such as synapse assembly, transmembrane transporter activity, TORC1 signaling, DNA damage response- signal transduction resulting in transcription, and ceramide metabolic process were upregulated in both 12-month-old Elovl2^C234W and 18-month-old wildtype retinas when compared to 12-month-old wildtype retinas (FIG.5J and FIG.6G). [0073]Restoration of vision in aged animals by intravitreal fatty acid injection: Without wishing to limit the present invention to any theory or mechanism, it is believed that the lack of ELOVL2 product, 24:5n-3, in the aging retina is one of the main culprits of age-related visual decline and that supplementation with this fatty acid may improve vision in aged animals. First, an intravitreal supplementation strategy was pursued, which, in contrast to oral gavage, enabled precise control of the amount of lipid administered to the eye. To assess the retinal toxicity of 24:5n-3, intravitreal injections of the compound were administered unilaterally to 3-month-old mice, and ERG responses were measured 2 days and 5 days post-injection (FIG. 8A). Compared to vehicle-injected eyes, 24:5n-3- injected eyes did not show any significant change in scotopic ERG a- or b-wave amplitudes on day 2 (FIG. 8B). Additionally, no significant changes were observed in either scotopic or photopic responses on day 5 (FIG.8C). In summary, intravitreal injection of 24:5n-3 did not cause retinal toxicity or affect rod- or cone-driven retinal responses in 3-month-old mice. [0074]To investigate the effect of 24:5n-3 supplementation on visual function in aged mice, the compound was administered intravitreally into one eye of each 18-month-old animal, while the contralateral eye received an injection of a vehicle as a control. After 5 days, a multimodal approach was used to test the impact on visual function, and the retinas were subsequently collected for analysis of fatty acid and lipid composition, and investigation of molecular changes (FIG. 7A). First, different doses of 24:5n-3 were tested to determine the optimal levels that could improve vision in 18-month-old mice. Interestingly, it was observed that 24:5n-3-treated eyes showed an improvement in both scotopic and photopic ERG a- and b-wave responses with an injection dose of 0.36 nmol (FIG. 7B, right), and no changes were observed when other doses were used (FIG.8E). Then, to test whether PUFAs up- or downstream of 24:5n-3, such as 20:5n-3, 22:6n-3 (DHA), and 32:6n-3, have similar effects, the procedure was replicated using the optimal dose (0.36 nmol). Intravitreal injections of 20:5n-3, 22:6n-3 and 32:6n-3 Skowronska-Krawczyk and Gao September 24, 2024 were administered, followed by functional analysis (FIG. 7B, left, and FIG. 8F). Strikingly, only eyes that received intravitreal supplementation of 24:5n-3 exhibited statistically significant improvement in both scotopic and photopic ERG a- and b-wave responses when assessed 5 days post-injection. In contrast, injections of 22:6n-3 and 20:5n-3 resulted in no functional improvement (FIG. 7B and FIG. 8F, left). A marginal improvement in photopic green and blue light-generated ERG b-wave responses, but not in scotopic responses, was also observed in eyes treated with 32:6n-3 (FIG. 8F, right). [0075]The effect of intraocular 24:5n-3 supplementation on rod dark adaptation was then assessed by in vivo ERG. In 17-month-old mice, this treatment improved the final post-bleach fraction of recovered rod Amax response by ~ 30% (FIG. 7C, left), as compared to that in vehicle-injected contralateral eyes, without affecting their sensitivity recovery (FIG. 7C, right). In contrast, a similar administration of 24:5n-3 did not affect the recovery of either of the two parameters in 3.5-month-old animals (FIG. 8D), thus indicating the specificity of the therapeutic effect of the lipid to aged mice. [0076]Following the observed improvement in ERG responses, which measure the function of the photoreceptors and inner retina in supplemented retinas, whether this improvement was associated with increased signaling from the retina to the brain by recording visually evoked potentials (VEPs) was next investigated. The recordings in the superior colliculus showed a significant increase in VEP peak-to-peak amplitude in 18-month-old animals supplemented with 24:5n-3 compared to the vehicle-treated eyes (FIG. 7D). Specifically, the VEP amplitude increased from 44.15 ± 5.95 μV in 18-month-old animals injected with a vehicle (n=4 animals; n=14 recording sites) to 86.67 ± 8.61 μV in supplemented eyes (n=4; n=16; p=0.00082). Thus, these results demonstrate that improvement of photoreceptor visual function after intravitreal supplementation of 24:5n-3 is robustly transmitted to the brain. [0077]Molecular rejuvenation of the retina following 24:5n-3 supplementation: To gain insight into the potential mechanism underlying the improvement in mouse vision following 24:5n-3 treatment, whether the lipid supplementation could cause any changes in the lipid composition of the retina was first investigated. Retina tissues were collected 5 days after lipid or vehicle injection, and lipids were extracted using the Bligh and Dyer method. The levels of all possible VLC-PUFA-containing phospholipids were analyzed Skowronska-Krawczyk and