Treatment of retinal degeneration

Abstract

The present disclosure provide methods and compositions for the treatment of retinal degeneration.

Description

PATENT Attorney Docket No.081906-1524446-321110PC Client Ref. No. SF-2024-195-2-PCT TREATMENT OF RETINAL DEGENERATION CROSS-REFERENCES TO RELATED APPLICATIONS

[0001] This application claims the benefit and priority to U.S. Provisional Application No. 63 / 704,776, filed on October 8, 2024, and U.S. Provisional Application No.63 / 872,053, filed on August 28, 2025, each of which is incorporated herein by reference for all purposes. BACKGROUND OF THE INVENTION

[0002] The retinal pigment epithelium (RPE) performs several essential functions to support the neural retina and the choriocapillaris and ensure healthy crosstalk between these tissues. Dysfunction of the RPE is a primary insult that triggers photoreceptor degeneration and eventual vision loss in various retina disease states, such as inherited and age-related macular degenerations (AMD). Although there are therapies available for the treatment of disorders involving retinal degeneration, there is a need for therapies that are effective in preserving healthy vision and can be more readily administered. BRIEF SUMMARY

[0003] This disclosure is based, at least in part, on the inventor’s investigations of the role of casein kinase 1 alpha (CK1α) in pathological changes in the RPE of human donors with non-neovascular “dry” AMD and mouse models of Stargardt’s disease, in particular the role of CK1α in RPE atrophy and subretinal inflammation. Experimental evaluation demonstrated that inactivation of CK1α compromises RPE health and function, and that this promotes RPE dedifferentiation and pro-inflammatory signaling in the retina. Specifically, the inventor observed that activating CK1α prevents RPE structural changes, decreases inflammation, and rescues vision loss in mouse models of Stargardt disease.

[0004] Pyrvinium pamoate is an FDA-approved anti-helminthic drug that activates CK1α. The experimental data further detailed herein demonstrated that pyrvinium restored RPE structural integrity and decreased inflammation in a mouse model of Stargardt’s disease. Further the inventors determined that the nitrogen-containing bisphosphonate zoledronic acidand pyrvinium in combination exhibited synergistic therapeutic benefits in mouse models. The following summarizes certain aspects of the present invention.

[0005] In one aspect, the disclosure provides a method of treating a retinal condition in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a nitrogen-containing bisphosphonate (NBP) and a casein kinase 1α (CK1α) agonist. In some instances, the NBP is zoledronic acid and / or the CK1α agonist is pyrvinium. In some embodiments, the retinal condition is Stargardt macular dystrophy or dry age-related macular degeneration (AMD). In some embodiments, the retinal disease is selected from the group consisting of Batten's Disease, Bietti’s crystalline dystrophy, Niemann-Pick disease Type C, Doyne’s honeycomb dystrophy, Farber disease, and Best vitelliform macular dystrophy. The NBP and CK1α agonist can be administered sequentially in either order or administered concurrently. In some embodiments, the NBP and CK1α agonist are administered as a pharmaceutical composition that comprises the therapeutically effective amount of NBP and CK1α agonist. In some instances, the pharmaceutical composition is formulated for parenteral delivery or ocular delivery. In other instances, the pharmaceutical composition is formulated for intramuscular injection, intravenous injection, or subcutaneous delivery. In some embodiments, the pharmaceutical composition is contained in a syringe for injection. In additional embodiments, the pharmaceutical composition is formulated for topical delivery to the eye. In some instances, the pharmaceutical composition is incorporated within an implant; drug-eluting device, structure, or material; polymeric drug-eluting wafer; injectable hydrogel; or implantable hydrogel scaffold. In some embodiments, the therapeutically effective amount of NBP is administered in a first pharmaceutical composition and the therapeutically effective amount of CK1α agonist is administered in a second pharmaceutical composition. In some instances, the NBP is administered at a dose of about 100 ng / kg body weight to about 50 μg per kg body weight and CK1α agonist is administered at a dose of about 100 ng / kg to about 50 μg per kg body weight. In some instances, the NBP is administered at a dose of about 100 ng / kg body weight to about 100 μg per kg body weight and CK1α agonist is administered at a dose of about 100 ng / kg to about 500 μg per kg body weight. Administration can be at various intervals, such as a frequency once per year, once per month, twice per month, weekly, twice weekly, every other day, or daily.

[0006] In a further aspect, the disclosure provides a pharmaceutical composition comprising a therapeutically effect amount of NBP and a therapeutically effective amount of a CK1α agonist. In some embodiments, the NBP is zoledronic acid and the CK1α agonist is pyrvinium. In some embodiments, the CK1α agonist is at a concentration of about 100 ng / kg to about 2.0 mg / kg body weight and the NBP is at a concentration of about 100 ng / kg to about 100 μg per kg body weight.

[0007] In further aspects, the disclosure provides a syringe containing a CK1α agonist and an NBP at a dose of CK1α agonist at a concentration of about 100 ng / kg to about 2.0 mg / kg body weight and a dose of NBP at a concentration of about 100 ng / kg to about 100 μg per kg body weight. In a further aspect, the disclosure provides an intravitreal, suprachoroidal, intracameral, or transscleral implant comprising a pharmaceutical composition comprising a CK1α agonist in amount of about 0.0001 to about 0.3 mg and an NBP in an amount of about 0.0001 to about 0.2 mg; or provides a topical ophthalmic preparation comprising a CK1α agonist in an amount of about 0.0001 to about 0.3 mg / dose and an NBP in an amount of about 0.0001 to about 0.2 mg / dose. In some embodiments, the CK1α agonist is pyrvinium and / or the NBP is zoledronic acid.

