WO2023212273A1 - Treatments for age-related macular degeneration - Google Patents

Abstract

Described herein are methods for treating and/or preventing, or slowing the progression, of age- related macular degeneration (AMD), as well as gene therapies and pharmaceutical compositions for use in the treatment or prevention of AMD. Also described herein are biomarkers of AMD and methods of diagnosing AMD, predicting patient outcomes from treatment, and selecting patients for treatment.

Description

TREATMENTS FOR AGE-RELATED MACULAR DEGENERATION

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Patent Application Ser. No. 63/337004, filed April 29, 2022, and U.S. Provisional Patent Application Ser. No. 63/419959, filed October 27, 2022, the entire contents of each of which is incorporated herein by reference.

FIELD

[0002] The present disclosure relates to treatments and preventions for age-related macular degeneration (AMD), as well as gene therapies and pharmaceutical compositions for use in the treatment or prevention of AMD. Also described herein are biomarkers of AMD and methods of diagnosing AMD, predicting patient outcomes from treatment, and selecting patients for treatment.

BACKGROUND

[0003] The following discussion is provided to aid the reader in understanding the disclosure and is not admitted to describe or constitute prior art thereto.

[0004] Age-related macular degeneration (AMD) is the most common cause of severe loss of eyesight among people 50 and older. AMD affects central vision, and with it, the ability to see fine details. In AMD, a part of the retina called the macula is damaged. In advanced stages, people lose their ability to drive, to see faces, and to read smaller print. In its early stages, AMD may have no signs or symptoms, so people may not suspect they have it. There are two types of advanced AMD: wet and dry.

[0005] Wet AMD is less common and usually leads to more severe vision loss in patients than dry AMD. Wet AMD happens when abnormal blood vessels start to grow beneath the retina. The primary treatment for wet AMD is the injection of anti -vascular endothelial growth factor (VEGF) agents in a patient’s eye. [0006] Dry AMD accounts for roughly 80% of AMD cases and its exact cause is unknown. In dry AMD the light-sensitive cells in the macula slowly break down, and loss of vision is usually slow and gradual. Currently, there is no treatment for dry.

[0007] There remains a need for treatments for AMD, in particular dry AMD. There likewise remains a need for identifying whether an individual is likely to develop AMD or likely to respond to treatment.

SUMMARY

[0008] Described herein are treatments and preventions for age-related macular degeneration (AMD), as well as gene therapies for use in the treatment or prevention of AMD. Also described herein are biomarkers of AMD and methods of diagnosing AMD, predicting patient outcomes from treatment, and selecting patients for treatment.

[0009] In one aspect, the present disclosure provides methods for identifying a subject at risk for developing Age-Related Macular Degeneration (AMD), and/or at risk of rapid progression of AMD, and/or diagnosing a predisposition to developing AMD, comprising: (a) obtaining a biological sample comprising genomic DNA from a subject; and (b) detecting in the biological sample the presence or absence of a rs77968014 variant in an SLC16A8 gene, wherein the presence of the rs77968014 variant indicates that the subject has AMD or has a predisposition to develop AMD.

[0010] For all of the methods disclosed herein, a subject may have been diagnosed with AMD, is suspected of having AMD, is at risk of developing AMD, or has a predisposition for developing AMD but has not yet developed AMD.

[0011] In another aspect, the present disclosure provides methods of diagnosing age-related macular degeneration (AMD) or a predisposition to developing AMD in a subject, comprising (i) obtaining a biological sample comprising genomic DNA from a subject, and (ii) detecting the presence or absence of a rs77968014 single nucleotide polymorphism (SNP) in SLC16A8, wherein the presence of a rs77968014 SNP risk allele indicates thatthe subject has AMD or a predisposition to develop AMD.

[0012] In another aspect, the present disclosure provides methods of detecting the presence or absence of a rs77968014 single nucleotide polymorphism (SNP) in SLC16A8, comprising (i) obtaining a biological sample comprising genomic DNA from a subject having or suspected of having age-related macular degeneration (AMD), and (ii) detecting the presence or absence of the rs77968014 SNP in the biological sample.

[0013] In some embodiments, the biological sample is obtained from the subject’ s eye. In some embodiments, the biological sample is blood, plasma, or serum.

[0014] In some embodiments, detecting the presence or absence of the rs77968014 comprises dynamic allele-specific hybridization, molecular beacons, SNP microarray analysis, gene chip analysis, restriction fragment length polymorphism analysis, flap endonuclease analysis, 5’- nuclease analysis, oligonucleotide ligation assay, single strand conformation polymorphism analysis, temperature gradient gel electrophoresis, capillary electrophoresis, reversed-phase high performance liquid chromatography (HPLC) detection, denaturing HPLC, high-resolution melting analysis, DNA mismatch-binding protein analysis, SNPlex analysis, surveyor nuclease assay, or sequencing.

[0015] In some embodiments, the disclosed methods result in early diagnosis of AMD in a subject, or diagnosis of risk of AMD in a subject, meaning that prior to the method the subject was not diagnosed with AMD. In some embodiments, early diagnosis of AMD results in initiating AMD treatment, thereby improving the subject’s quality of life and/or prolonging or improving the subject’s visual acuity as compared to the quality of life and/or visual acuity expected in the absence of treatment.

[0016] In some embodiments, the disclosed methods of identifying a subject at risk for developing AMD, and/or at risk of rapid progression of AMD, and/or diagnosing a predisposition to developing AMD, may further comprise, when the variant is present, administering to the subject a therapy to treat, prevent, or slow the progression of AMD. In some embodiments, the therapy comprises (a) photodynamic therapy (PDT), (b) laser surgery; (c) one or more injections of a Vascular Endothelial Growth Factor (VEGF) inhibitor such as faricimab-svoa (Vabysmo®) (Genentech), bevacizumab (Avastin®) (Genentech), and ranibizumab (Susvimo®) (Genentech); (d) administration of one or more angiogenesis inhibitors such as brolucizumab (Beovu®) (Novartis), aflibercept (Eylea®) (Regeneron), ranibizumab (Lucentis®) (Genentech), pegaptanib sodium (Macugen®) (Gilead); (e) administration of one or more nutritional supplements (e.g., about 500 mg vitamin C, about 400 IUS vitamin E, about 10 mg Luteain, about 2 mg zeaxanthin, about 80 mg zinc, and about 2 mg copper); (f) gene therapy described herein; or (g) any combination thereof.

[0017] In another aspect, the present disclosure provides methods of treating or preventing age-related macular degeneration (AMD) in a subject, comprising administering a gene therapy to a subject that has AMD, is suspected of having AMD, is at risk of developing AMD, or has a predisposition for developing AMD but has not yet developed AMD, wherein the gene therapy provides to the subject a nucleic acid encoding a functioning SLC16A8 protein.

[0018] In another aspect, the present disclosure provides methods of selecting a subject with age-related macular degeneration (AMD) for treatment with a gene therapy that restores, augments, or increases activity of a SLC16A8 protein, comprising (i) obtaining a biological sample comprising genomic DNA from a subject having or suspected of having age-related macular degeneration (AMD), and (ii) detecting in the sample the presence or absence of the rs77968014 SNP in a SLC16A8 gene.

[0019] In another aspect, the present disclosure provides methods of treating or preventing age-related macular degeneration (AMD) or restoring function of SLC16A8 in a subject, comprising administering to a subject with a rs77968014 single nucleotide polymorphism (SNP) risk allele in SLC16A8 a gene therapy that restores, augments, or increases activity of a SLC16A8 protein.

[0020] In another aspect, the present disclosure provides methods of treating or preventing age-related macular degeneration (AMD) or restoring function of SLC16A8 in a subject, comprising administering a gene therapy that restores, augments, or increases activity of a SLC16A8 protein to a subject with a single nucleotide polymorphism (SNP) or mutation in SLC16A8 that results in a loss of function or decreased expression of the SLC16A8 protein.

[0021] The gene therapies may also be given in conjunction with other treatments for AMD.

[0022] In some embodiments, the gene therapy is selected from a plasmid encoding wild-type human SLC16A8, a viral vector encoding wild-type human SLC16A8, a bacterial vector encoding wild-type human SLC16A8, a mRNA encoding wild-type human SLC16A8, a CRISPR/Cas gene editing system, a zinc finger nuclease (ZFN) gene editing system, a transcription activator-like effector nuclease (TALEN) gene editing system, and a patient-derived cellular gene therapy. In some embodiments, the VEGF inhibitor is ranibizumab. In some embodiments, the therapy may be administered before AMD onset, or after AMD onset.

[0023] In some embodiments, the subject has a rs77968014 SNP risk allele in SLC16A8. Thus, for any of the methods described herein, the subject can be selected for treatment with the gene therapy by determining, prior to commencement of treatment, that the subject has a rs77968014 SNP risk allele.

[0024] In some embodiments, the nucleic acid comprises a copy of SLC16A8 that does not comprise any SNPs or mutations, SEQ ID NO: 1, or SEQ ID NO: 2. In some embodiments, the nucleic acid encodes a protein comprising SEQ ID NO: 3 or an amino acid with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity thereto.

[0025] In some embodiments, the gene therapy provides to the subject with a nucleic acid that encodes a functioning SLC16A8 protein. In some embodiments, the nucleic acid comprises a copy of SLC16A8 that does not comprise any SNPs or mutations, SEQ ID NO: 1, or SEQ ID NO: 2. In some embodiments, the nucleic acid encodes a protein comprising SEQ ID NO: 3 or an amino acid with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity thereto. [0026] For any of the foregoing aspect and embodiments, the gene therapy can be administered to the subject’s eye. For example, in some embodiments, the gene therapy is administered to the subject’s eye via subretinal, intravitreal, or suprachoroidal injection. Alternatively, for any of the foregoing aspects or embodiments, the gene therapy is administered systemically.

[0027] The present disclosure also provides uses of a gene therapy that restores, augments, or increases activity of a SLC16A8 protein for treating or preventing age-related macular degeneration (AMD) or restoring function of SLC16A8 in a subject with a rs77968014 single nucleotide polymorphism (SNP) risk allele in SLC16A8.

[0028] The present disclosure also provides gene therapies that restore, augment, or increase activity of a SLC16A8 protein for use in treating or preventing age-related macular degeneration (AMD) in a subject that has a single nucleotide polymorphism (SNP) or mutation in SLC16A8 that results in a loss of function or decreased expression of the SLC16A8 protein.

[0029] The present disclosure also provides gene therapies that provide a wild-type copy of a nucleic acid encoding SLC16A8 or a nucleic acid encoding a functioning SLC16A8 protein for use in treating or preventing age-related macular degeneration (AMD) or restoring function of SLC16A8 in a subject with a rs77968014 single nucleotide polymorphism (SNP) risk allele in SLC16A8.

[0030] For any of the disclosed gene therapies, the gene therapy provides to the subject with a nucleic acid that encodes a functioning SLC16A8 protein. For example, in some embodiments, the nucleic acid comprises a copy of SLC16A8 that does not comprise any SNPs or mutations, SEQ ID NO: 1, or SEQ ID NO: 2. In some embodiments, the nucleic acid encodes a protein comprising SEQ ID NO: 3 or an amino acid with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity thereto.