Gao September 24, 2024 from isolated POS, and all were elevated in the 24:5n-3 injected group. Among them, VLC-PUFA incorporated 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), 40% (p=0.007), in the 24:5n-3- supplemented group, respectively (FIG.9A). Overall, the level of phospholipids containing VLC-PUFAs was elevated by ~25% (p=0.033) in the POS receiving fatty acid supplementation. Analysis of the total FA levels, however, indicated no notable increase in total levels of VLC-PUFAs in the retina following supplementation, suggesting that either other forms of VLC-PUFAs, such as free VLC-PUFAs, or the level of VLC-PUFAs in retinal compartments other than POS, may compensate for the observed changes (FIGs.10A and 10B). [0078]To understand the molecular changes occurring in the retinas of 18-month-old mice after fatty acid supplementation, bulk RNAseq analysis was performed of 24:5n-3- vs. vehicle- injected retinas isolated five days following intravitreal injection. The changes in the transcriptome were identified using Bioconductor package DESeq229. First, the expression of key enzymes involved in the elongation of PUFAs— Elovl2, Elovl4, Elovl5— was not changed after supplementation (FIG. 10C). 234 significantly changed genes (fold change > |1.5|, p-adj < 0.05) were identified. Interestingly, almost all differentially expressed genes (DEGs) were downregulated in the 24:5n-3-supplemented eyes (FIG.9B). Gene set enrichment analysis (GSEA) indicated significant downregulation of complement (NES = -1.77, FDR q-value = 0.00) and oxidative stress and redox pathways (NES = -1.70, FDR q-value = 0.006) in 24:5n-3-supplemented retinas (FIG. 9C). The visualization of enriched terms as a network using Metascape demonstrated downregulation of pathways involved in immune response, inflammation, microglial phagocytosis, and cell migration (FIG. 9D and FIG.10D). [0079]To further identify transcription factors (TFs) responsible for the observed gene expression changes following 24:5n-3 supplementation, ChIP-X Enrichment Analysis 3 (ChEA3), a tool that predicts TFs associated with DEGs based on TF-target gene set libraries curated from human, mouse, and rat was used. The Mean Rank output was used, which averaged integrated ranks across libraries, for downstream analysis and created global TF co-expression networks. The RNA sequencing data demonstrated downregulation of Sp100, Lyl1, Sp140, Irf5, Stat6, Fli1, Ikzf1, Spi1, Relb, Elf4, Sp110 Skowronska-Krawczyk and Gao September 24, 2024 and upregulation of Trafd1. To visualize protein-protein interactions among these associated TFs, a functional interaction network of the top 20 TFs was created using STRING (v12.0) (FIG. 9E). Low expressing TFs, as defined by normalized read count (DESeq2 baseMean) < 10, were removed, and the difference in mean Z-scores after 24:5n-3 supplementation of the remaining 16 TFs were calculated. This data demonstrated striking downregulation of nearly all top TFs, including several interferon regulatory factors (IRFs), Irf1, Irf2, Irf5, Irf7, Irf8, responsible for activating immune response. [0080]Further analysis demonstrated significant downregulation of genes in the “aging cerebellum” gene set (NES = -1.82, FDR = 5.83E-4) in retinas from 18-month-old mice after supplementation with 24:5n-3 compared to control, including significant downregulation of apolipoprotein E (Apoe) transcript (log2FC = -0.79, p-adj = 0.001). To corroborate the RNA sequencing data showing decreased expression of Apoe and genes related to the complement cascade after supplementation, immunohistochemical analysis was performed of retinal cross-sections (FIG.9F). First, a marked reduction of complement component C3, a key inflammatory protein activated in AMD35, in the RPE of 24:5n-3-supplemented eyes was observed. A decreased accumulation of APOE, a major component of age-related sub-RPE drusenoid deposits, was also observed. In addition, the level of HTRA1, a serine protease secreted by RPE that is increased in AMD, was reduced following treatment with 24:5n-3. Lastly, decreased levels of C5b-9, a subunit of the membrane attack complex, the terminal complex of the complement cascade, was observed, indicating decreased complement activation in 24:5n-3- supplemented eyes. [0081]Animals: All animal procedures were approved by the Institutional Animal Care Committee (IACUC) at the University of California, Irvine. Elovl2^C234W 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 vivarium at the University of California, Irvine on a standard 12hr/12hr light (<150 lux)/dark cycle, and were maintained on a standard soy protein-free rodent chow diet (Envigo Teklad 2020X) ad libitum. Levels of fatty acids in the diet are included in Table 1 below. Table 1: Dietary fatty acid content in Envigo Teklad 2020X Skowronska-Krawczyk and Gao September 24, 2024 Fatty Acids % Fatty Acids %