[0008] In an additional aspect, the disclosure provides a method of treating a retinal condition in a subject, the method comprising administering to the subject a therapeutically effective amount of zoledronic acid and a therapeutically effective amount of pyrvinium, wherein the zoledronic acid and pyrvinium are administered concurrently. In some instances, the zoledronic acid and pyrvinium are administered in the same pharmaceutical composition at the same doses.

[0009] In another aspect, the disclosure provides a method of treating a retinal condition in a subject, the method comprising administering to the subject a therapeutically effective amount of a CK1α agonist. In some embodiments, the CK1α agonist is pyrvinium. Alternatively, the CK1α agonist is SSTC3 or SSTC104. In some embodiments, the retinal condition is Stargardt macular dystrophy. In other embodiments, the retinal condition is dry age-related macular degeneration. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] FIG.1A-C provides bar graphs from bulk RNAseq analyses of the RPE from Abca4- / -mice treated with either vehicle or pyrvinium. Pyrvinium (PP) administered at 0.3mg / kg, 3 times / week for 8 weeks decreases inflammatory signaling (1A), upregulates autophagy (1B), and modulates the expression of genes that regulate RPE differentiation in aged Abca4- / -mice (1C).

[0011] FIG.2 provides electroretinogram (ERG) recordings showing effects of pyrvinium treatment. Pyrvinium at 0.5 mg / kg, 3 times / week for 8 weeks improves visual function in aged Abca4- / -mice. Visual function was measured using electroretinograms (ERGs). A-waves are a measure of photoreceptor function; B-waves are a measure of retinal circuitry; and C- waves represent RPE function. Data are presented as Mean ± SEM, n = 4 mice per group. *, p < 0.05; **, p < 0.01, one-way ANOVA with Dunnet’s post-test.

[0012] FIG.3A-C provides illustrative data showing effects of pyrvinium treatment. Pyrvinium restored RPE structural integrity in the Abca4- / -mouse model of Stargardt inherited macular degeneration. RPE in 18-month-old Abca4- / -mice are enlarged and dysmorphic compared to the ordered cobblestone-like appearance of RPE in wildtype mice (3A). Pyrvinium treatment restored RPE structure and decreased the number of enlarged dysmorphic cells in 18-month-old Abca4- / -mice (3B) and (3C).

[0013] FIG.4A-D provides illustrative data showing effects of pyrvinium and zoledronic acid (ZA) treatment. Concomitant administration of lower doses of pyrvinium (0.25 mg / kg) and zoledronic acid (5 μg / kg) improve mitochondrial function in Abca4- / -mice RPE. Mitochondrial energetics were measured using the Seahorse Extracellular Flux analyzer.4A, Volumetric reconstructions of TOM20-stained mitochondria in wildtype and Abca4- / -mice RPE.4B, Quantitative live imaging of mouse RPE flatmounts showed significantly less ATP in 6-mo Abca4- / -mice compared to age-matched wildtypes indicative of impaired oxidative phosphorylation (OXPHOS).4C, Bulk RNAseq analyses showed upregulation of oxidative stress pathways in 6-month-old Abca4- / -mice RPE.4D, combination therapy of lower doses of ZA and pyrvinium pamoate (PP) (combination indicated by ZAPP designation) restored mitochondrial bioenergetics in Abca4- / -mice RPE. Data are presented as Mean ± SEM, n = 3- 5 mice per group. *, p < 0.05; **, p < 0.01, ***, p < 0.001, one-way ANOVA with Dunnet’s post-test.

[0014] FIG.5 provides illustrative data showing effects of pyrvinium and zoledronic acid treatment. Concomitant administration of lower doses of pyrvinium (0.25 mg / kg) and zoledronic acid (5 μg / kg) restored visual function in aged Abca4- / -mice. Visual function was measured using electroretinograms (ERGs). a-waves are a measure of photoreceptor function,b-waves a measure of retinal circuitry, and c-waves represent RPE function. Data are presented as Mean ± SEM, n = 6 mice per group. *, p < 0.05; **, p < 0.01, ***, p < 0.001, one-way ANOVA with Dunnet’s post-test.

[0015] FIG.6 provides illustrative data showing effects of pyrvinium and zoledronic acid treatment. Concomitant administration of lower doses of pyrvinium (0.25 mg / kg) and zoledronic acid (5 μg / kg) restore visual function in aged Abca4- / -mice fed a high-calorie diet (HFD). a wave, photoreceptor function; b wave, retinal circuitry; c wave, RPE function. Mean ± SEM, n = 4 mice per group. *, p < 0.05; ***, p < 0.001, one-way ANOVA with Dunnet’s post-test.

[0016] FIG.7A-D provides in 7A and 7B, in silico modeling of interactions between TTR, RBP4, and ZA, and in 7C and 7D, representative fluorescence images of Alexa647-ZA (red) in mouse RPE flatmounts after intraperitoneal administration.7C shows live imaging and 7D shows formalin fixed tissue. The data presented in 7A and 7B showed that ZA binds TTR with a strong binding affinity (-6.7 kCal / mol) comparable to that of vitamin A, and that this occurs at a different binding site than RBP4. The imaging data presented in C and D showed significant ZA signal in the RPE, predominantly near the apical surface, after intraperitoneal administration (50 μg / kg, three times). DETAILED DESCRIPTION OF THE INVENTION

[0017] As used herein, “a”, “an”, and “the” include aspects with one member, but also include aspects with more than one member unless the context clearly dictates otherwise.