[0031] In some embodiments, a therapy in accordance with the disclosed methods improves the quality of life of the subject, wherein the improvement in quality of life comprises one or more of: (a) delaying the need for additional therapeutic interventions; (b) preventing or reducing the need for additional therapeutic interventions; and (c) reversing, halting, or reducing the rate of vision loss. In some embodiments, the improvement in quality of life comprises reversing, halting, or reducing the rate of vision loss.

[0032] In some embodiments, the AMD disease onset and/or progression is slowed by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%, as measured by any pharmaceutically acceptable method. In some embodiments, the pharmaceutically acceptable method comprises a clinical evaluation.

[0033] The foregoing general description and following detailed description are exemplary and explanatory and are intended to provide further explanation of the disclosure as claimed. Other objects, advantages, and novel features will be readily apparent to those skilled in the art from the following brief description of the drawings and detailed description of the disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

[0034] FIG. 1 shows drosophila fluorescent deep pseudopupil (DPP) and optic neutralization of the cornea (ONC) imaging of photoreceptors.

[0035] FIGs. 2A and 2B shows that RNAi knockdown (vl09464) of the SLC16A8 Drosophila ortholog sin (silnoon) leads to progressive changes in photoreceptor EGFP expression patterns by day 14. (95% mean loss of DPP). In particular, FIG. 2A shows, and FIG 2B shows graphs of % flies with fluorescent DPP vs days at 25°C for RNAi negative controls, SLC16A8 RNAi ortho screen #1, and SLC16A8 RNAi ortho screen #2.

[0036] FIGs. 3A and 3B show that SLC16A8 expression in humans is highly specific for retinal pigment epithelial (RPE) cells. In particular, FIG. 3A shows a matrix of scaled mean expression of the following genes in various cell types: ATL2 (Atlastin GTPase 2), CFH (Complement Factor H), PR0M1 (Prominin-1), RGS7 (Regulator of G Protein Signaling 7), SLC10A3 (Solute Carrier Family 10 Member 3), and SLCJ6A8 (Solute Carrier Family 16 Member 8). Cell types evaluated included vascular, RPE, Rods, RGC (retinal ganglion cell), NA, myeloid, Muller (principal glial cell of the retina), retinal horizontal cells, cones, retinal bipolar cells, astrocyte, and amacrine cells. The matrix clearly shows significant expression of SLC16A8 in RPE cells. FIG. 3B shows RPKM vs SLC16A8 in various tissues, including retina (macula and nonmacula), and RPE (macula and non-macula).

[0037] FIG. 4 shows that knockout of Sic 16a8 (MCT3) in C57BL/6J mice leads to significant functional ERG a/b/c-wave defects at 10 weeks of age. Two-way RM ANOVA, comparison between genotypes within time points, data shown as mean ± SD, n > 12 eyes/group.

[0038] FIG. 5 shows an overview of the Slcl6a8 rodent light exposure model.

[0039] FIG. 6 shows that heterozygous knockout of Slcl6a8 in C57BL/6J mice leads to significantly decreased photoreceptor function as compared to WT and homozygous Slcl6a8 knockout mice, as illustrated by significant ERG A- wave amplitude defects after 7 days of constant light exposure (CLE) at 100k lux. Two-way RM ANOVA, comparison between groups within time points, Tukey post-hoc, data shown as mean ± SD, n > 12 eyes/group.

[0040] FIG. 7 shows that heterozygous knockout of Slcl6a8 in C57BL/6J mice leads to significantly decreased retinal pigment epithelia (RPE) function as compared to WT and homozygous Slcl6a8 knockout mice, as illustrated by significant ERG C-wave amplitude defects after 7 days of constant light exposure (CLE) at 100k lux. Two-way RM ANOVA, comparison between groups within time points, Tukey post-hoc, data shown as mean ± SD, n > 12 eyes/group.

DETAILED DESCRIPTION

[0041] Age-related macular degeneration (AMD) is a complex neurodegenerative disease commonly afflicting the elderly and is one of the leading causes of visual impairment worldwide. There are several clinical subtypes of AMD. Early AMD is also known as “intermediate AMD,” and this can progress to Geographic Atrophy (GA) or dry AMD, for which there are no treatments, or Classic Neovascular (CNV) or wet AMD, for which anti-VEGF treatments are typically utilized. Polygenic predisposition to AMD enables strategies to define the complex pathogenesis of this disease and identify therapeutic targets, as well as identifying patient populations that may benefit from early intervention.

[0042] In one aspect, the present disclosure provides methods for identifying a subject at risk for developing Age-Related Macular Degeneration (AMD), and/or at risk of rapid progression of AMD, and/or diagnosing a predisposition to developing AMD. In an exemplary aspect, the method comprises: (a) obtaining a biological sample comprising genomic DNA from a subject; and (b) detecting in the biological sample the presence or absence of a rs77968014 variant in an SLC16A8 gene, wherein the presence of the rs77968014 variant indicates that the subject has AMD or has a predisposition to develop AMD. Identification of subjects or patient populations at risk may be followed by therapeutic and/or preventative measures, as detailed herein, to prevent, minimize, and/or slow progression of AMD.

[0043] In another aspect, the present disclosure provides methods of diagnosing age-related macular degeneration (AMD) or a predisposition to developing AMD in a subject, comprising (i) obtaining a biological sample comprising genomic DNA from a subject, and (ii) detecting the presence or absence of a rs77968014 single nucleotide polymorphism (SNP) in SLC16A8, wherein the presence of a rs77968014 SNP risk allele indicates thatthe subject has AMD or a predisposition to develop AMD.

[0044] In another aspect, the present disclosure provides methods of detecting the presence or absence of a rs77968014 single nucleotide polymorphism (SNP) in SLC16A8, comprising (i) obtaining a biological sample comprising genomic DNA from a subject having or suspected of having age-related macular degeneration (AMD), and (ii) detecting the presence or absence of the rs77968014 SNP in the biological sample.

[0045] In some embodiments, the disclosed methods result in early diagnosis of AMD in a subject, or diagnosis of risk of AMD in a subject, meaning that prior to the method the subject was not diagnosed with AMD. In some embodiments, early diagnosis of AMD results in initiating AMD treatment, thereby improving the subject’s quality of life and/or prolonging or improving the subject’s visual acuity as compared to the quality of life and/or visual acuity expected in the absence of treatment.

[0046] In some embodiments, the disclosed methods of identifying a subject at risk for developing AMD, and/or at risk of rapid progression of AMD, and/or diagnosing a predisposition to developing AMD, may further comprise, when the variant is present, administering to the subject a therapy to treat, prevent, or slow the progression of AMD. In some embodiments, the therapy comprises (a) photodynamic therapy (PDT), (b) laser surgery; (c) one or more injections of a Vascular Endothelial Growth Factor (VEGF) inhibitor such as faricimab-svoa (Vabysmo®) (Genentech), bevacizumab (Avastin®) (Genentech), and ranibizumab (Susvimo®) (Genentech); (d) administration of one or more angiogenesis inhibitors such as brolucizumab (Beovu®) (Novartis), aflibercept (Eylea®) (Regeneron), ranibizumab (Lucentis®) (Genentech), pegaptanib sodium (Macugen®) (Gilead); (e) administration of one or more nutritional supplements (e.g., about 500 mg vitamin C, about 400 IUS vitamin E, about 10 mg Luteain, about 2 mg zeaxanthin, about 80 mg zinc, and about 2 mg copper); (f) gene therapy described herein; or (g) any combination thereof.

[0047] In another aspect, the present disclosure provides gene therapies for treating and/or preventing age-related macular degeneration (AMD). In particular, the present disclosure has identified loss of function of Solute Carrier Family 16 Member 8 SLC16A NCBI Entrez Gene: 23539 Ensembl: ENSG00000100156 OMIM®: 610409 UniProtKB/Swiss-Prot: 095907) as a primary driver of AMD development. Accordingly, provided herein are treatments for AMD that restore, augment, or increase activity of an SLC16A8 protein in a subject in need (e.g., a subject with AMD, reduced SLC16A8 activity, reduced SLC16A8 expression, or a risk allele of rs77968014) Further, a risk allele of the single nucleotide polymorphism (SNP) rs77968014 gives rise to a splice donor variant with loss of function that (a) may be predictive of development of AMD, (b) may be predictive of subjects that will respond the disclosed AMD treatments (e.g., gene therapies that restore function to SLC16A8), and (c) may serve as a biomarker for selection of patients that should be treated with the disclosed AMD treatments. Based on the in vivo data provided herein, it can be extrapolated that a loss of function of SLC16A8, in general (i.e., regardless of SNP status), can be used to predict development of AMD, responsiveness to certain therapies, and serve as a biomarker for AMD and AMD treatment.

[0048] Experimental Data: As described in the examples below, it was surprisingly discovered that the loss of Slcl6a8 lactate transport disrupts retinal homeostasis in multiple organisms (e.g., Drosophila and mice), and that two key cell types (RPE and PR) have functional defects with loss of Slcl6a8 in mouse models. In addition, RPE and PR cell functions are significantly more decreased in Slcl6a8-Hets vs. WT/KO after exposure to light induced damage. Thus, an unbiased, genome-wide analysis of rare coding variants provides support of SLC16A8 (MCT3) significantly associated with AMD, including advanced AMD, risk. The data additionally demonstrate that loss of function of the monocarboxylate transporter in both drosophila (sin) and mice (Slcl6a8-KO and Hets) disrupts retinal homeostasis. Finally, meta-analyses of combined data sets for shared SLC16A8 high impact protein altering variants identify rs77968014 donor splice-site intronic variant as the most significant driver (p-val = 2.76E-08) for SLC16A8 signal in rare variant burden tests.

[0049] Other aspects of the disclosure are described herein.

I. Definitions

[0050] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0051] Technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art, unless otherwise defined. Unless otherwise specified, materials and/or methodologies known to those of ordinary skill in the art can be utilized in carrying out the methods described herein, based on the guidance provided herein.

[0052] As used herein, the singular terms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Reference to an object in the singular is not intended to mean “one and only one” unless explicitly so stated, but rather “one or more.” [0053] As used herein, “about” when used with a numerical value means the numerical value stated as well as plus or minus 10% of the numerical value. For example, “about 10” should be understood as both “10” and “9-11.”

[0054] Also as used herein, “and/or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”).

[0055] As used herein, a phrase in the form “A/B” or in the form “A and/or B” means (A), (B), or (A and B); a phrase in the form “at least one of A, B, and C” means (A), (B), (C), (A and B), (A and C), (B and C), or (A, B, and C).

[0056] As used herein, the term “increase” when used with respect to expression or activity of a SLC16A8 gene or SLC16A8 protein refers to a level that is 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 100% or greater than the expression level or activity level of a reference value. The reference value may be the corresponding level of expression or activity prior to a subject being treated with a gene therapy as disclosed here.