[0082]Electroretinography (ERG): Animals were dark-adapted overnight and anesthetized by IP injection of ketamine (100 mg/kg) and xylazine (4 mg/kg). During recordings, a heating pad maintained body temperature at 37–38°C. The eyes were dilated using 1% atropine sulfate (rod dark adaptation experiments) or 1% tropicamide and 2.5% phenylephrine (regular scotopic and photopic recordings). The eyes were lubricated with a corneal gel. The reference electrode needle was inserted under the skin at the skull. The responses for eleven stimuli of increasing intensities were recorded and averaged. The a- and b-wave responses were plotted from the averaged ERG waveform. Scotopic and photopic ERG recordings were performed using commercially available platforms (Celeris, Diagnosys LLC). For photopic recordings, mice were kept in a lighted vivarium for 10 minutes prior to the experiment. M-cone and S-cone specific function was measured. Stimulation was performed by means of alternating UV and green light with flashes of increasing intensities. Green light stimulation had steps of increasing intensities of −0.5, 0.5, 1.5, and 2.5 log cd·s/m². UV light stimulation had increasing intensities of −1, 0, 1, and 2 log cd·s/m². The responses for treated and non-treated eyes were grouped respectively and averaged together. The a- and b-wave tracings represent the averaged ERG waveform. The data was analyzed with Espion v.6 software (Diagnosys). GraphPad Prism was used for graph preparation and statistical analysis. [0083]Dark Adaptation measurements: For rod dark adaptation experiments, full-field ERGs were recorded using a UTAS BigShot apparatus (LKC Technologies). Scotopic responses to calibrated green (530 nm) LED light were recorded from both eyes. Measurements from several trials were averaged, and the intervals between trials were adjusted so that responses did not decrease in amplitude over the series of trials for each step. Skowronska-Krawczyk and Gao September 24, 2024 [0084]Rod ERG a-wave fractional flash sensitivity (Sf) was calculated from the linear part of the intensity-response curve as follows: ՛_Վ = ^{^} ^_ՕՉ^·Է

the amplitude of the rod a-wave dim flash response, Amax is the maximum amplitude of the rod a-wave response for that eye (determined at 23.5 cd∙s m^-2), and I is the flash strength. The maximal response and sensitivity of rods were first determined in the dark. To monitor the post-bleach recovery of A_max and S_f, more than 90% of rhodopsin was bleached with a 35-sec exposure to 520-nm LED light focused at the surface of the cornea. The fraction of a bleached pigment was calculated with the following equation: Դ = 1 − Ս^{(−1·Ծ·՜)} where F is the fraction of rhodopsin bleached, t is the duration of the light exposure (s), I is the bleaching light intensity of 520-nm LED light (1.3 x 108 photons μm^-2 s^-1), and P is the photosensitivity of mouse rods at the wavelength of peak absorbance (5.7 x 10^-9 μm2). Mice were re-anesthetized once after 30 min post-bleach with a lower dose of ketamine (~1/3 of the initial dose). If needed, a small drop of PBS solution was gently applied to their eyes with a plastic syringe to protect them from drying and to maintain contact with the recording electrodes. For each time point, the A_max and S_f were normalized to the corresponding A_max ^DA and S_f ^DA values. [0085]Optomotor responses (OMR): Optomotor responses (OMRs) were recorded using a commercially available OMR setup. (PhenoSys GmbH, Berlin, Germany). The software automatically tracks animal head movement in the direction of rotating gratings stimulus (optomotor reflex) and calculates correct/incorrect tracking behavior presented as OMR (optomotor) index. The mouse was placed on an elevated platform in OMR arena. Gratings stimuli (rotating at 12°/s) were presented for ~12 min per trial. The spatial frequency of the gratings was set at 0.2 cycles per degree of visual angle. The stimulus was presented at differing contrasts between the light and dark stripes: 3, 6, 10, 12.5, 15, 17.5, 20, 25, 37.5, 50, 75, 100 contrast. The stimulation at each contrast level lasted for 60 s. For the scotopic part of the experiment (nighttime light levels) the animals were dark-adapted for 12h before the experiments. The OMR arena was dimmed using 4 neutral density filters in front of the stimulus displays (to ~0.03 lux). Skowronska-Krawczyk and Gao September 24, 2024 Results for 100% contrast were excluded due to adjustment (mice adjustment to the system). OMR results that yielded correct/incorrect ratio lower than 0.8 were excluded. For each mouse, the results from several trials were averaged for analysis. Each data point represents the average of all mice tested. Graphpad prism was used for statistical analysis and to generate curves. [0086]Neurophysiology (Visual Evoked Potential - VEP): Animals were initially anesthetized with 2% isoflurane in a mixture of N2O/O2 (70%/30%) then placed into a stereotaxic apparatus, followed by analgesia (Flunixin Meglumine, 2.5 mg/kg SC every 8 hours) and local subcutaneous injection of lidocaine (0.5%). A small, custom-made plastic chamber was secured to the exposed skull using dental acrylic. After one day of recovery, re-anesthetized animals were placed in a custom-made hammock, maintained under isoflurane anesthesia (1-2% in N₂O/O₂), a craniotomy was performed, and multiple single tungsten electrodes were inserted into V1 layers II-VI. Following electrode placement, the chamber was filled with sterile agar and sealed with sterile bone wax. Animals were then sedated with chlorprothixene hydrochloride (1 mg/kg; IM) and kept under light 