[0018] The terms “about” and “approximately” as used herein with respect to a given value generally mean a deviation from the stated value that is typically within 30% or within 20% of the stated value. For example, “about” with respect to doses or amounts is recognized by one of ordinary skill in the art to provide a pharmacological effect equivalent to that obtained from the specified dose or amount. In certain embodiments, the terms “about” and “approximately,” when used in this context, includes a dose or amount within 20%, within 15%, within 10%, or within 5%, of the specified dose or amount.

[0019] The present disclosure provides methods and compositions as detailed below for the treatment of retinal conditions. Such methods comprise administering a casein kinase 1α (CK1α) agonist, e.g., pyrvinium, to a subject in need of treatment, e.g., a subject that has a degenerative retinal disease, a symptom of a degenerative retinal disease, or is at risk, e.g.,has a genetic risk factor for developing a degenerative retinal disease. In a related embodiment, the present disclosure further provides a pharmaceutical composition comprising a CK1α activator e.g., pyrvinium, for use in a method of treating a retinal disease in a subject, wherein the method comprises administering the CK1α activator, e.g., pyrvinium, to a patient that has a retinal disorder or a symptom of a retinal disorder. In another aspect, the disclosure provides a use of a CK1α activator e.g., pyrvinium, in a method of making a medicament for the treatment of a retinal disorder.

[0020] The present disclosure additionally provides methods and compositions also as detailed below for treatment of a retinal condition. Such methods comprise administering a CK1α activator, e.g., pyrvinium, in conjunction with a nitrogen-containing bisphosphonate (NBP) that inhibits acid sphingomyelinase (ASM) and farnesyl diphosphate synthase (FDPS), e.g., zoledronic acid, to a subject in need of treatment e.g., a subject that has a degenerative retinal disease, a symptom of a degenerative retinal disease, or is at risk, e.g., has a genetic risk factor for developing a degenerative retinal disease. In a related embodiment, the present disclosure further provides a pharmaceutical composition comprising a CK1α activator e.g., pyrvinium, and an NBP, e.g., zoledronic acid, for use in a method of treating a retinal disease in a subject, wherein the method comprises administering the pharmaceutical compositions to a patient that has a retinal disorder or a symptom of a retinal disorder. In another aspect, the disclosure provides a use of a CK1α activator e.g., pyrvinium, and an NBP, e.g., zoledronic acid, in a method of making a medicament for the treatment of a retinal disorder. Zoledronic acid

[0021] Zoledronic acid, (1-Hydroxy-2-imidazol-1-yl-phosphonoethyl) phosphonic acid monohydrate, also called zoledronate, is a nitrogen-containing bisphosphonate (NBP), comprisingAmong its various biological effects, zoledronic acid is an inhibitor of acid sphingomyelinase (ASM) activity and farnesyl diphosphate synthase (FDPS) activity. Therapeutic interventions based upon these activities for the treatment of retinal disease have been described, for example, in US20150366876 and WO2023 / 220136. The term “ASM / FDPS inhibitor” as used herein thus refers to an NBP compound that inhibits both acid sphingomyelinase (ASM) activity and farnesyl diphosphate synthase (FDPS) activity. Illustrative NBP ASM / FDPS inhibitors are provided in US20150366876. In some embodiments, alendronate, risedronate, or ibandronate may be used as an NBP in the methods and compositions of the present disclosure. Casein kinase 1α agonist

[0022] In some aspects, the present disclosure provides methods of treating a retinal condition using a CK1α agonist (also referred to herein as a CK1α activator), e.g., alone or in conjunction with zoledronic acid or another NPB. In typical embodiments, the CK1α activator is pyrvinium.

[0023] Pyrvinium is a quinolinium ion that is 1-methylquinolinium substituted by dimethylamino group at position 6 and a (E)-2-(2,5-dimethyl-1-phenyl-1H-pyrrol-3- yl)ethenyl at position 2 (IUPAC name 2-[(E)-2-(2,5-dimethyl-1-phenylpyrrol-3-yl)ethenyl]- N,N,1-trimethylquinolin-1-ium-6-amine). Salts of pyrvinium can be used as anti-helminthic drugs and anti-cancer agents. Several forms of pyrvinium have been prepared with variable counter anions, such as halides, e.g., chloride, tosylate, triflate and pamoate. Pyrvinium is typically administered as a pamoate salt, pyrvinium-pamoate. The structure of pyrvinium is provided below (PubChem CID 5281035).

[0024] Other CK1α activators are known. These include SST compounds SSTC3 and SSTC104 (e.g., Shen et al, Int. J. Mol. Sci 21(16):5940, 2020 ; and Li et al., Sci Signal10(485):eaak9916, 2017), which bind to CK1 in a manner that is competitive to pyrvinium,which indicates that they bind to a similar site on CK1 .Treatment of Retinal Conditions.

[0025] The methods and compositions of the invention are employed for the treatment of a retinal condition in a subject in need thereof.