[0057] As used herein, the term “restore” when used with respect to expression or activity of a SLCJ6A8 gene or SLC16A8 protein refers to achieving, in a subject being treated with a gene therapy, a level of expression or activity that is not statistically significantly different from a reference level. The reference level can be obtained from individual that does not have a mutation, SNP, or alteration in SLC16A8 that results in a loss of function or from a population of individuals that do not have a mutation, SNP, or alteration in SLC16A8 that results in a loss of function. The reference level is expected to be a range of expression or activity, and does not necessarily refer to a single, definitive value. In other words, once the subject’s SLC16A8 expression or activity is “restored,” it should not be substantially different from the reference level. For these purposes, “substantially different” denotes a sufficiently high degree of difference between two numeric values such that one of skill in the art would consider the difference between the two values to be of statistical significance within the context of the biological characteristic measured by said values. [0058] As used herein, the phrase “therapeutically effective amount” with reference to a gene therapy means that dose of the gene therapy that provides the specific pharmacological effect for which the drug is administered in a subject in need of such treatment (e.g., treating AMD or restoring/supplementing the function, activity, or expression of SLC16AS). A therapeutically effective amount may be effective to reduce, ameliorate, or eliminate vision loss associated with AMD. It is emphasized that a therapeutically effective amount of a gene therapy will not always be effective in treating AMD in every individual subject, even though such dose is deemed to be a therapeutically effective amount by those of skill in the art. Those skilled in the art can adjust what is deemed to be a therapeutically effective amount in accordance with standard practices as needed to treat a specific subject. A therapeutically effective amount may vary based on, for example, the age and weight of the subject, and/or the subject’s overall health, and/or the severity of the subject’s AMD.

[0059] The terms “treat,” “treatment” or “treating” as used herein with reference to AMD refer to reducing, ameliorating, or eliminating vision loss associated with AMD.

[0060] A “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result.

[0061] The terms “prevent,” “preventing” or “prevention” as used herein with reference to AMD refer to precluding development or reducing the risk of developing AMD. Thus, in some embodiments, “preventing” can refer to stopping vision loss associated with AMD before it occurs or reducing a subject’s risk of experiencing vision loss associated with AMD.

[0062] The terms “individual,” “subject,” and “patient” are used interchangeably herein, and refer to any individual mammalian subject, e.g., bovine, canine, feline, equine, or human. In specific embodiments, the subject, individual, or patient is a human.

IL Age-Related Macular Degeneration (AMD) and SLC16A8

[0063] Age-related macular degeneration (AMD) is an eye disease that is a leading cause of vision loss in older people in developed countries. Subtle abnormalities indicating changes in vision may occur in a person's forties or fifties. Distorted vision and vision loss usually become noticeable in a person's sixties or seventies and tend to worsen over time. While it is believed that AMD is caused by a combination of genetic and environmental factors, a definitive cause was never previously determined.

[0064] For example, the CFH gene is thought to contribute to a person’s risk of developing AMD, but it is unclear how genetic changes in this gene are related to the retinal damage and vision loss characteristic of AMD. Changes on the long (q) arm of chromosome 10 in a region known as 10q26 are also associated with an increased risk of AMD, and, in particular, it is believed the ARMS2 o HTRAl genes in this region may be of interest. However, causative mutations have not been definitively determined. Other genes that are associated with AMD include genes involved in transporting and processing high-density lipoproteins, such as apolipoprotein E (APOE), but to date none of these target genes has successfully been targeted for treatment, and there are still no clinically approved gene therapies available to AMD patients, particularly those with dry AMD. Even in wet AMD, gene therapies directed to clear putative targets like vascular endothelial growth factor (VEGF) have not been successful, and early stage clinical trials for anti- VEGF gene therapies were discontinued for lack of efficacy. In sum, identifying an effective therapeutic target for gene therapy in AMD has remained elusive.

[0065] SLC16A8 (also known as monocarboxylate transporter 3 or MCT3) is a member of a family of proton-coupled monocarboxylate transporters that mediate lactate transport across cell membranes, and it is encoded by the SLC16A8 gene. Expression of SLC16A8 is confined to the retinal pigment epithelium and choroid plexus epithelia, where it is located on the basal membrane. The present disclosure is the first to confirm in vivo that loss of function of SLC16A8 and, more specifically, a risk allele for a single nucleotide polymorphism (SNP) rs77968014 are drivers of AMD developments, and therefore can serve as therapeutic targets and biomarkers or predictors of the disease.

[0066] The genetic analysis presented herein (see Example 1) indicates that a risk allele of rs77968014 (alleles A/B are C/G; with risk allele being G) gives rise to a splice donor variant that is more prevalent and more important than previously believed. This splice variant results in a loss of function in the SLC16A8 protein, and RNAi knockdown analysis in an in vivo drosophila model confirmed that knockdown of SLC16A8 (or the drosophila ortholog) resulted in the development of AMD-like changes to the eye. Moreover, the knockdown studies also indicated that some genes that were previously thought to be linked to AMD (e.g., CFH) had no impact on AMD development when expression was decreased.

[0067] In experiments presented herein, it was observed that SLC16A8 protein is important for both photoreceptor and retinal pigment epithelium (RPE) function in an in vivo mouse model. Loss-of-function of SLC16A8 resulted in disrupted retinal homeostasis in mice. Signatures of the physiological health of the photoreceptors in the outer retina, the health of the bipolar cells of the retina, and the heath of RPE cells revealed defects in all three categories of cells at 10 weeks of age in heterozygous and homozygous Sic 16a8 knockout mice. In addition, photoreceptor and RPE function were significantly more diminished in Sic 16a8 heterozygous mice compared to WT and Slcl6a8 homozygous knockout mice after exposure to light-induced retinal damage.

[0068] Prior to the present disclosure, there was no uniform consensus in the art regarding the involvement or impact of SLC16A8 expression or activity in the developments of AMD. For example, other groups have recently indicated that SLC16A8/MCT3 inhibition can treat AMD (U.S. Patent Pub. 2021/0002637), thus suggesting the precise opposite of the date include here. The data provided herein also highlights the deficiencies and lack of predictability of previous studies, such as Fritsche et al., Nature Genetics, 2015, 48:134-143. Fritsche speculated that the SLC16A8 gene could be linked to AMD, but the analysis was limited and even Fritsche indicated that its results could not explain proportions of AMD risk. Moreover, Fritsche identified CFH as a high priority gene with a potentially causal role in their in silico studies, but the present inventors found in their in vivo model that no significant changes were observed following knockdown of CFH, further underscoring that Fritsche’s studies were not necessarily a reliable predictor of what will occur in vivo. [0069] Based on the surprising findings detailed herein, the present disclosure provides treatments and preventions of AMD that function by augmenting, restoring, or increasing the function, activity, or expression of SLC16A8 protein in a subject’s eye or, more specifically, the retina pigment epithelial (RPE). Similarly, present disclosure provides methods of patient selection, and diagnostic/prognostic methods based on the identification of rs77968014 in the SLC16A8 gene or loss of function/expression of the SLC16A8 protein.

III. Treatments for AMD

[0070] The present disclosure provides methods of treating or preventing AMD in a subject having AMD or at risk of developing AMD.

[0071] In some embodiments, the disclosed methods of identifying a subject at risk for developing AMD, and/or at risk of rapid progression of AMD, and/or diagnosing a predisposition to developing AMD, may further comprise, when the variant is present, administering to the subject a therapy to treat, prevent, or slow the progression of AMD. In some embodiments, the therapy comprises (a) photodynamic therapy (PDT), (b) laser surgery; (c) one or more injections of a Vascular Endothelial Growth Factor (VEGF) inhibitor such as faricimab-svoa (Vabysmo®) (Genentech), bevacizumab (Avastin®) (Genentech), and ranibizumab (Susvimo®) (Genentech); (d) administration of one or more angiogenesis inhibitors such as brolucizumab (Beovu®) (Novartis), aflibercept (Eylea®) (Regeneron), ranibizumab (Lucentis®) (Genentech), pegaptanib sodium (Macugen®) (Gilead); (e) administration of one or more nutritional supplements (e.g., about 500 mg vitamin C, about 400 IUS vitamin E, about 10 mg Luteain, about 2 mg zeaxanthin, about 80 mg zinc, and about 2 mg copper); (f) gene therapy described herein; or (g) any combination thereof.

[0072] The disclosed methods can comprise, consist of, or consist essentially of administering a gene therapy to the subject that has AMD or is at risk of developing AMD (e.g., a subject with a rs77968014 SNP in his or her SLC16A8 gene. Also disclosed herein are uses of gene therapies for treating or preventing AMD. Additionally, particular subgroups of subjects may be especially suitable for treatment or prevention according to the disclosed methods and uses (e.g., patients with a rs77968014 SNP in the SLC16A8 gene), and therefore the present disclosure likewise discloses method of patient selection.

[0073] In one aspect, the disclosed methods of treating or preventing age-related macular degeneration (AMD) or restoring function of SLC16A8 in a subject comprise administering a gene therapy that restores normal function of SLC16A8 to a subject with a rs77968014 single nucleotide polymorphism (SNP) in SLC16A8. The subject may have AMD, be suspected of having AMD (but, e.g., has not yet been diagnosed), is at risk of developing AMD, or has a predisposition for developing AMD (e.g., as a result of environmental or genetic factors).

[0074] The present disclosure also provides methods of restoring, augmenting, or increasing function of SLC16A8 or expression of SLC16A8 in a subject comprising administering a gene therapy that encodes a functioning SLC16A8 protein to a subject with a rs77968014 single nucleotide polymorphism (SNP) in SLC16A8. The subject may have AMD, be suspected of having AMD (but, e.g., has not yet been diagnosed), is at risk of developing AMD, or has a predisposition for developing AMD (e.g., as a result of environmental or genetic factors).

[0075] In another aspect, the disclosed methods of treating or preventing age-related macular degeneration (AMD) in a subj ect comprise administering a gene therapy to a subject that has AMD, is suspected of having AMD (but, e.g., has not yet been diagnosed), is at risk of developing AMD, or has a predisposition for developing AMD (e.g., as a result of environmental or genetic factors), wherein the gene therapy provides to the subject a nucleic acid encoding a functioning SLC16A8 protein (e.g., a nucleic acid encoding wild-type SLC16A8). The subject may have a rs77968014 SNP in SLC16A8, or the subject may possess another SNP, mutation, or alteration that results in a loss of function SLC16A8 or decreased expression of SLC16A8.