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, MA). [0087]Data was acquired using a multi-channel Scout recording system (Ripple, UT, USA). Local field potentials (LFP) from multiple locations at matching cortical depths were band-pass filtered from 0.1 Hz to 250 Hz and stored along with spiking data at a 1 kHz sampling rate. LFP signal was aligned to stimulus time stamps and averaged across trials for each recording depth in order to calculate visually evoked potentials (VEP). Single neuron spike signals were band-pass filtered from 500 Hz to 7 kHz and stored at a 30 kHz sampling frequency. Spikes were sorted online in Trellis (Ripple, UT, USA) while performing visual stimulation. Visual stimuli were generated in Matlab (Mathworks, USA) using Psychophysics Toolbox and displayed on a gamma-corrected LCD monitor (55 inches, 60 Hz; 1920 x 1080 pixels; 52 cd/m2 mean luminance). Stimulus onset times were corrected for monitor delay using an in-house designed photodiode system. Visual responses were assessed according to previously published methods. For recordings of visually evoked responses, animals were tested with 100 repetitions of a 500 ms bright flash of light (105 cd/m²). Skowronska-Krawczyk and Gao September 24, 2024 [0088]Optical coherence tomography (OCT): Mice were anesthetized via intraperitoneal injection with ketamine/xylazine (10mg/kg), followed by pupil dilation using 1% tropicamide and 2.5% phenylephrine. Optical coherence tomography (OCT) was performed using a Bioptigen spectral-domain OCT device (Leica Microsystems Inc., Buffalo Grove, IL), capturing four frames of OCT b-scan images from a series of 1200 a-scans. Outer nuclear layer (ONL) thickness was assessed 500 μm from the optic nerve head (ONH) at the nasal, temporal, superior, and inferior quadrants of each eye, and these values were averaged to determine the average ONL thickness. [0089]POS isolation: Photoreceptor outer segments (POS) were isolated by OptiPrep™ density gradient centrifugation described previously with minor modification. Briefly, 1 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 min. The samples were centrifuged at 200 x g for 1 min at 4°C and the supernatant containing the ROS was collected gently. The vortexing and sedimentation sequence was repeated six times. The supernatant was combined for lipid extraction. [0090] Intravitreal Injections: All animals were anesthetized with Isoflurane; their eyes were anesthetized with proparacaine (0.5%, Bausch-Lomb) and followed by dilation of pupils with a drop of tropicamide (1%, Alcon Laboratories) and of phenylephrine (2.5%, Akorn Pharmaceuticals, Lake Forest, IL). All drops were applied with pipette. Injections were performed under a surgical microscope (Zeiss). First, a drop of Gonak (Akorn Pharmaceuticals, Lake Forest, IL) was applied to the corneal surface for better visibility and prevention of back-leak from the eye. Next, a 33-gauge hypodermic needle was used to create a tunnel reaching into vitreous. A 33-gauge blunt-end needle (World Precision Instruments), connected to an RPE-KIT (World Precision Instruments) by SilFlex tubing (World Precision Instruments; SILFLEX-2), was advanced through the tunnel. Each mouse received 1 μl injection of a compound per eye. [0091]Lipid formulation: Fatty acids were mixed with 0.22μm membrane-filtered 5% bovine serum albumin (BSA) in saline at a concentration of 36 mmol/L as stock solutions, and then incubated on a shaker at 40oC for 1 h to allow binding of fatty acids and BSA. Stock solutions were diluted with saline to obtain required concentrations. Vehicle was saline containing matching amounts of BSA. Skowronska-Krawczyk and Gao September 24, 2024 [0092]Lipid extraction: Lipid extractions were performed according to the methodology of Bligh and Dyer. In brief, the tissue was homogenized in 200 μL water, transferred to a glass vial, and 750 μL 1:2 (v/v) CHCl₃: MeOH was added and vortexed. Then 250 μL CHCl₃ was added and vortexed. Finally, 250 μL ddH2O was added and vortexed. The samples were centrifuged at 3000 RPM for 5 min at 4°C. The lower phase was transferred to a new glass vial and dried under nitrogen stored at -20°C until subsequent lipid analysis. [0093]LC-MS/MS: Separation of lipids was performed on an Accucore C30 column (2.6 μm, 2.1 mm × 150 mm, Thermo Scientific). The Q Exactive MS was operated in a full MS scan mode (resolution 70,000 at m/z 200) followed by ddMS2 (17,500 resolution) in both positive and negative mode. The AGC target value was set at 1E6 and 1E5 for the MS and MS/MS scans, respectively. The maximum injection time was 200 ms for MS and 50 ms for MS/MS. HCD was performed with a stepped collision energy of 30 ± 10% for negative and 25%, 30% for positive ion mode with an isolation window of 1.5 Da. [0094]Data analysis and post-processing: Data were analyzed with LipidSearch 4.2.21 software. Only peaks with molecular identification grade: A or B were accepted (A: lipid class and fatty acid completely identified or B: lipid class and some fatty acid identified). The relative abundance of each lipid species was obtained by normalization to the total lipids intensity. VLC-PUFAs incorporated into PCs were analyzed using Lipid Data Analyzer with a customized database. Lipid pathways