[0026] The subject may be a mammalian subject of any species in need of treatment for a retinal condition. In one embodiment, the subject is a human. In other embodiments, thesubject is a non-human primate. In some instances, the zoledronic acid and CK1 inhibitorare ministered to a mouse, rat, pig, horse, cow, dog, cat, rabbit, or other mammal.

[0027] In some embodiments, the subject has been diagnosed with a retinal condition as described herein. In some instances, the subject may be suspected of having a retinal condition. In one embodiment, the subject is at risk of developing a retinal condition, e.g., AMD or Stargardt macular dystrophy. In one embodiment, the subject is a human subject of at least 50 years, at least 55 years, at least 60 years, or at least 65 years of age. In some instances, the subject has one or more genetic markers indicative of risk of a retinal condition, or has a family history of a retinal condition. In other embodiments, the subject isa human child, teenager, or young adult, e.g., 25 years of age or younger at risk for, suspected of having, or diagnosed with a retinal disease.

[0028] In some instances, the subject is at risk, suspected of having, or suffering from Stargardt macular dystrophy, also referred to herein as Stargardt disease. In some embodiments, the subject is an adult above the age of 25 that is at risk for Stargardt macular dystrophy or is suspected of having, or has, Stargardt macular dystrophy. In some embodiments, the subject has autosomal dominant Stargardt disease. In some instances, the subject has autosomal recessive Stargardt disease.

[0029] The retinal disease or conditions can be any disease or condition of the retina wherein RPE dysfunction, RPE atrophy, microglial activation, or photoreceptor deficits are known or suspected, including conditions characterized or involving mitochondrial defects and autophagy in RPE cells, conditions involving drusen accumulations or drusen-like deposits above or beneath the RPE, complement mediated injury of mitochondria in the RPE, presence of subretinal microglia, or loss of photoreceptors or functional deficits in photoreceptors. In one embodiment, the condition to be treated is Dry AMD. In other embodiments, the condition to be treated is Stargardt macular dystrophy, for example, including autosomal dominant or autosomal recessive Stargardt disease; Doyne’s honeycomb dystrophy; and conditions involving lipid storage, autophagy, or mitochondrial defects, such as Niemann Pick Type C disease, Best vitelliform macular dystrophy; retinitis pigmentosa; and Bietti’s crystalline dystrophy.

[0030] Any of the retinal conditions noted above can be treated with pyrvinium or a combination of pyrvinium and zoledronic acid as described in the present disclosure. As used herein, “treatment” encompasses achieving any number of therapeutic effects and outcomes with respect to the selected retinal condition, including, for example: ameliorating symptoms associated of the selected retinal condition; slowing the progression of the selected retinal condition; preventing further damage to the RPE by the selected retinal condition; improving RPE function; maintaining, improving, or restoring RPE structural integrity; maintaining, improving or restoring photoreceptor health; maintaining, improving or restoring vision; or any other reduction in morbidity associated with the selected retinal condition.

[0031] In some instances, “treating” comprises preventing the onset of one or more symptoms of a retinal disease; or preventing or delaying the onset of the selected retinalcondition in an at-risk subject; maintaining normal vision or function of the RPE; or otherwise preventing onset of the selected retinal condition. “Treatment”, as used herein, additionally encompasses prevention of progression to geographic atrophy, slowing progression of geographic atrophy, reduction of number and / or frequency of other treatments. For example, reducing the number and / or frequency of treatments for AMD (such as anti- VEGF injections, complement inhibitor(s), etc.), change in total geographic atrophy (GA) area based on fundus autofluorescence, reduction in photoreceptor loss (e.g. as measured by ellipsoid zone attenuation area), change in reading speed under standard and low light, change in contrast sensitivity, change in retinal sensitivity on perimetry, change in patient- reported outcomes, change in dark adaptation, change in drusen volume, changes in pigment mottling, frank macular atrophy, bull’s eye maculopathy, or fundus flecks, changes in electroretinogram (ERG), and / or structural changes as measured by optical coherence tomography (OCT).

[0032] Treatment further encompasses any inhibition of pathological processes underlying the selected retinal condition. Exemplary treatment effects include, for example, decreasing accumulation of cholesterol, ceramide, and other lipids, improving or restoring autophagic capacity, activity, and flux in RPE cells, improving mitochondrial function, reducing the formation of drusen accumulations or like deposits, reducing complement-mediated injury of mitochondria in the RPE, preventing or rescuing RPE structural changes, dedifferentiation or atrophy, preventing or rescuing microglial migration into the subretinal space, or preventing or rescuing photoreceptor loss and functional deficits. Pharmaceutical Compositions.

[0033] The NPB, e.g., zoledronic acid and the CK1α agonist, e.g., pyrvinium, can be administered as separate pharmaceutical compositions or can be prepared in a single pharmaceutical composition. A pharmaceutical composition can comprise any other number of agents including, for example, excipients, carriers, diluents, release formulations, drug delivery or drug targeting vehicles, as well as additional active therapeutic agents. The pharmaceutical compositions of the invention may be formulated to be compatible with the selected route of administration, for example, injected systemically, e.g., by intramuscular, intravenous, or subcutaneous delivery or ocular delivery. Liquid pharmaceutical compositions include, for example, a solution suitable for parenteral administration or administration directly to the eye. The solution may be packaged for use as is, or lyophilized.