[0076] The present disclosure also provides methods of restoring, augmenting, or increasing function of SLC16A8 or expression of SLC16A8 in a subject comprising administering a gene therapy that encodes a functioning SLC16A8 protein to a subject has AMD, is suspected of having AMD (but, e.g., has not yet been diagnosed), is at risk of developing AMD, or has a predisposition for developing AMD (e.g., as a result of environmental or genetic factors). [0077] For the purposes of the disclosed methods, the gene therapy should function to augment the activity of SLC16A8 protein, increase expression of a functioning SLC16A8 protein, or restore normal (i.e., non-pathological) levels of activity of SLC16A8. As such, the gene therapy may, for example, provide to the subject one or more copies of a nucleic acid encoding a wild-type copy of SLC16A8 (i.e., a copy of the SLC16A8 gene that does not comprise any SNPs or mutations). Additionally or alternatively, the gene therapy may provide to the subject at least one sequence selected from:

ACACACTCTAAGCTCCAGGCCTGGGGCAGGGAGCGTCAGGCGGGAGTCGGCCAGAG GCTCAGCCCAGGCTGGTGGGAAGCTGGGGTGTCTGGCAGCCCCCATCCGGGTAAGC TTCACCGGGAGCTGCGGCGGCGGCCCCTCCCTGCAGGCCTCAGGAGATGTGAGGCT GGACCCCCAAACCCACGAGGCAGGCTCAGGCCGCCTGGTGGATGTGTTGAAGAACT ATGAGATCATCTTCTACCTGGCCGGCTCTGAGGTGGCCCTGGCTGGGGTCTTCATGG CTGTCGCCACCAACTGCTGCCTGCGTTGTGCTAAAGCTGCCCCGTCAGGCCCAGGCA CTGAGGGCGGAGCCAGTGACACTGAGGACGCTGAGGCTGAAGGGGACTCTGAGCCC CTGCCTGTTGTTGCAGAGGAACCCGGCAACCTGGAGGCCCTGGAGGTGCTCAGCGC CCGGGGCGAGCCCACAGAACCAGAAATAGAGGCGAGGCCGAGGCTGGCTGCCGAG TCTGTATAACTCAGGGTGCTCTGGGTGGGGTGGCCCAGTGACTTGGGAACACAGCTT CTTGTCTCAGAGAGCCTGGTACGCTGGGAGGCTGGTCTGGGGTGGTCACTCCCGGGG TCCACGTCTGGGCTCCAGTTGATCCCCTGGGTGTCTGGGAACTGCCTCCCTCATCTGT GCCCCAAGTTCCGCCAGGCCCTGCCCCATCGCCAGTGAAGCTGCTATGGAGCAACA GGAAACTGGTGATAAAGCTCAGCTGAACGACA (SEQ ID NO: 1; NCBI Reference Sequence: NM_001394131.1);

ACACACTCTAAGCTCCAGGCCTGGGGCAGGGAGCGTCAGGCGGGAGTCGGCCAGAG GCTCAGCCCAGGCTGGTGGGAAGCTGGGGTGTCTGGCAGCCCCCATCCGGGTAAGC TTCACCGGGAGCTGCGGCGGCGGCCCCTCCCTGCAGGCCTCAGGAGATGTGAGGCT GGACCCCCAAACCCACGAGGCAGGCTCAGTCATTTGTGATCACTGAGAAACATTAG GAACATGGCGGAAGGCTAAAGAGGAAAATCACGCACAGCGGCCCTTTGCAGCCGGC ATCTGCCCCTCTGCTGTTTGTTCTGTTGTCCCGAGACCCTCACGCGTACGGGGCGACT TTGGTGCTGGACGCCCACCTCCAGGCACATACCTGCTCCACTTCCCGACGCCTTCCA CCTGAGGCCCTGAGGGGCCAGCAGTTGTCCTTCAGAGGGAGCCTGCAGAGGTGCAG

AGTCAGGTGGGACCCGTCGTCCTCCCCTCGTCCTTCAGCGCCCTTTGCAGGAGAAGG

AGACTTGGGAGGCAGCGATGGGCGCTGGCGGCCCCCGGCGGGGCGAGGGCCCCCCA

GACGGCGGCTGGGGCTGGGTGGTGCTGGGCGCCTGCTTTGTGGTCACCGGCTTCGCC

TACGGCTTCCCCAAAGCCGTGAGCGTCTTCTTCCGCGCGCTCATGCGCGACTTCGAC

GCCGGCTACAGCGACACGGCCTGGGTGTCCTCCATCATGCTAGCCATGCTCTACGGC

ACGGGCCCCGTGTCCAGCATCCTCGTGACCCGCTTTGGCTGTCGCCCGGTGATGCTG

GCGGGTGGGCTGCTGGCTTCCGCGGGCATGATCCTAGCTTCCTTTGCCACGCGCCTC

CTGGAGCTCTACCTGACCGCTGGGGTGCTCACAGGCCTGGGCCTGGCCCTCAACTTC

CAGCCGTCGCTCATCATGCTGGGGCTGTACTTCGAGCGGCGGCGGCCTCTGGCCAAC

GGGCTGGCGGCGGCGGGCAGCCCCGTGTTCCTGTCCGCGCTGTCGCCGCTCGGCCAG

CAGCTGCTGGAGCGCTTCGGCTGGCGCGGCGGCTTCCTGCTGCTCGGCGGGCTCCTG

CTGCACTGCTGCGCCTGCGGGGCTGTCATGAGGCCGCCGCCCGGGCCGGGCCCGCG

ACCGCGCAGGGACAGCGCCGGCGACCGCGCCGGGGACGCTCCGGGCGAGGCGGAG

GCTGACGGTGCGGGGCTGCAGCTGCGCGAGGCATCCCCCAGGGTCCGGCCCCGCCG

GCGCCTGCTGGACTTGGCAGTGTGCACCGACCGCGCCTTCGCCGTGTACGCCGTCAC

CAAGTTCCTGATGGCGCTCGGGCTCTTCGTCCCCGCCATCCTGCTGGTGAACTACGC

CAAGGACGCGGGCGTGCCCGACACCGACGCCGCCTTCCTGCTGTCCATCGTGGGCTT

CGTGGACATCGTGGCGCGCCCGGCGTGCGGCGCCCTGGCGGGCCTGGCGCGTCTGC

GGCCGCACGTCCCGTATCTGTTCAGCCTGGCCCTGCTGGCCAATGGGCTCACAGACC

TGAGCAGCGCACGCGCGCGCTCCTACGGCGCCCTCGTCGCCTTCTGCGTCGCCTTCG

GCCTCTCCTACGGCATGGTGGGCGCGCTGCAGTTCGAGGTGCTCATGGCGGCTGTGG

GCGCGCCCCGCTTCCCCAGTGCGCTGGGCCTGGTGTTGCTCGTGGAGGCCGCGGCTG

TGCTCATCGGACCGCCCTCTGCCGGCCGCCTGGTGGATGTGTTGAAGAACTATGAGA

TCATCTTCTACCTGGCCGGCTCTGAGGTGGCCCTGGCTGGGGTCTTCATGGCTGTCGC

CACCAACTGCTGCCTGCGTTGTGCTAAAGCTGCCCCGTCAGGCCCAGGCACTGAGGG

CGGAGCCAGTGACACTGAGGACGCTGAGGCTGAAGGGGACTCTGAGCCCCTGCCTG

TTGTTGCAGAGGAACCCGGCAACCTGGAGGCCCTGGAGGTGCTCAGCGCCCGGGGC

GAGCCCACAGAACCAGAAATAGAGGCGAGGCCGAGGCTGGCTGCCGAGTCTGTATA ACTCAGGGTGCTCTGGGTGGGGTGGCCCAGTGACTTGGGAACACAGCTTCTTGTCTC AGAGAGCCTGGTACGCTGGGAGGCTGGTCTGGGGTGGTCACTCCCGGGGTCCACGT CTGGGCTCCAGTTGATCCCCTGGGTGTCTGGGAACTGCCTCCCTCATCTGTGCCCCA AGTTCCGCCAGGCCCTGCCCCATCGCCAGTGAAGCTGCTATGGAGCAACAGGAAAC TGGTGATAAAGCTCAGCTGAACGACA (SEQ ID NO: 2; NCBI Reference Sequence: NM_013356.3); and NC_000022.11. In some embodiments, the gene therapy may provide to the subject a nucleic acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NO: 1, SEQ ID NO: 2, or NC_000022.11. The nucleic acid sequence may vary and may be, for example, codon optimized, so long as the encoded protein is a functioning SLC16A8 protein.

[0078] Thus, treatment with the gene therapy should ideally result in the expression of a functioning SLC16A8 protein in the eye (e.g., the RPE cells) of the subject being treated. The SLC16A8 protein may comprise, for example, the amino acid sequence: MGAGGPRRGEGPPDGGWGWVVLGACFVVTGFAYGFPKAVSVFFRALMRDFDAGYSD TAWVS SIMLAMLYGTGPVS SILVTRFGCRPVMLAGGLLAS AGMILASF ATRLLELYLTA GVLTGLGLALNFQPSLIMLGLYFERRRPLANGLAAAGSPVFLSALSPLGQQLLERFGWR GGFLLLGGLLLHCCACGAVMRPPPGPGPRPRRDSAGDRAGDAPGEAEADGAGLQLREA

SPRVRPRRRLLDLAVCTDRAFAVYAVTKFLMALGLFVPAILLVNYAKDAGVPDTDAAF LLSIVGFVDIVARPACGALAGLARLRPHVPYLFSLALLANGLTDLSSARARSYGALVAFC VAFGLSYGMVGALQFEVLMAAVGAPRFPSALGLVLLVEAAAVLIGPPSAGRLVDVLKN YEIIFYLAGSEVALAGVFMAVATNCCLRCAKAAPSGPGTEGGASDTEDAEAEGDSEPLP VVAEEPGNLEALEVLSARGEPTEPEIEARPRLAAESV (SEQ ID NO: 3; NP 037488.2) or a sequence that shares at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity, so long as the encoded protein is a functioning SLC16A8 protein. In some embodiments, the gene therapy comprises a nucleic acid that encodes a protein comprising SEQ ID NO: 3 or an amino acid with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity thereto.

[0079] The type of gene therapy that can be used for the disclosed methods is not limited. Rather, the gene therapy needs only to be capable of providing a functioning copy of SLC16A8 or a nucleic acid that otherwise encodes a functioning SLC16A8 protein. Suitable forms of gene therapy include, but are not limited to, a plasmid, a viral vector, a bacterial vector, a mRNA, a CRISPR/Cas gene editing system, a zinc finger nuclease (ZFN) gene editing system, a transcription activator-like effector nuclease (TALEN) gene editing system, and a patient-derived cellular gene therapy. Each of the foregoing categories of gene therapies is described in more detail below, and those skilled in the art will understand that the following descriptions are provided for illustration only and do not limit the scope of possible ways in which the gene therapy could be prepared or formulated.

A. Plasmid expression vectors

[0080] Plasmid DNA expression vectors are fundamental to all forms of non-viral gene transfer. Plasmid vectors can, in some instances, possess many advantages over other types of gene therapy in terms of safety and ease of use, and many clinical studies have been performed using plasmids. Plasmids are a circular, double-stranded DNA molecule varying in size from <1000 to >200 000 bp, and they may comprise an origin of replication, a promoter, one or more multiple cloning sites, a transgene, and, optionally, a selection marker. In some embodiments, a plasmid used for the disclosed methods may comprise only one nucleic acid sequence encoding a SLC16A8 protein. In some embodiments, a plasmid used for the disclosed methods may be polycistronic and encode multiple proteins.