analysis was performed using MetaboAnalyst whereas BioPAN software (www.lipidmaps.org/biopan). Pathways having a p-value < 0.05 and fold change >1.5 were regarded as significant. The most active and most suppressed lipid reaction was taken as significant (p-value < 0.05 equivalent to Z-score >1.645)21. Significantly changed lipid species (FC>1.5. P-value<0.05) were submitted to Lipid Ontology (LION) for lipid ontology analysis. Data visualization was performed on Prism 7 software (GraphPad Software, Inc.). [0095]FA analysis: Separation of VLC-PUFAs was achieved on an Acquity UPLC® BEH C18 column (1.7 μm, 2.1 mm × 100 mm, Waters Corporation). The Q Exactive MS was operated in a full MS scan mode (resolution 70,000 at m/z 200) in negative mode. For the compounds of interest, a scan range of m/z 250–800 was chosen. The identification of fatty acids was based on retention time and formula. Skowronska-Krawczyk and Gao September 24, 2024 [0096]RNA sequencing [0097]Sample collection and preparation: Fresh retina tissue was dissected from each mouse eye. Total RNA was extracted from the retinas using the RNeasy Plus Mini Kit (Qiagen) following the manufacturer’s instructions. RNA quantity and quality were assessed 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 on the NovaSeq 6000 System using the Flow Cell Type S4, generating paired-end reads with a length of 100 base pairs, with approximately 30 million reads per sample. [0099]Bioinformatics analysis: Raw sequencing reads were preprocessed to remove adapter sequences and low-quality bases using Trimmomatic. Cleaned reads were mapped to the mm10 assembly mouse reference genome using the aligner tool HISAT2. Gene-level expression counts were quantified using feature Counts. Differential expression analysis was performed using DESeq2, and genes with an adjusted p-value < 0.05 and |fold change| > 1.5 were considered differentially expressed. [00100] Single-nuclei RNA sequencing (snRNA-seq) [00101] Sample collection and preparation: Retinas (n=3 per age group) were dissected and snap-frozen in liquid nitrogen. Deep frozen samples were sent to Active Motif for further processing. Active Motif is a 10x Genomics Certified Service Provider of Single-Cell Multiome to measure genome-wide gene expression & open chromatin. Samples were processed according to the Illumina “Isolation of Nuclei for Single Cell RNA Sequencing & Tissues for Single Cell RNA Sequencing” procedure and subjected to the snRNAseq following the Illumina protocol. [00102] Quality control, integration, and clustering: 91bp sequencing reads were generated by Illumina sequencing. Reads were mapped to the (reference genome) and counts at features were identified using the Cell Ranger for Single Cell Gene Expression software with default parameters (mkfastq and count functions). Quality control was performed, and cellular barcodes that matched the following three criteria Skowronska-Krawczyk and Gao September 24, 2024 were kept: 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 the percentage of reads mapping to mitochondrial genes under 15%. The expression of approximately 1000 genes per nucleus was 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 optimal cluster resolution was determined using the clustering-tree method. [00103] To visualize cells onto a two-dimensional space, Uniform Manifold Approximation and Projection (UMAP) was performed. Cell identities were assigned using known markers established in previous studies and markers identified in the study. The Wilcoxon Rank Sum test was used to perform differential gene expression analysis between the clusters. Genes with a p-value of less than 0.05 and a log fold change greater than 1 were considered as differentially expressed. Additionally, the Wilcoxon Rank Sum test was used to perform differential gene expression analysis between young and aged conditions within each cell type. Genes with a p-value of less than 0.05 and a log fold change greater than 0.1 were considered as differentially expressed. The FeaturePlot of Elovl2 expression levels was normalized to TBP. [00104] Cell-cell communication analysis: Cell-cell communication analysis was performed using the CellChat package (v.2.0.0). Only cells in cones, rods and Müller glia were used to infer the cell–cell communication. CellChat was first applied to young (3-month-old) and aged (18-month-old) datasets separately to infer cell-cell communication followed by comparison analysis via merging the CellChat objects from young and aged datasets. In brief, the official workflow and loaded the normalized counts into CellChat was followed and applied the preprocessing functions ‘identifyOverExpressedGenes’, ‘identifyOverExpressedInteractions’ and ‘projectData’ with standard parameters set. For the main analyses the core functions ‘computeCommunProb’, ‘computeCommunProbPathway’ and ‘aggregateNet’ were applied using standard parameters. [00105] Gene set enrichment analysis (GSEA): Gene Set Enrichment Analysis (GSEA) was conducted using the GSEA software (v4.2.3)76. Differentially expressed genes identified by DESeq2 were ranked using the signal-to-noise ratio rank metric. Enrichment analysis was performed using predefined gene sets from databases such as Skowronska-Krawczyk and