[0034] The pharmaceutical compositions may further comprise drug delivery components to facilitate delivery of the therapeutic agent(s). Drug delivery compositions encompass any moieties, materials, and the like that facilitate the delivery of the NBP, e.g., zoledronic acid and / or the CK1α agonist, e.g., pyrvinium, to the RPE. In some embodiments, the drug delivery composition facilitates targeting of the NBP, e.g., zoledronic acid to facilitate transport across the blood-retinal barrier.

[0035] In some embodiments, the delivery compositions comprise carriers to which the NBP, e.g., zoledronic acid, is conjugated, encapsulated within, or otherwise combined with to facilitate delivery to target RPE cells. Illustrative carriers include: liposomes; extracellular vesicles or synthetic mimetics thereof, such as exosomes microspheres, such as poly(lactic- co-glycolic acid) (PLGA) microspheres; and other drug delivery nanoparticles such as PLGA-PEG nanoparticles, alginate or chitosan nanoparticles, silica nanoparticles, and iron oxide nanoparticles.

[0036] In one embodiment the delivery composition comprises or is incorporated within an implant, for example, a drug-eluting implant placed within the target tissue, for example, the eye. Exemplary implants include, for example, implants made of a biodegradable material, such as PLGA, polymeric drug-eluting wafers, polymeric drug-eluting rods, injectable hydrogels, implantable hydrogel scaffolds, hydrophilic microsphere-based systems, cyclodextrin-based systems, polymeric micelle-based systems, and other drug-eluting implants known in the art. Exemplary implants for delivery of agents to the RPE include those described in: WO2012177968, “A scaffold for subretinal cell transplantation and drug delivery,” by Tao et al. Illustrative polymers and implants for ocular drug delivery are additionally described by Allyn et al, Frontiers in Medicine, 8: Article 787644, January 2022; Cao et al, Drug Discovery Today 24(8), 1694-1700 (2019).

[0037] In some embodiments, the NBP, e.g., zoledronic acid, and CK1α agonist, e.g., pyrvinium are administered by a route comprising any of intravenous delivery, intramuscular delivery, intraperitoneal delivery, or subcutaneous delivery. In some embodiments, the NBP, e.g., zoledronic acid, and CK1α agonist, e.g., pyrvinium are administered at a frequency selected from the group consisting of: once per year, once per month, twice per month, weekly, twice weekly, every other day, daily, twice per day, and thrice per day. In some embodiments, a pharmaceutical composition comprising an NBP, e.g., zoledronic acid,and / or a CK1α agonist, e.g., pyrvinium, comprises a pharmaceutical excipient, carrier, diluent, or other component of pharmaceutical compositions.

[0038] CK1α agonist, e.g., pyrvinium, and / or the NBP, e.g., zoledronic acid, can be administered in conjunction with other therapies to treat the retinal disorder. These include, for example, administration of: anti-VEGF agents (including but not limited to rolucizumab, aflibercept, ranibizumab, pegaptanib sodium, faricimab-svoa and bevacizumab), complement inhibitors (including but not limited to pegcetacoplan and avacincaptad pegol), MCO 010, deuterated retinol, emixustat, MA09 hRPE, STG-001, tinlarebant, QR1011 and REV 0100. The timing of such treatments may be determined by one of skill in the art. For example, a patient therapeutic agents may be administered any contemporaneously, sequentially, or alternating.

[0039] The pharmaceutical compositions of the invention may be formulated in any number of dosage forms. Exemplary dosage forms include: liquid solutions; suspensions, emulsions, lyophilized preparation that can be reconstituted and other dosage forms known in the art. In one embodiment the dosage form is formulated for topical application to the eye, including eye drops, ophthalmic ointments or gels. In other embodiments, the dosage form is formulated for systemic delivery, e.g., intramuscular injection. Illustrative dosing regimens

[0040] In some embodiments, the CK1α agonist, e.g., pyrvinium, is administered systemically, e.g., intramuscularly, at a dose between 0.005 and 2.0 mg / kg, at a dose between 0.1 and 1.0 mg / kg, or at a dose between 0.2 and 0.5 mg / kg for the treatment of a retinal condition. In some embodiments, the CK1α agonist, e.g., pyrvinium, is administered systemically, e.g., intramuscularly, at a dose from about 0.005 mg / kg, 0.05 mg / kg, 0.1 mg / kg, 0.5 mg / kg, 1.0 mg / kg, or 2.0 mg / kg for the treatment of a retinal condition.

[0041] In some embodiments, the CK1α agonist, e.g., pyrvinium, is administered by intravitreal, suprachoroidal, transscleral, or intracameral implant to deliver a dose of about 50 ng / day to about 200 μg / day to an eye (e.g., a dose of about 50 ng / day to about 100 μg / day, or from about 100 ng / day to about 50 μg / day to an eye). In some embodiments, the CK1α agonist, e.g., pyrvinium, is administered as an eye drop solution or suspension, or ophthalmic ointment or gel at a dose of about 50 ng / day to about 200 μg / day to an eye (e.g., a dose of about 50 ng / day to about 100 μg / day, or from about 100 ng / day to about 50 μg / day to aneye). In some embodiments, the CK1α agonist, e.g., pyrvinium, is administered by suprachoroidal injection at about 50 ng / day to about 200 μg / day to an eye (e.g., a dose of about 50 ng / day to about 100 μg / day, or from about 100 ng / day to about 50 μg / day to an eye).