[0081] Plasmid vectors (and non-viral vector, in general) tend to have a low immunogenicity and therefore may be suitable for the disclosed methods. Moreover, the modular nature of plasmids also allows for straightforward molecular cloning, making them easy to manipulate and design for therapeutic use. Plasmid vectors have been pursued in clinical trials for treating diseases and conditions including, but not limited to, cancer, HIV, heart disease, hepatitis B and C, diabetes, asthma, and others.

[0082] In some embodiments, the plasmid may comprise an origin of replication, a promoter, one or more multiple cloning sites, or a combination thereof. In some embodiments, the plasmid comprises at least one gene encoding a functioning SLC16A8 protein.

[0083] In some embodiments, the plasmid may be selected from an operator repressor titration plasmid (pORT), a conditional origin of replication plasmid (pCOR), plasmid free of antibiotic resistance (pF AR), or other known forms of therapeutic plasmids (Hardee et al., Genes, 2017, 8(2):65, which is incorporated herein by reference).

B. Viral vectors

[0084] Established viral vector systems that are suitable for the disclosed methods include, but are not limited to, adeno-associated viruses (AAVs), adenoviruses, and retroviruses (e.g., lentiviruses). Each of these systems have proven capacity to introduce genetic material into mammalian cells, and other system are known. In some embodiments, the viral vector is a vector of adenovirus, adeno-associated virus (AAV), alphavirus, flavivirus, herpes simplex virus, lentivirus, measles virus, picornavirus, poxvirus, retrovirus, or rhabdovirus. In some embodiments, the viral vector is a recombinant viral vector.

[0085] AAV vectors can include, but are not limited to, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrhlO, PHP.B, PHPeB, and PHP.S. Adenovirus vectors may be conditionally replicating or they may be “gutless” or “helper-dependent,” in which all viral sequences are deleted, except for the inverted terminal repeats (ITRs) and the packaging signal. Suitable retroviral vectors include lentiviral vectors, such as first, second, or third generation lentiviral vectors.

[0086] In some embodiments, the viral vector may be encoded by DNA. In some embodiments, the viral vector may be encoded by RNA, optionally viral RNA. The viral vector can be single stranded or double stranded. In some embodiments, the structural format of the viral vector (i.e., DNA or RNA, single stranded or double stranded) can be determined by those skilled in the art. For example, a lentiviral vector comprises a single stranded RNA genome. An AAV vector comprises either a single stranded DNA vector genome or a duplex DNA vector genome, which may depend on the size of the vector genome and/or vector genome design. When a lentiviral vector genome is reverse transcribed during transduction, the RNA sequence is converted into the corresponding DNA sequence.

[0087] AAV vectors may be particularly attractive for the disclosed methods of treatment and prevention. Advances in developing clinically desirable AAV capsids, optimizing genome designs and harnessing revolutionary biotechnologies have contributed substantially to the growth of the gene therapy field. Various different suitable AAV platforms are known in the art and can be utilized in the disclosed methods. See Wang et al., Nature Reviews Drug Discovery, 2019, 18:358- 378, which is incorporated herein by reference.

[0088] For example, in some embodiments an AAV may be selected from the group consisting of: AAV2, AAV5, AAV6, AAV7, AAV8, AAV9, AAV6.2, AAVrh.64Rl, AAVhu.37, AAVrh.8, AAVrh.32.33, AAV3B, and LK03. The type of AAV gene therapy vector used is not particularly limited, and may include AAVs from various serotypes, as well as recombinant or chimeric AAVs.

[0089] AAV gene therapy vectors may encode multiple components (e.g., capsid proteins, ITRs, etc.), which may be the same or different serotypes, and the vectors may encode one or more copies or variants of a gene (or genes) encoding a SLC16A8 protein.

[0090] AAV gene therapy vectors may comprise an AAV capsid and a polynucleotide. The polynucleotide may encode a therapeutic protein (i.e., SLC16A8 . The serotype of the AAV gene therapy vector is not particularly limited and may include, but is not limited to, AAV serotype 1 (AAV1), AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11. In some embodiments, the AAV is chimeric, meaning it comprises components from at least two AAV serotypes, such as the inverted terminal repeats (ITRs) of an AAV2 and the capsid protein of an AAV5. In some embodiments, gene therapy may comprise administration of a plurality of AAV gene therapy vectors, and the vectors may be the same or different serotypes. [0091] In some embodiments, the AAV capsid is not a wild-type capsid but is a recombinant AAV (rAAV), such as a rAAV2/5, which comprises at least a portion of AAV2 and AAV5. For example, the VP1 capsid protein may consist of a hybrid amino acid sequence between AAV2 and AAV5, whereas the VP2 and VP3 capsids may be derived from the AAV5 serotype (e.g., Urabe et al., J Virol., 2006, 80(4): 1874-85, which is incorporated herein by reference). In some embodiments, the AAV is a chimeric AAV (AAV^ch), such as a chimeric serotype 1 (AAVl^ch), serotype 2 (AAV2^ch), serotype 3 (AAV3^ch), serotype 4 (AAV4^ch), serotype 5 (AAV5^ch), serotype 6 (AAV6^ch), serotype 7 (AAV7^ch), serotype 8 (AAV8^ch), serotype 9 (AAV9^ch), serotype 10 (AAV10^ch), serotype 11 (AAVl l^ch), serotype 12 (AAV12^ch), or serotype 13 (AAV13^ch).

[0092] When a plurality of AAV gene therapy vectors are administered to a subject, at least two of the plurality of AAV gene therapy vectors may be the same type of AAVs, while in some embodiments, at least two of the plurality of AAV gene therapy vectors may be different types of AAVs.

C. Bacterial Vectors

[0093] Bacteria can be used for gene therapy via two strategies - either by transfection of eukaryotic host cells using bacteria (bactofection) or by alternative gene therapy that does not alter the host genome, but uses the prokaryotic expression system, which can be controlled or stopped from outside. While bactofection is particularly suited for the disclosed methods, an alternative gene therapy is suitable for in situ delivery of proteins and treatment with intracellular bactochondria.

[0094] Various suitable bacterial vectors are known in the art and can be used for the purposes of the disclosed methods. See Celec and Gardik, Front. Biosci., 2017, 22(l):81-95, which is incorporated herein by reference. Bacterial may be modified to deliver a therapeutic gene, such as SLC16A8. Suitable bacteria include, but are not limited to, Salmonella typhi, Salmonella typhimurium, Salmonella choleraesuis, Salmonella enteritidis, Escherichia coli (e.g., K-12 0157:H7), Shigella dysenteriae, Shigella flexneri, Shigella boydii, Shigella sonnei, Yersinia pestis, Yersinia pseudotuberculosis, or Yersina enter ocolitica. [0095] Such bacteria may comprise an expression cassette that encodes an SLC16A8 protein under the control of a promoter. The promoter may, optionally, comprise an inducer binding site. In some embodiments, the promoter may be selected from the group including, but are not limited to, EFla, PGK1 (human or mouse), Ubc, human beta actin, CAG, TRE, UAS, Ac5, Polyhedrin, CaMKIIa, GALI and GAL10 (either independently or together), TEF1, GDS, ADH1, CaMV35S, Ubi, Hl, U6, EF- la promoter, CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein-I..

D. mRNAs

[0096] The use of mRNAs as gene therapy has also been established. In general, the mRNA of interest (e.g., an mRNA encoding a functioning SLC16A8 protein) is formulated with lipids (e.g., ionizable or cationic lipids) to protect and stabilize the mRNA. Often, the mRNA and lipids are formulated as lipid nanoparticles (LNPs), and various suitable LNPs are known in the art (Hou et al., Nature Reviews Materials, 2021, 6: 1078-1094; Damase et al, Front. Bioeng. Biotechnol., 2021, 18 March, both of which are incorporated herein by reference).

[0097] In some embodiments, a mRNA gene therapy may comprise additional elements beyond the therapeutic gene sequence (i.e., a sequence encoding SEQ ID NO: 3 or an amino acid sequence comprising at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity thereto. For example, the mRNA may comprise 3’ and/or 5’ untranslated regions, a 5’ cap, a 3’ polyA tail, or a combination thereof.

E. Gene editing platforms and enzymes

[0098] Gene editing platforms or enzymes, such as CRISPR/Cas gene editing systems, zinc finger nuclease (ZFN) gene editing systems, and transcription activator-like effector nuclease (TALEN) gene editing systems are also suitable for the disclosed methods. These systems rely on targeted enzymatic excision of a sequence within the genome and replacement with a target sequence (e.g., nucleic acid sequence encoding a functioning SLC16A8 protein). For example, CRISPR/Cas, ZFN, or TALEN could be used to replace a non-functioning SLC16A8 gene (e.g., one comprising a rs77968014 SNP risk allele or another mutation causing a loss of expression or function) with a nucleic acid sequence encoding a functioning SLC16A8 protein.

[0099] Those skilled in the art will understand that the dose and route of delivery will vary depending on the type of gene therapy being used. The foregoing gene therapies (e.g., plasmids, viral vectors, bacterial vectors, mRNA, CRISPR/Cas, ZFN, and TALEN) may be administered directly into a subject’s eye. For example, administration may comprise subretinal, intravitreal, or suprachoroidal injection. Alternatively, in some embodiments, the gene therapy may be administered systemically, such as a subcutaneous, intramuscular, intravenous, or intraperitoneal injection. In some embodiments, the methods disclosed herein relate to a gene therapy for treating or preventing AMD using a CRISPR-Cas system. In some embodiments, the CRISPR-Cas system comprises a Class-2 Cas protein and a guide RNA. A Class-2 Cas protein refers to a single-chain polypeptide with RNA-guided DNA binding activity.

[0100] In some embodiments, the Class-2 Cas proteins is a Type-II Cas protein such as Cas9. See Cong et al., Science, 339:819-822 (2013) and Mali et al., Science, 339:823-826 (2013), each of which is incorporated herein by reference in its entirety.

[0101] In some embodiments, the Class-2 Cas protein is a Type-V Cas protein such as Casl2a/Cpfl, Casl2b/C2cl, Casl2c/C2c3, Casl2d/CasY, Casl2e/CasX, Casl2f/Casl4, Casl2i, Casl2j, or Cas . See Shmakov et al., Mol. Cell, 60:385-397 (2015); Makarova et al., Nature Reviews Microbiology, 18:67-83 (2020); Zetsche et al., Cell, 163:1-13 (2015); Strecker et al., Nature Communications, 10:212 (2019); Burstein et al., Nature, 542:237-241 (2017); Pausch et al., Science, 369:333-337 (2020); and Xu et al., Mol. Cell, 81(20):4333-4345 (2021), each of which is incorporated herein by reference in its entirety.

[0102] In some embodiments, the Class-2 Cas protein is a nuclease capable of cleaving both strands of a target DNA (e.g., wild-type SpCas9). In some embodiments, the Class-2 Cas protein is a nickases capable of cleaving one strand of a target DNA (e.g., SpCas9 D10A, H840A or N863A mutants). In some embodiments, the Class-2 Cas protein is a catalytically inactive Cas protein substantially lacking DNA cleavage activity (e.g., SpCas9 D10A+H840A or D10A+N863A mutants).