Gao September 24, 2024 Gene Ontology (GO), Molecular Signatures Database hallmark gene set, Reactome, and Wikipathways. Statistical significance of enrichment was determined using a weighted (p=1) scoring calculation scheme with 1000 permutations. Gene sets with size larger than 500 and smaller than 5 were excluded from the analysis. [00106] Metascape gene enrichment and functional analysis: Differentially expressed genes (DEGs) (adjusted p-value < 0.05, |fold change| > 1.5) were inputted into the Metascape platform, where pathway and process enrichment analysis were carried out with selected ontology sources including GO Biological Processes, KEGG Pathway, Reactome Gene Sets, and WikiPathways. Terms with a p-value < 0.01, a minimum count of 3, and an enrichment factor > 1.5 (the enrichment factor is the ratio between the observed counts and the counts expected by chance) were collected and grouped into clusters based on their membership similarities. Then, to further capture the relationships between the terms, a network plot was constructed for a subset of enriched terms, where terms with a similarity > 0.3 were connected by edges. This network was visualized using Cytoscape (v3.10.0), where each node represents an enriched term and is proportional to the number of input genes in each term and is colored by its Cluster ID or p-value. [00107] Transcription factor enrichment analysis: Transcription factor (TF) enrichment analysis was performed using the ChEA3 (ChIP-X Enrichment Analysis 3) web tool for differentially expressed genes (DEGs) (adjusted p-value < 0.05, |fold change| > 1.5). TFs were ranked using the Mean Rank metric, which averaged integrated ranks across gene set libraries. A global transcription co-expression network was constructed from GTEx expression data for the top 20 TFs and clustered by GO enrichment term. Protein-protein interaction analysis was performed for the top 20 associated TFs using the Search Tool for the Retrieval of Interaction Genes/Protein (STRING) database. After filtering out TFs with low expression levels (normalized read count DESeq2 baseMean < 10), protein-protein interactions were visualized in Cytoscape (v3.10.0) as nodes and edges, and each node was colored by the difference in mean Z-score. [00108] Bisulfite sequencing for DNA methylation analysis: The methylation profile of the DNA was determined using PCR amplification followed by DNA Sanger sequencing. Methylated DNA was converted using EZ DNA Methylation-Direct™ Kit Skowronska-Krawczyk and Gao September 24, 2024 (Zymo Research, USA) following the instructions of the manufacturer. The converted DNA was amplified using primers that do not overlap with CpGs. The amplified product was sequenced to detect the methylation status of individual cytosines within the amplified region. The methylation percentage at each CpG site was computed as the ratio of methylated cytosines to the total level signal of cytosines. [00109] Retina cryosection and immunohistochemistry: Mouse eyes were enucleated following euthanasia and fixed in 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS, pH 7.4) for 2 hours at 4°C. The cornea, lens, and vitreous were removed, and the eyecups were fixed in 4% PFA overnight at 4°C. Following fixation, eyecups were cryoprotected by immersion in a sucrose gradient (10% and 20% sucrose for 1 hour at room temperature, and 30% sucrose overnight at 4°C). Eyecups were embedded in Tissue-Tek OCT (Sakura, Torrance, CA) and frozen on a conductive metal block placed on dry ice. Cryosectioning was performed using a cryostat, and retina sections were cut into 10 μm-thick serial sections. [00110] For immunohistochemistry, sections were blocked in 0.3% TritonX-100 in 5% BSA for 1 hour at room temperature to minimize nonspecific binding. Sections were then incubated with primary antibodies diluted in 0.1% TritonX-100 in 5% BSA (see Table 1) overnight at 4°C. Following three washes with PBS, sections were incubated with fluorescently-labeled secondary antibodies diluted in 0.1% TritonX-100 in 5% BSA (see Table 1) for 1 hour at room temperature. Following three washes with PBS, nuclei were counterstained with Hoescht 33342 (Thermo), and sections were mounted using ProLong Gold Antifade (Thermo). Immunostained sections were imaged on a Keyence All-in-One Fluorescence microscope (BZ-X810, Keyence, Itasca, IL) at 40X and 100X magnification. [00111] RNAscope: In situ hybridization was performed using the RNAscope® Multiplex Fluorescent Assay v2 (ACD Diagnostics). Probes used were designed by the manufacturer. Briefly, fresh frozen histologic sections of mouse eyes were pretreated per manual using hydrogen peroxide and target retrieval reagents, including protease IV. Probes were then hybridized according to the protocol and then detected with TSA Plus® Fluorophores fluorescein, cyanine 3, and cyanine 5. Sections were mounted with ProLong Gold Antifade (Thermo Fisher) and imaged (Keyence BZ-X810). Skowronska-Krawczyk and Gao September 24, 2024 [00112] Proteomic analysis: Mouse retinas were harvested and suspended in a Urea buffer containing 8 M Urea, 0.1 M Tris-HCl (pH 8.5), protease inhibitor cocktail (cOmplete™ Protease Inhibitor Cocktail, Roche). The suspension was sonicated on ice for 4 