[0042] In a further aspect, the disclosure provides an intravitreal implant comprising the CK1α agonist, e.g., pyrvinium, loaded with an amount of from 0.001 to 0.3 mg (e.g., from 0.005 to 0.01 mg, from 0.05 to 0.1 mg, or from 0.1 to 0.3 mg). In some embodiments, the implant is loaded with 0.005 to 2.5 mg. In some embodiments, the implant is loaded with 0.005, 0.01 mg, 0.1 mg, 0.3 mg, 0.5 mg, 1.0 mg, 2.0 mg, or 2.5 mg.

[0043] In another aspect, the disclosure provides a topical ophthalmic preparation comprising 0.001-0.2 mg / dose of a CK1α agonist, e.g., pyrvinium. In some embodiments, the ophthalmic preparation comprises 0.005-0.05 mg / dose. In some embodiments, the ophthalmic preparation comprises from about 0.001 mg / dose, 0.005 mg / dose, 0.05 mg / dose, 0.1 mg / dose, or 0.2 mg / dose.

[0044] In an additional aspect, the disclosure provides an injectable ophthalmic preparation comprising the CK1α agonist, e.g., pyrvinium, at a concentration of 1 μg / ml-10 mg / ml (e.g., 1 μg / ml-5 mg / ml, 10 μg / ml-1 mg / ml, or 100 μg / ml-1 mg / ml). In some embodiments, the injectable ophthalmic preparation comprises pyrvinium at a concentration of from 10 μg / ml-1 mg / ml (e.g., 10 μg / ml-1 mg / ml, 100 μg / ml-1 mg / ml, or 100 μg / ml-0.5 mg / ml). In some embodiments, the ophthalmic preparation comprises pyrvinium conjugated to dendrimers or formulated as nano particles.

[0045] In some embodiments, an NBP, e.g., zoledronic acid, and CK1α agonist, e.g., pyrvinium, are administered at the same time in the same pharmaceutical preparation. In some embodiments, the NBP, e.g., zoledronic acid, is administered systemically at a dose of from 10 ng / kg body weight to 1000 μg per kg body weight with the CK1α agonist, e.g., pyrvinium a dose of from about 100 ng / kg-3 mg / kg body weight.

[0046] Alternatively, NBP, e.g., zoledronic acid, and CK1α agonist, e.g., pyrvinium, can be administered sequentially, in either order.

[0047] An NBP, typically zoledronic acid, can be administered at a range of doses, including at ultra-low doses in conjunction with the CK1α agonist, e.g., pyrvinium.

[0048] In some embodiments, an NBP, e.g., zoledronic acid is administered by intravitreal implant to deliver a dose of about 1 ng / day to about 200 μg / day to an eye. In some embodiments, an NBP, e.g., zoledronic acid is administered by intravitreal implant to deliver a dose of about 1 ng / day, 10 ng / day, 100 ng / day, 1 μg / day, 100 μg / day, or 200 μg / day to an eye. In some embodiments, the NBP, e.g., zoledronic acid, is administered as an eye drop solution or suspension, or ophthalmic ointment or gel at a dose of about 1 ng / day to about 200 μg / day to an eye. In some embodiments, the NBP, e.g., zoledronic acid, is administered as an eye drop solution or suspension, or ophthalmic ointment or gel at a dose of about 1 ng / day, 10 ng / day, 100 ng / day, 1 μg / day, 100 μg / day, or 200 μg / day to an eye. In some embodiments, the NBP, e.g., zoledronic acid, is administered by suprachoroidal injection at about 1 ng / day to about 200 μg / day to an eye. In some embodiments, the NBP, e.g., zoledronic acid, is administered by suprachoroidal injection at about 1 ng / day, 10 ng / day, 100 ng / day, 1 μg / day, 100 μg / day, or 200 μg / day to an eye.

[0049] In some embodiments, the NBP, e.g., zoledronic acid, is administered systemically at a dose of from 10 ng / kg body weight to 500 μg per kg body weight, at a dose of between 1.0 and 7.0 μg per kg body weight, or at a dose of about 5.0 μg to 20 μg kg per kg body weight.

[0050] In a further aspect, the disclosure provides an intravitreal implant comprising an NBP, e.g., zoledronic acid, loaded with an amount of from 0.0001 to 0.3 mg. In some embodiments, the implant is loaded with 0.005 to 2.5 mg. In some embodiments, the implant is loaded with 0.005 mg, 0.05 mg, 0.1 mg, 0.3 mg, 0.5 mg, 1.0 mg, 2.0 mg, or 2.5 mg.

[0051] In another aspect, the disclosure provides a topical ophthalmic preparation comprising 0.001-0.2 mg / dose of an NBP, e.g., zoledronic acid. In some embodiments, the ophthalmic preparation comprises 0.005-0.05 mg / dose.

[0052] In an additional aspect, the disclosure provides an injectable ophthalmic preparation comprising an NBP, e.g., zoledronic acid, at a concentration of 1 μg / ml-10 mg / ml. In some embodiments, the injectable ophthalmic preparation comprises zoledronic acid at a concentration of from 10 μg / ml-1 mg / ml. In some embodiments, the injectable ophthalmic preparation comprises zoledronic acid at a concentration of 10 μg / ml, 50 μg / ml, 100 μg / ml, 500 μg / ml, 0.1 mg / ml, 0.5 mg / ml, or 1 mg / ml. In some embodiments, the ophthalmicpreparation comprises zoledronic acid conjugated to dendrimers or formulated as nano particles.