[0103] The guide RNA is capable of forming a CRISPR complex with the Class-2 Cas protein. The guide RNA comprises a guide sequence (also called a spacer) that is complementary to a target sequence within a target DNA and functions to direct the CRISPR-complex to the target DNA for binding or editing (e.g., cleavage). A guide sequence can be 20 base pairs in length, e.g., in the case of SpCas9 and related Cas9 homologs/orthologs. Shorter or longer sequences can also be used as guides, e.g., 15-, 16-, 17-, 18-, 19-, 21-, 22-, 23-, 24-, or 25-nucleotides in length. In some embodiments, the degree of complementarity or identity between a guide sequence and its corresponding target sequence may be about or at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the guide sequence and the target sequence may be 100% complementary or identical over a region of at least 15, 16, 17, 18, 19, or 20 contiguous nucleotides. In other embodiments, the guide sequence and the target sequence may contain at least one mismatch. For example, the guide sequence and the target sequence may contain 1, 2, 3, or 4 mismatches, where the total length of the target sequence is at least 17, 18, 19, 20 or more base pairs. In some embodiments, the guide sequence and the target sequence may contain 1-4 mismatches where the guide sequence comprises at least 17, 18, 19, 20 or more nucleotides. In some embodiments, the guide sequence and the target sequence may contain 1, 2, 3, or 4 mismatches where the guide sequence comprises 20 nucleotides.

[0104] In some embodiments, the guide RNA is a dual-molecule guide RNA, such as a crRNA in combination with a tracrRNA. Examples of dual-molecule guide RNAs are disclosed in Cong et al., Science, 339:819-822 (2013); Strecker et al., Nature Communications, 10:212 (2019). In some embodiments, the guide RNA is a single-molecule guide RNA, such as a Cas9 sgRNA engineered by artificially fusing all or parts of a crRNA and a tracrRNA. Examples of singlemolecule guide RNAs are disclosed in Mali et al., Science, 339:823-826 (2013); Zetsche et al., Cell, 163: 1-13 (2015); and Strecker et al., Nature Communications, 10:212 (2019). [0105] In some embodiments, the CRISPR-Cas system is conjugated or linked to a heterologous protein domain to acquire additional functionality. In some embodiments, the CRISPR-Cas system is a transcriptional activator or repressor comprising a Class-2 Cas protein conjugated or linked to a transcriptional activator or repressor domain. See Shalem et al., Nature Reviews Genetics, 16:299-311 (2015), which is incorporated herein by reference in its entirety.

[0106] In some embodiments, the CRISPR-Cas system is a base editor comprising a Class-2 Cas protein conjugated or linked to a deaminase. See Gaudelli et al., Nature, 551 :464-471 (2017) and Li et al., Nature Biotechnology, 36:324-327 (2018), each which is incorporated herein by reference in its entirety. In some embodiments, the base editor is engineered to convert a SLC16A8 risk allele (e.g., G at rs77968014) to a major allele (e.g., C at rs77968014).

[0107] In some embodiments, the CRISPR-Cas system is a prime editor comprising a Class-2 Cas protein conjugated or linked to a reverse transcriptase. See Anzalone et al., Nature, 576: 149- 157 (2019), which is incorporated herein by reference in its entirety. In some embodiments, the prime editor is engineered to convert a SLC16A8 risk allele (e.g., G at rs77968014) to a major allele (e.g., C at rs77968014).

[0108] In some embodiments, the CRISPR-Cas system further comprises a repair template polynucleotide for homology-directed repair. In some embodiments, the repair template polynucleotide is engineered to convert a SLC16A8 risk allele (e.g., G at rs77968014) to a major allele (e.g., C at rs77968014) based on homology-directed repair.

[0109] In some embodiments, the CRISPR-Cas system is packaged in a viral vector for delivery to a patient, such as a lentiviral, retroviral, or adeno-associated viral (AAV) vector. In some embodiments, the AAV vector comprises a DNA encoding a Class-2 Cas protein and a gRNA. See Maeder et al., Nature Medicine, 25:229-233 (2019), which is incorporated herein by reference in its entirety.

[0110] In some embodiments, the CRISPR-Cas system is packaged in a lipid nanoparticle (LNP) for delivery to a patient. In some embodiments, the LNP comprises an mRNA encoding a Class-2 Cas protein, and a gRNA. See Gillmore et al., N. Engl. J. Med., 385:493-502 (2021), which is incorporated herein by reference in its entirety.

[OHl] In some embodiments, the CRISPR-Cas system is packaged in a viral-like particle (VLP) for delivery to the patient. In some embodiments, the VLP comprises a ribonucleoprotein (RNP) formed by a Class-2 Cas protein and a gRNA. See Banskota et al., Cell, 185:250-265 (2022), which is incorporated herein by reference in its entirety.

F. Patient-derived cellular gene therapy

[0112] Patient-derived cellular gene therapy generally comprise methods in which cells are removed from a patient’s body, genetically modified, and then returned to the patient. Such therapies may rely on viral vectors, CRISPR/Cas, ZFN, TALEN, or plasmids to effectuate this form of ex vivo gene editing. For example, one or more RPE cells could be removed from a subject’s eye, transfected with a viral vector encoding a functioning SLC16A8 protein, and then returned to the subject’s eye.

G. Administration, Dosing, and Patient Selection

[0113] Some forms of gene therapy may require only a single administration of the therapeutic, while other may require multiple rounds of administration spread out over a designated dosing schedule. Thus, in some embodiments, the gene therapy may be administered once. In some embodiments, the gene therapy may be administered 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more times. In some embodiment requiring multiple administrations, the administrations may be separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 days; 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 weeks; 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 months; or 1, 2, 3, 4, or 5 years. In some embodiments, the gene therapy may be administered daily, every other day, weekly, bi-weekly, once every three week, monthly, once every other month, once every three months, or once every six months.

[0114] While the present disclosure establishes that rs77968014 is a SNP with a risk allele that can drive development of AMD, this SNP may not be present at all and the subject may instead have another SNP, mutation, or alteration that result in a loss of function of SLC16A8 or a decrease in expression of SLC16A8. Additionally, the subject may possess a rs200624792 SNP in SLC16A8, a rsl 13748161 SNP in SLC16A8, or a combination thereof. Additionally, the subject may possess a mutation or SNP in one of more genes selected from CFI, CFH, CFHR5, AAED1, RGS7, MAP IS, and OSGIN2.

[0115] For the purposes of the disclosed methods, the gene therapy of choice for a given method is generally administered in a therapeutically effective amount or a prophylactically effect amount, as needed based on the method and the subject being treated. Those skilled in the art will recognize that the specific dose may vary based on the type of gene therapy being administered, the route of administration, the timing of administration, the severity of the subject’s AMD, the subject’s overall health, the subject’s age, the subject’s weight, and other such factors.

[0116] For the purposes of the disclosed methods of treatment and prevention, any of these methods may further comprise an optional patient screening/selection step in which the subject (e.g., a subject that has AMD, is suspected of having AMD, is at risk of developing AMD, or has a predisposition for developing AMD) may undergo a screening to determine whether the subject has a SNP (e.g., rs77968014), risk allele, or other mutation or alteration that results in a loss of function or decreased expression of the SLC16A8 protein. Such screenings may be genetic screenings, such as gene sequencing of SLC16A8, and various means of determining SNP and mutation status are known in the art. If the subject possesses a SNP, mutation, or alteration that results in a loss of function or decreased expression of the SLC16A8 protein, then the subject may be selected for any one of the disclosed methods. Thus, in some embodiments, any of the disclosed methods of treatment or prevention may further comprise a step of selecting a patient for treatment with a gene therapy after determining that the subject possesses a SNP, mutation, or alteration that results in a loss of function or decreased expression of the SLC16A8 protein. In some embodiments, the SNP may be rs77968014.

[0117] A subject who may undergo screening to determine whether the subject has a SNP (e.g., rs77968014), risk allele, or other mutation or alteration that results in a loss of function or decreased expression of the SLC16A8 protein can be a subject who has AMD, is suspected of having AMD, is at risk of developing AMD, or has a predisposition for developing AMD but has not yet developed AMD.

IV. Diagnosis and Prognosis of AMD

[0118] The data provided in this disclosure (see Examples 1 and 2) indicate that SNPs, mutations, or alterations in SLC16A8 that lead to a loss of function or decreased expression of the SLC16A8 protein are likely drivers of AMD development and/or progression. As a result, SLC16A8 not only serves as a therapeutic target, as indicated above, it may also serve as a marker for diagnosing AMD and predicting disease development and suitability for treatment.

[0119] In one aspect, the present disclosed provides methods of diagnosing age-related macular degeneration (AMD) or a predisposition to developing AMD in a subject. The disclosed methods of diagnosing age-related macular degeneration (AMD) or a predisposition to developing AMD in a subject comprise (i) obtaining a biological sample comprising genomic DNA from a subject, and (ii) detecting the presence or absence of a rs77968014 single nucleotide polymorphism (SNP) in SLC16A8, wherein the presence of the rs77968014 SNP risk allele indicates that the subject has AMD or a predisposition to develop AMD. Additionally or alternatively, the disclosed methods of diagnosing age-related macular degeneration (AMD) or a predisposition to developing AMD in a subject may comprise more broadly (i) obtaining a biological sample comprising genomic DNA from a subject, and (ii) detecting the presence or absence of a SNP or mutation in SLC16A8 that causes a loss of function or decreased expression of a SLC16A8 protein, wherein the presence of the SNP or mutation indicates that the subject has AMD or a predisposition to develop AMD.

[0120] In one aspect, the present disclosed provides methods of detecting the presence or absence of a rs77968014 single nucleotide polymorphism (SNP) in SLC16A8. The disclosed methods comprise (i) obtaining a biological sample comprising genomic DNA from a subject having or suspected of having age-related macular degeneration (AMD), and (ii) detecting the presence or absence of the rs77968014 SNP in the biological sample.

[0121] In one aspect, the present disclosure provides methods of identifying a subject at risk for developing Age-Related Macular Degeneration (AMD), and/or at risk of rapid progression of AMD, and/or diagnosing a predisposition to developing AMD. In some embodiments, methods for identifying a subject at risk for developing Age-Related Macular Degeneration (AMD), and/or at risk of rapid progression of AMD, and/or diagnosing a predisposition to developing AMD, can include detecting in a biological sample obtained from the subject the presence or absence of a rs77968014 variant in an SLC16A8 gene, wherein the presence of the rs77968014 variant indicates that the subj ect has AMD or has a predisposition to develop AMD. In some embodiments, methods for identifying a subject at risk for developing Age-Related Macular Degeneration (AMD), and/or at risk of rapid progression of AMD, and/or diagnosing a predisposition to developing AMD, can include obtaining a biological sample comprising genomic DNA from a subject, and detecting in the biological sample the presence or absence of a rs77968014 variant in an SLC16A8 gene, wherein the presence of the rs77968014 variant indicates that the subject has AMD or has a predisposition to develop AMD.