min, followed by centrifugation at 12,000 g for 10 min at 4℃. The supernatant was collected and digested by the filter-aided sample preparation (FASP) method83. Briefly, the supernatant was transferred into a spin filter column (30 kDa cutoff). Proteins were reduced with 10 mM DTT for 1 hr at 56℃, and alkylated with 20 mM iodoacetic acid for 30 min at room temperature in the dark. Next, the buffer was exchanged with 50 mM NH₄HCO₃ by washing the membrane three times. [00113] For quantification of Rho, PED6A, PDE6B, GNAT1, GBB1, stable isotope–labeled (SIL) peptides were spiked into protein samples, and then free trypsin was added into the protein solution at trypsin to protein ratio of 1:50 and incubated overnight at 37℃. The tryptic digests were recovered in the supernatant after centrifugation and an additional wash with water. The combined supernatants were vacuum-dried and then dissolved in 20 μL 0.1% FA in H₂O. [00114] As used herein, the term “about” refers to plus or minus 10% of the referenced number. [00115] Although there has been shown and described the preferred embodiment of the present invention, it will be readily apparent to those skilled in the art that modifications may be made thereto which do not exceed the scope of the appended claims. Therefore, the scope of the invention is only to be limited by the following claims. In some embodiments, the figures presented in this patent application are drawn to scale, including the angles, ratios of dimensions, etc. In some embodiments, the figures are representative only and the claims are not limited by the dimensions of the figures. In some embodiments, descriptions of the inventions described herein using the phrase “comprising” includes embodiments that could be described as “consisting essentially of” or “consisting of”, and as such the written description requirement for claiming one or more embodiments of the present invention using the phrase “consisting essentially of” or “consisting of” is met.

Claims

Skowronska-Krawczyk and Gao September 24, 2024 WHAT IS CLAIMED IS: 1. A method of treating age-related eye conditions in a subject in need thereof, the method comprising administering a composition comprising tetracospentaenoic acid [24:5n-3] to the subject. 2. The method of claim 1, wherein the composition is administered to an eye of the subject. 3. A method of treating age-related eye conditions in a subject in need thereof, the method comprising administering a composition comprising tetracospentaenoic acid [24:5n-3] into an eye of the subject. 4. The method of any one of claims 1–3, wherein the tetracospentaenoic 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 of any one of claims 1–4, wherein the composition further comprises docosahexaenoic fatty acid (DHA). 6. The method of claim 5, wherein the DHA is administered as a free fatty acid, a fatty acid ethyl ester (FAEE), or a fatty acid methyl ester (FAME). 7. The method of any one of claims 1–6, wherein the composition is administered via an intravitreal injection, an intravenous injection, intraperitoneal injection, or topically. 8. The method of any one of claims 1–7, wherein the age-related eye condition is age-related macular degeneration and/or age-related vision loss/decline. 9. The method of any one of claims 1–8, wherein a therapeutically effective dose is administered for the disease. 10.A method of treating age-related liver conditions in a subject in need thereof, the method comprises administering a composition comprising tetracospentaenoic acid [24:5n-3] to the subject. 11. The method of claim 10, wherein the tetracospentaenoic 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 of claim 10 or claim 11, wherein the composition further comprises docosahexaenoic fatty acid (DHA). 13.The method of claim 12, wherein the DHA is administered as a free fatty acid, a fatty acid ethyl ester (FAEE), or a fatty acid methyl ester (FAME). Skowronska-Krawczyk and Gao September 24, 2024 14.The method of any one of claims 10–13, wherein the composition is administered via an intravenous injection. 15.The method of any one of claims 10–14, wherein the age-related liver condition is nonalcoholic fatty liver disease, alcoholic liver disease, hepatitis C, fibrosis, or cirrhosis. 16.The method of any one of claims 10–15, wherein a therapeutically effective dose is administered for the disease. 17.A method of treating age-related brain conditions in a subject in need thereof, the method comprising administering a composition comprising tetracospentaenoic acid [24:5n-3] to the subject. 18.The method of claim 17, wherein the composition is administered to a brain of the subject. 19.A method of treating age-related brain conditions in a subject in need thereof, the method comprising administering a composition comprising tetracospentaenoic acid [24:5n-3] into a brain of the subject. 20.The method of any one of claims 17–19, wherein the tetracospentaenoic 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 of any one of claims 17–20, wherein the composition further comprises docosahexaenoic fatty acid (DHA). 22.The method of claim 21, wherein the DHA is administered as a free fatty acid, a fatty acid ethyl ester (FAEE), or a fatty acid methyl ester (FAME). 