[0053] In some embodiments, doses for humans are based on animal doses, e.g., based on dose-scaling conversions that are frequently used in the art. (see, for example Nair & Jacob, J. Basic and Clin. Pharmac 2016; 7:27-31). Thus, for example, to convert an animal dose in mg / kg to a human equivalent dose in mg / kg, an animal dose may be divided by 12.3. Linked NBP-CK1α agonist

[0054] In some embodiments, an NBP, e.g., zoledronic acid and a CK1α agonist, e.g., pyrvinium, are linked for administration to a patient. In some instances, the linker that connects the two agents is a cleavable linker. The following discussion of types of linkages that can be employed to link the NBP and CK1α agonist is presented in the context of zoledronic acid and pyrvinium. One of skill understands, however, that such linkages can be employed to link alternative NBP and CK1α agonists for administration to a patient.

[0055] A "linker" or “linking unit”, as used herein refers to a chemical structural fragment or bond that connects compound 1 (e.g., zoledronic acid) at one end and compound 2 (e.g., pyrvinium) at the other end, thus thereby acting as a bridge to link the two compounds together. A “linker” may include spacers and amino acid units, and can be synthesized by methods known in the art. As used herein, “linking unit” or “linker” can be considered as two categories: non-cleavable linkers and cleavable linkers.

[0056] A “cleavable” linker, refers to any linker which can be cleaved physically or chemically. Examples for physical cleavage may be cleavage by light, radioactive emission or heat, while examples for chemical cleavage include cleavage by redox-reactions, hydrolysis, pH-dependent cleavage or cleavage by enzymes.

[0057] In some instances, chemically labile linkers can be selectively cleaved under particular environmental conditions, including pH, concentration of glutathione, etc. pH- sensitive linkers are typically relatively stable in the neutral environment of blood (pH 7.3- 7.5), but will be hydrolyzed in weakly acidic environments, e.g., (pH 4.5-5.0). Glutathione- sensitive linkers are also referred to as disulfide linkers. Thus, for example, drug release is based on the difference between a high glutathione concentration in the cells (in a millimolar range) and a relatively low glutathione concentration in the blood (in a micromolar range). As is the case with antibody-drug conjugates, common linkers, such as hydrazones,carbonates, acetals, and ketals can also be used in the drug conjugates of the present invention.

[0058] Enzymatically labile linkers, such as peptide linkers may also be employed. Such linkers can be employed for better control of drug release. Peptide linkers can be efficiently cleaved by cellular proteases, such as Cathepsin B or plasmin. Additional enzymatically labile linkers include glycosidase-sensitive linkers, and phosphatase cleavable linkers. Enzymatically labile linkers are widely used as cleavable linkers due to their high plasma stability, good intracellular cleavage selectivity and effectiveness.

[0059] In some instances, a linker may be attached to the drugs by functional groups independently selected from an ester bond, disulfide, thioether, amide, acylhydrazone, ether, carbamate, carbonate, carbon-carbon bond, and urea. Alternatively, the linker moiety can be attached to either the targeting moiety or the active drug by a non-cleavable group such as provided by the conjugation between a thiol and a maleimide, or between an azide and an alkyne. In some instance, the linker is independently selected from the group consisting alkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl, wherein each of the alkyl, alkenyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl groups optionally is substituted with one or more groups, each independently selected from halogen, cyano, nitro, hydroxyl, carboxyl, carbamoyl, ether, alkoxy, aryloxy, amino, amide, carbamate, alkyl, alkenyl, alkynyl, aryl, arylalkyl, cycloalkyl, heteroaryl, heterocyclyl, wherein each of the carboxyl, carbamoyl, ether, alkoxy, aryloxy, amino, amide, carbamate, alkyl, alkenyl, alkynyl, aryl, arylalkyl, cycloalkyl, heteroaryl, or heterocyclyl is optionally substituted with one or more groups, each independently selected from halogen, cyano, nitro, hydroxyl, carboxyl, carbamoyl, ether, alkoxy, aryloxy, amino, amide, carbamate, alkyl, alkenyl, alkynyl, aryl, arylalkyl, cycloalkyl, heteroaryl, and heterocyclyl.

[0060] In some embodiments the alkyl chain of the linker moiety may optionally be interrupted by one or more atoms or groups selected from–O-, -C(=O)-, -NR, -O-C(=O)-NR-, -S-, and -S-S-. For example, Compound A may be linked, via a free phosphate group to a linking moiety shown below:In some embodiments, a linking group may be used to link compound A to compound B, via a linking group L as shown below.wherein A is Zoledronic acid, L is a linking moiety as described herein, and B is Pyrvinium. In some embodiments, Zoledronic acid may be linked to the linker via a free phosphate group as shown above in the binding of Zoledronic acid to ethyl-dimethyl- aminopropylcarbodiimide (EDC) to form an intermediate as shown, which can, in turn, be coupled to B, i.e., Pyrvinium, through a functional group on the compound.

[0061] Additional linkers and method of attaching such linkers to drug are well known in the art and may be used as alternatives to the linkers described herein. Technical section Example 1. Pyrvinium modulates the expression of genes that regulate inflammation, autophagy, and differentiation in a preclinical model of Stargardt disease.