[0122] The subject can be a subject who has AMD, is suspected of having AMD, is at risk of developing AMD, or has a predisposition for developing AMD but has not yet developed AMD. The subject can be a subject who has a family history of AMD, or no family history of AMD.

[0123] The biological sample used for the disclosed methods may vary. For example, the biological sample may be blood, plasma, or serum. Alternatively, the biological sample may be obtained from the subject’s eye, such as fluid or tissue from the eye. Samples from the eye may be obtained by methods such as fine needle biopsy and other procedures known in the art.

[0124] The disclosed methods are also amendable to various established protocols for detecting the presence or absence of a SNP or mutation. For example, detecting the presence or absence of a SNP, such as rs77968014, may comprises dynamic allele-specific hybridization, molecular beacons, SNP microarray analysis, gene chip analysis, restriction fragment length polymorphism analysis, flap endonuclease analysis, 5’-nuclease analysis, oligonucleotide ligation assay, single strand conformation polymorphism analysis, temperature gradient gel electrophoresis, capillary electrophoresis, reversed-phase high performance liquid chromatography (HPLC) detection, denaturing HPLC, high-resolution melting analysis, DNA mismatch-binding protein analysis, SNPlex analysis, surveyor nuclease assay, or sequencing.

[0125] Additionally, the disclosed methods of diagnosing, predicting, and detecting are not solely limited to the detection of rs77968014. Rather, further SNPs that cause SNP to the SLC16A8 (e.g., further splice variants, missense, nonsense, etc.) may be of interest and can be detected concurrently. Thus, the disclosed methods may further comprise detecting the presence or absence of additional SNPs beyond rs77968014. For example, in some embodiments, the disclosed methods may further comprise detecting the presence or absence of a rs200624792 SNP in SLC16A8, a rsl 13748161 SNP in SLC16A8, or a combination thereof.

[0126] The disclosed methods of diagnosing, predicting, and detecting are likewise not limited to detecting only aberrations in SLC16A8. The disclosed methods may further comprise detecting the presence or absence of a mutation or SNP in one of more genes that may also be associated with AMD, such as one or more genes selected from CFI, CFH, CFHR5, AAED1, RGS7, MARIS, and OSGIN2.

[0127] As indicated above, the presence of a SNP or mutation that causes a loss of function or decreased expression of the SLC16A8 protein, including a rs77968014 risk allele, can serve as a biomarker for selecting a subject that is likely to respond well to the disclosed gene therapies. Thus, any of the foregoing methods of diagnosis, prognosis, or detection may further comprising, when a SNP or mutation that causes a loss of function or decreased expression of the SLC16A8 protein is present, administering to the subject a gene therapy that provides a wild-type copy of a nucleic acid encoding SLC16A8 to treat or prevent AMD. In some embodiments, the methods may further comprising, when the rs77968014 SNP risk allele is present, administering to the subject a gene therapy that provides a wild-type copy of a nucleic acid encoding SLC16A8 to treat or prevent AMD. Any of the foregoing methods of diagnosis, prognosis, detection, or identifying may further comprise, when the variant is present, administering to the subject a therapy to treat, prevent, or slow the progression of AMD. In some embodiments, the therapy can include a gene therapy and/or an injection (e.g., an injection into the eye) of a Vascular Endothelial Growth Factor (VEGF) inhibitor, such as Ranibizumab. The therapy can be administered at any stage of AMD disease progression. The therapy can be administered before the onset of AMD or after the onset of AMD. In any such method, the AMD disease onset and/or progression can be slowed by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%, as measured by any pharmaceutically acceptable method, such as by clinical evaluation. [0128] In some embodiments, early diagnosis of AMD results in initiating AMD treatment, thereby improving the subject’s quality of life as compared to the quality of life expected in the absence of treatment. Improvement in quality of life can include, without limitation, delaying the need for additional therapeutic interventions, preventing or reducing the need for additional therapeutic interventions, and/or reversing, halting, or reducing the rate of vision loss experienced by the subject.

[0129] The following examples are given to illustrate the present disclosure. It should be understood that the invention is not to be limited to the specific conditions or details described in these examples.

EXAMPLES

Example 1 - Whole genome sequencing for rare variants and imaging analyses in model organisms identify SLC16A8 as a significant contributor for AMD risk

[0130] Purpose

[0131] Age-related macular degeneration (AMD) is a complex neurodegenerative disease commonly afflicting the elderly and is one of the leading causes of visual impairment worldwide. Polygenic predisposition to AMD enables strategies to define the complex pathogenesis of this disease and identify therapeutic targets. This example combined human genetics and in vivo functional studies to elucidate genes and pathways implicated in AMD.

[0132] Results

[0133] WGS for RV burden tests pinpointed multiple genes, including the top hits for complement factor I (CFI; P=3.13E-14; OR=4.57), complement factor H (CFH; P=2.14E-06; OR=2.05), and solute carrier family 16 member 8 (SLC16A8; / .50E-05; OR=1.78). Based largely on functional analyses for gene expression in the human eye and Drosophila RNAi knockdowns, further experimentation was focused on the proton-coupled monocarboxylate transporter SLC16A8 which is expressed only in the retinal pigment epithelia (RPE) of humans and rodents. Knockdown of the Drosophila ortholog silnoon (sin) led to progressive loss of retinal homeostasis in the adult fly, as visualized by changes in photoreceptor EGFP expression patterns at day 14 (95% mean loss of DPP), but not by day 1, 7, or in comparison to negative controls (n=10 for each line and time point; reproduced in two assays).

[0134] Human Genetics Studies'. Human genetics studies relied on the population described in Table 1. Briefly, population demographics for whole genome sequenced (WGS) included advanced AMD cases and non-AMD diseased controls of European ancestry. WGS were obtained from patients in various Genentech/Roche and external clinical trials including: AREDS1/2, CHROMA, DAWN, HARBOR, LAMP A, MAHALO, PROXIMA A/B, and SPECTRI.

[0135] Certain human genes were associated with AMD (P<5.0E-04) due to predicted nonsense and/or missense mediated loss of function by rare variant burden tests. The three most significant p-value genes observed in this study were consistent with earlier studies relating to genes involved in AMD.

[0136] Meta-analyses of combined data sets for shared SLC16A8 high impact protein altering variants identify rs77968014 donor splice-site intronic variant as the most significant driver (p-val = 2.76E-08) for SLC16A8 signal in rare variant burden tests. rs77968014 case frequency (0.0142) and control frequency (0.0076).

[0137] Drosophila Orthologs and Imaging'. To determine the impact of loss of function of identified genes associated with AMD, drosophila orthologs were identified (see Table 4) and knockdown of these genes was performed. Drosophila fluorescent deep pseudopupil (DPP) and optic neutralization of the cornea (ONC) imaging of photoreceptors were used for AMD associated RNAi screenings of orthologous genes. Only photoreceptors #1 - 6 express Rhl (rhodopsin /).

See FIG. 1

[0138] RNAi knockdown (vl 09464) of the SLC16A8 Drosophila ortholog sin (silnoon) led to progressive changes in photoreceptor EGFP expression patterns by day 14. (95% mean loss of DPP), but not by day 1, 7, or in comparison to negative controls (n=10 for each line and time point). See FIGs. 2A and 2B. No significant changes were observed following knockdown for other orthologous genes of interest associated with AMD risk (data not shown). This was a significant finding, as it indicated that some genes that were thought to play a role in the development of AMD may not, in fact, by causative. [0139] Human Eye Expression'. Next, expression of SLC16A8 was confirmed to be highly selective for RPE cells. Briefly, single cell RNA-NucSeq and bulk RNA-Seq in controls were used to assess both macular and non-macular tissues. As shown in FIGs. 3A and 3B, SLC16A8 was confirmed at high levels in both macular and non-macular RPE, but not in the retina.

[0140] Mouse Model'. Sic 16a8 (Mct4) loss of function was assessed in C57BL/6J mice, and knockout of this gene led to significant functional electroretinogram (ERG) a/b/c-wave defects at 10 weeks of age, as shown in FIG. 4.

[0141] The a-wave reflects the general physiological health of the photoreceptors in the outer retina, while the b-wave reflects the health of the bipolar cells of the retina. The c-wave is indicative of RPE cell function. The observed defects presented in FIG. 4 are consistent with those seen in AMD. Further constant light exposure (CLE) stress model studies can be performed between baseline and 7 days of continuous light exposure in the knockout mice as further confirmation of loss of function of SLC16A8 as a cause of AMD.

Example 2

[0142] The purpose of this example was to evaluate whether the loss of Sic 16a8 under a light induced AMD model alter retinal homeostasis leads to retinal defects.

[0143] For the experiment, 8-9 week old C57BL/6J mice comprised of Slcl6a8^wt/wt (n=8), Slcl6a8^wt/ko (N= 13), and Slcl6a8^ko/ko (N = 6). First, baseline electroretinogram (ERG measurements were taken. Next, the mice were exposed to constant light exposure (CLE) for 7 days. Finally, ERG measurements were taken post -CLE. An overview of the Slcl6a8 rodent light exposure model is shown in FIG. 5.

[0144] Following baseline ERG measurements in Slcl6a8 WT, heterozygous, and knockout mice (as shown in FIG. 4), the mice were subjected to CLE to assess whether loss of Slcl6a8 under a light-induced AMD model alters retinal homeostasis, leading to retinal defects. Mice were subjected to CLE at 100k lux for 7 days. Mice were dark-adapted overnight before the post-CLE ERG measurements were acquired. [0145] It was surprisingly observed that Slcl6a8 heterozygous mice have decreased photoreceptor function compared to WT and Slcl6a8 homozygous KO mice following CLE, as evidenced by the reduced A-wave amplitude. See FIG. 6. Moreover, it was also surprisingly observed that Slcl6a8 heterozygous mice have decreased retinal pigment epithelia (RPE) function as compared to WT and Slcl6a8 homozygous KO mice following CLE, as evidenced by the reduced C-wave amplitude. See FIG. 7. Thus, photoreceptor and RPE function were significantly more diminished in Sic 16a8 heterozygous animals as compared to WT and Slcl6a8 homozygous KO animals after exposure to light-induced retinal damage.

[0146] Conclusion: An unbiased, genome-wide analysis of rare coding variants provides support of SLC16A8 (MCT3) significantly associated with AMD, including advanced AMD, risk. These examples demonstrate that loss of function of the monocarboxylate transporter in both drosophila sin) and mice (Slcl6a8-KO and Hets) disrupts retinal homeostasis.

[0147] The data described herein also demonstrates that the loss of Slcl6a8 lactate transport disrupts retinal homeostasis in multiple organisms, and that two key cell types (RPE and PR) have functional defects with loss of Slcl 6a8 in mouse models. Finally, RPE and PR cell functions are significantly more decreased in Slcl6a8-Hets vs. WT/KO after exposure to light induced damage.