23.The method of any one of claims 17–22, wherein the composition is administered via an intravenous and/or stereotactic direct injection. 24.The method of any one of claims 17–23, wherein the age-related brain condition is selected from the diseases consisting of Alzheimer’s Disease, Amyotrophic Lateral Sclerosis (ALS), age-related cognitive decline, dementia, and Parkinson’s disease. 25.The method of any one of claims 17–24, wherein a therapeutically effective dose is administered for the disease. 26.A method of treating age-related bone conditions in a subject in need thereof, the method comprises administering a composition comprising tetracospentaenoic acid [24:5n-3] to the subject. Skowronska-Krawczyk and Gao September 24, 2024 27.The method of claim 26, wherein the tetracospentaenoic 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 of claim 26 or claim 27, wherein the composition further comprises docosahexaenoic fatty acid (DHA). 29.The method of claim 28, wherein the DHA is administered as a free fatty acid, a fatty acid ethyl ester (FAEE), or a fatty acid methyl ester (FAME). 30.The method of any one of claims 26–29, wherein the composition is administered via an intravenous and/or intravenous injection. 31.The method of any one of claims 26–30, wherein the age-related bone condition is multiple myeloma and other Myelodysplastic Syndromes (MDS) or anemia. 32.The method of any one of claims 26–31, wherein a therapeutically effective dose is administered for the disease. 33.A composition for use in treating age-related eye conditions in a subject in need thereof, wherein the composition comprises tetracospentaenoic acid [24:5n-3]. 34.The composition of claim 33, wherein the tetracospentaenoic 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 of claim 33 or claim 34, wherein the composition further comprises docosahexaenoic fatty acid (DHA). 36.The method of claim 35, wherein the DHA is administered as a free fatty acid, a fatty acid ethyl ester (FAEE), or a fatty acid methyl ester (FAME). 37.The composition of any one of claims 33–36, wherein the composition is administered to an eye of the subject. 38.The composition of any one of claims 33–37, wherein the composition is administered via an intravitreal injection, an intravenous injection, intraperitoneal injection, or topically. 39.The composition of any one of claims 33–38, wherein the age-related eye condition is age-related macular degeneration and/or age-related vision loss/decline. 40.A composition for use in treating age-related brain conditions in a subject in need thereof, wherein the composition comprises tetracospentaenoic acid [24:5n-3]. Skowronska-Krawczyk and Gao September 24, 2024 41.The composition of claim 40, wherein the tetracospentaenoic 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 of claim 40 or claim 41, wherein the composition further comprises docosahexaenoic fatty acid (DHA). 43.The method of claim 42, wherein the DHA is administered as a free fatty acid, a fatty acid ethyl ester (FAEE), or a fatty acid methyl ester (FAME). 44.The composition of any one of claims 40–43, wherein the composition is administered via an intravenous and/or stereotactic direct injection.. 45.The composition of any one of claims 40–44, wherein the composition is administered to a brain of the subject. 46.The composition of any one of claims 40–45, wherein the age-related brain condition is selected from the diseases consisting of Alzheimer’s Disease, Amyotrophic Lateral Sclerosis (ALS), age-related cognitive decline, dementia, and Parkinson’s disease. 47.A composition for use in treating age-related liver conditions in a subject in need thereof, wherein the composition comprises tetracospentaenoic acid [24:5n-3]. 48.The composition of claim 47, wherein the tetracospentaenoic 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 of claim 47 or claim 48, wherein the composition further comprises docosahexaenoic fatty acid (DHA). 50.The method of claim 49, wherein the DHA is administered as a free fatty acid, a fatty acid ethyl ester (FAEE), or a fatty acid methyl ester (FAME). 51.The composition of any one of claims 47–50, wherein the composition is administered via an intravenous injection. 52.The composition of any one of claims 47–51, wherein the age-related liver condition is nonalcoholic fatty liver disease, alcoholic liver disease, hepatitis C, fibrosis, or cirrhosis. 53.A composition for use in treating age-related bone conditions in a subject in need thereof, wherein the composition comprises tetracospentaenoic acid [24:5n-3]. Skowronska-Krawczyk and Gao September 24, 2024 54.The composition of claim 53, wherein the tetracospentaenoic 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 of claim 53 or claim 54, wherein the composition further comprises docosahexaenoic fatty acid (DHA). 56.The method of claim 55, wherein the DHA is administered as a free fatty acid, a fatty acid ethyl ester (FAEE), or a fatty acid methyl ester (FAME). 57.The composition of any one of claims 53–56, wherein the composition is administered via an intravenous and/or intravenous injection. 58.The composition of any one of claims 53–57, wherein the age-related bone condition is multiple myeloma and other Myelodysplastic Syndromes (MDS) or anemia.