EQUIVALENTS

[0148] The present technology is not to be limited in terms of the particular embodiments described in this application, which are intended as single illustrations of individual aspects of the present technology. Many modifications and variations of this present technology can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. Functionally equivalent methods and apparatuses within the scope of the present technology, in addition to those enumerated herein, will be apparent to those skilled in the art from the foregoing descriptions. Such modifications and variations are intended to fall within the scope of the present technology. It is to be understood that this present technology is not limited to particular methods, reagents, compounds compositions or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0149] In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0150] As will be understood by one skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as “up to,” “at least,” “greater than,” “less than,” and the like, include the number recited and refer to ranges which can be subsequently broken down into subranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 cells refers to groups having 1, 2, or 3 cells. Similarly, a group having 1-5 cells refers to groups having 1, 2, 3, 4, or 5 cells, and so forth.

INCORPORATION BY REFERENCE

[0151] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and/or take precedence over any such contradictory material.

Claims

What is claimed:

1. A method of detecting the presence or absence of a rs77968014 single nucleotide polymorphism (SNP) in SLC16A8 in a subject, comprising:

(a) obtaining a biological sample comprising genomic DNA from a subject having or suspected of having age-related macular degeneration (AMD), and

(b) detecting the presence or absence of the rs77968014 SNP in the biological sample.

2. A method of diagnosing age-related macular degeneration (AMD) or a predisposition to developing AMD in a subject, comprising:

(a) obtaining a biological sample comprising genomic DNA from a subject, and

(b) detecting the presence or absence of a rs77968014 single nucleotide polymorphism (SNP) in SLC16A8 in the subject; wherein the presence of the a rs77968014 SNP risk allele indicates that the subject has AMD or a predisposition to develop AMD.

3. A method for identifying a subj ect at risk for developing Age-Related Macular Degeneration (AMD), and/or at risk of rapid progression of AMD, and/or diagnosing a predisposition to developing AMD, comprising:

(a) obtaining a biological sample comprising genomic DNA from a subject; and

(b) detecting in the biological sample the presence or absence of a rs77968014 variant in an SLC16A8 gene, wherein the presence of the rs77968014 variant indicates that the subject has AMD or has a predisposition to develop AMD.

4. The method of any one of claims 1-3, wherein the subject has been diagnosed with AMD, is suspected of having AMD, is at risk of developing AMD, or has a predisposition for developing AMD but has not yet developed AMD. The method of any one of claims 1-4, wherein the biological sample comprises the rs77968014 variant in the SLC16A8 gene. The method of any one of claims 1-4, wherein the biological sample:

(a) comprises SLC16A8 that does not comprise any SNPs or mutations; or

(b) does not comprise a nucleic acid comprising SEQ ID NO: 1 or SEQ ID NO: 2. The method of any one of claims 1-6, wherein the biological sample is obtained from the subject’s eye. The method of any one of claims 1-7, wherein the biological sample is blood, plasma, or serum. The method of any one of claims 1-8, wherein detecting the presence or absence of the rs77968014 SNP comprises dynamic allele-specific hybridization, molecular beacons, SNP microarray analysis, gene chip analysis, restriction fragment length polymorphism analysis, flap endonuclease analysis, 5’-nuclease analysis, oligonucleotide ligation assay, single strand conformation polymorphism analysis, temperature gradient gel electrophoresis, capillary electrophoresis, reversed-phase high performance liquid chromatography (HPLC) detection, denaturing HPLC, high-resolution melting analysis, DNA mismatch-binding protein analysis, SNPlex analysis, surveyor nuclease assay, or sequencing. The method of any one of claims 1-9, wherein the method results in early diagnosis of AMD in a subject, or diagnosis of risk of AMD in a subject, meaning that prior to the method the subject was not diagnosed with AMD. The method of claim 10, wherein early diagnosis of AMD results in initiating AMD treatment, thereby improving the subject’s quality of life and/or extending the subject’s life span as compared to the quality of life and/or life span expected in the absence of treatment. The method of any one of claims 1-11, further comprising, when the rs77968014 SNP is present, administering to the subject a therapy to treat, prevent, and/or slow the onset and/or progression of AMD. The method of claim 12, wherein the therapy is selected from the group consisting of:

(a) photodynamic therapy (PDT);

(b) laser surgery;

(c) one or more injections of a Vascular Endothelial Growth Factor (VEGF) inhibitor;

(d) administration of one or more angiogenesis inhibitors;

(e) administration of one or more nutritional supplements;

(f) a gene therapy; or

(g) any combination thereof. The method of claim 12, wherein the VEGF inhibitor is selected from the group consisting of faricimab-svoa (Vabysmo®), bevacizumab (Avastin®), and ranibizumab (Susvimo®). The method of claim 12, wherein the angiogenesis inhibitor is selected from the group consisting of brolucizumab (Beovu®), aflibercept (Eylea®), ranibizumab (Lucentis®), and pegaptanib sodium (Macugen®). The method of claim 12, wherein the nutritional supplement comprises a combination of about 500 mg vitamin C, about 400 IUS vitamin E, about 10 mg Luteain, about 2 mg zeaxanthin, about 80 mg zinc, and about 2 mg copper. The method of claim 12, wherein the gene therapy provides a wild-type copy of a nucleic acid encoding SLC16A8 to treat and/or prevent AMD. The method of claim 12 or 17, wherein the gene therapy is selected from a plasmid encoding wild-type human SLC16A8, a viral vector encoding wild-type human SLC16A8, a bacterial vector encoding wild-type human SLC16A8, a mRNA encoding wild-type human SLC16A8, a CRISPR/Cas gene editing system, a zinc finger nuclease (ZFN) gene editing system, a transcription activator-like effector nuclease (TALEN) gene editing system, and a patient-derived cellular gene therapy. The method of any one of claims 12-18, wherein the therapy is administered before AMD onset. The method of any one of claims 12-18, wherein the therapy is administered after AMD onset. The method of any one of claims 12-20, wherein the therapy improves the quality of life of the subject, wherein the improvement in quality of life comprises one or more of:

(a) delaying the need for additional therapeutic interventions;

(b) preventing and/or reducing the need for additional therapeutic interventions; and/or

(c) reversing, halting, and/or reducing the rate of vision loss. The method of claim 21, wherein the improvement in quality of life comprises reversing, halting, and/or reducing the rate of vision loss. The method of any one of claims 12-22, wherein the AMD disease onset and/or progression is slowed by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%, as measured by any pharmaceutically acceptable method. The method of claim 23, wherein the pharmaceutically acceptable method comprises a clinical evaluation. A method of treating and/or preventing age-related macular degeneration (AMD) in a subject, and/or restoring function of SLC16A8 in a subject, comprising administering a gene therapy to a subject, wherein the gene therapy provides to the subject a nucleic acid encoding a functioning SLC16A8 protein, and wherein the gene therapy restores, augments, and/or increases activity of a SLC16A8 protein. The method of claim 25, wherein the subject has a rs77968014 single nucleotide polymorphism (SNP) risk allele in SLC16A8, wherein the presence of the rs77968014 SNP results in a loss of function and/or decreased expression of the SLC16A8 protein. A method of selecting a subject with age-related macular degeneration (AMD) for treatment with a gene therapy that restores, augments, and/or increases activity of a SLC16A8 protein, comprising:

(a) a first step of selecting a subject comprising:

(i) obtaining a biological sample comprising genomic DNA from a subject having or suspected of having age-related macular degeneration (AMD), and

(ii) detecting in the sample the presence or absence of a rs77968014 SNP in a SLC16A8 gene, wherein the presence of rs77968014 SNP results in a loss of function and/or decreased expression of the SLC16A8 protein, and which indicates that the subject is a candidate for gene therapy; and

(b) administering a gene therapy to the subject that restores, augments, and/or increases activity of the SLC16A8 protein. A gene therapy that provides a wild-type copy of a nucleic acid encoding SLC16A8 for use in treating and/or preventing age-related macular degeneration (AMD), and/or restoring function of SLC16A8 in a subject with a rs77968014 single nucleotide polymorphism (SNP) risk allele in SLC16A8, wherein the presence of the rs77968014 SNP results in a loss of function or decreased expression of the SLC16A8 protein, and wherein the gene therapy restores, augments, and/or increases activity of a SLC16A8 protein. The gene therapy of claim 28, wherein the subject is selected for treatment with the gene therapy by determining, prior to commencement of treatment, that the subject has the rs77968014 SNP risk allele, wherein the presence of the rs77968014 SNP results in a loss of function and/or decreased expression of the SLC16A8 protein. The method or therapy of any one of claims 25-29, wherein the subject has been diagnosed with AMD, is suspected of having AMD, is at risk of developing AMD, or has a predisposition for developing AMD but has not yet developed AMD. The method or therapy of any one of claims 25-30, wherein the gene therapy provides to the subject a nucleic acid that encodes a functioning SLC16A8 protein. The method or therapy of claim 31, wherein the nucleic acid comprises a copy of SLC16A8 that does not comprise any SNPs or mutations, SEQ ID NO: 1, or SEQ ID NO: 2. The method or therapy of any one of claims 27-32, wherein the biological sample is obtained from the subject’s eye. The method or therapy of any one of claims 27-33, wherein the biological sample is blood, plasma, or serum. The method or therapy of any one of claims 27-33, wherein detecting the presence or absence of the rs77968014 comprises dynamic allele-specific hybridization, molecular beacons, SNP microarray analysis, gene chip analysis, restriction fragment length polymorphism analysis, flap endonuclease analysis, 5’-nuclease analysis, oligonucleotide ligation assay, single strand conformation polymorphism analysis, temperature gradient gel electrophoresis, capillary electrophoresis, reversed-phase high performance liquid chromatography (HPLC) detection, denaturing HPLC, high-resolution melting analysis, DNA mismatch-binding protein analysis, SNPlex analysis, surveyor nuclease assay, or sequencing. The method or therapy of any one of claims 25-35, wherein the gene therapy is selected from a plasmid encoding wild-type human SLC16A8, a viral vector encoding wild-type human SLC16A8, a bacterial vector encoding wild-type human SLC16A8, a mRNA encoding wild-type human SLC16A8, a CRISPR/Cas gene editing system, a zinc finger nuclease (ZFN) gene editing system, a transcription activator-like effector nuclease (TALEN) gene editing system, and a patient-derived cellular gene therapy. The method or therapy of any one of claims 25-36, wherein the gene therapy is administered to the subject’s eye. The method or therapy of claim 37, wherein the gene therapy is administered to the subject’s eye via subretinal, intravitreal, or suprachoroidal injection. The method or therapy of any one of claims 25-38, wherein the gene therapy is administered systemically. Use of a gene therapy that restores, augments, and/or increases activity of a SLC16A8 protein for treating and/or preventing age-related macular degeneration (AMD). Use of a gene therapy that restores, augments, and/or increases activity of a SLC16A8 protein for restoring function of SLC16A8 in a subject with a rs77968014 single nucleotide polymorphism (SNP) risk allele in SLC16A8, wherein the presence of the rs77968014 SNP results in a loss of function or decreased expression of the SLC16A8 protein. Use of a gene therapy that restores, augments, and/or increases activity of a SLC16A8 protein for the manufacture of a medicament.