[0062] Bulk RNAseq analyses revealed that pyrvinium treatment (0.5 mg / kg i.p.3 times / week for 8 weeks) increased the expression of anti-inflammatory genes especially those that regulate NF-kB signaling in the RPE (FIG.1A). A multi-level disruption of autophagy was identified in Abca4- / -RPE (Tan et al., 2023; Toops et al., 2015). Pyrvinium treatment increased the expression of a subset of autophagy genes in Abca4- / -mice RPE (FIG.1B). Atrophy of the RPE is a key feature of both late-stage AMD and Stargardt disease. Pyrvinium decreased the expression of MMP3, which remodels the extracellular matrix to induce dedifferentiation, and increased the expression of AXIN2, PER1, and RAP1B, which inhibit Wnt / beta-catenin signaling (FIG.1C). Example 2. Pyrvinium improves photoreceptor function in the preclinical model of Stargardt disease.

[0063] Electroretinogram (ERG) recordings showed that pyrvinium treatment improved photoreceptor function in aged Abca4- / -mice compared to mice treated with vehicle alone (FIG.2). Pyrvinium improved photoreceptor function (A Wave), retinal circuitry (B Wave), and RPE function (C Wave). Example 3. Pyrvinium restores RPE structural integrity in the Abca4- / -mouse model of Stargardt inherited macular degeneration.

[0064] Imaging of RPE in 18-month-old Abca4- / -mice showed enlarged and dysmorphic RPE compared to the ordered cobblestone-like appearance of RPE in wildtype mice (FIG. 3A). Pyrvinium treatment restored RPE structure and decreased the number of enlarged dysmorphic cells in 18-month-old Abca4- / -mice (FIG.3B and 3C). Example 4. Zoledronic Acid and Pyrvinium exhibit synergistic therapeutic benefit in Stargardt disease mice.

[0065] Using the Abca4- / -mouse model of Stargardt disease, pathological accumulation of cholesterol in the RPE was shown to activate acid sphingomyelinase (ASM), the enzyme that hydrolyzes sphingomyelin to ceramide. Without being limited by theory, studies have confirmed ceramide accumulation in the RPE of human donors with dry AMD and in mice with RPE-specific deletion of Abca4- / -, a model of dry AMD. Excess ceramide compromises critical RPE homeostatic functions such as autophagy, mitochondrial energetics, and complement inhibition, leading to microglial activation and photoreceptor dysfunction. Intraperitoneal administration of the osteoporosis drug zoledronic acid, the only knownstructural inhibitor of ASM, safeguards RPE health and restores photoreceptor function in aged pigmented Abca4- / -mice. ZA was administered at 10 μg / kg for 8 weeks, which is 15- fold lower than that used for osteoporosis, because data showed that ZA also decreases RPE cholesterol by inhibiting farnesyl diphosphate synthase (FDPS) in the cholesterol biosynthetic pathway.

[0066] Although Pyrvinium improves autophagy, it does not appreciably decrease ceramide levels. Whether combining ZA and pyrvinium provided a synergistic benefit while minimizing potential side-effects was tested. To test this, half the dose of ZA and Pyrvinium each for 8 weeks was administered (5 μg / kg ZA and 0.3 mg / kg pyrvinium).

[0067] Mitochondrial injury and impaired autophagy in the RPE are features of both AMD and Stargardt disease (Ferrington et al., 2016; La Cunza et al., 2021; Tan et al., 2016; Toops et al., 2015). Using volumetric reconstructions of TOM20-stained mitochondria, many more fragmented mitochondria in Abca4- / -mice RPE compared to age-matched wildtypes were observed (La Cunza et al., 2021; FIG.4A). Quantitative live imaging of mouse RPE flatmounts showed significantly less ATP in 6-mo Abca4- / -mice compared to age-matched wildtypes indicative of impaired oxidative phosphorylation (OXPHOS) (FIG.4B). RNAseq analyses showed upregulation of oxidative stress pathways in 6-month-old Abca4- / -mice RPE (FIG.4C). The combination therapy of ZA and pyrvinium restored mitochondrial bioenergetics in Abca4- / -mice RPE as measured by the Seahorse extracellular flux analyzer (FIG.4D).

[0068] This combination therapy regimen also restored visual function in Abca4- / -mice comparable to that of wildtype mice (FIG.5) likely due to improved autophagy and mitochondrial function and decreased inflammatory signaling. Example 5. Zoledronic Acid and Pyrvinium exhibit synergistic therapeutic benefit in metabolically stressed Stargardt disease mice.

[0069] A high-calorie diet has been shown to exacerbate both Stargardt disease and AMD- related pathologies in mouse models (Landowski et al., 2019; Kim et al., 2023). Whether ZA and pyrvinium were therapeutically effective in Abca4- / -mice fed a high calorie diet (9% fat vs 4.5% in regular diet) for 12 months was tested. An 8-week treatment with the combination regimen significantly improved photoreceptor function in Abca4- / -mice fed the high calorie diet (FIG.6).

[0070] Zoledronic acid is a hydrophilic molecule that contains phosphonic acid groups, rendering it acidic. Based on studies showing remarkable efficacy of ZA in restoring RPE health and preventing vision loss in mouse models of macular degeneration, how ZA could be transported to the RPE and retina was tested. Vitamin A is delivered to the RPE by serum retinol binding protein 4 (RBP4) and transthyretin (TTR). In silico modeling of ZA binding to TTR and / or RBP4 showed that ZA binds TTR with a strong binding affinity (-6.7 kCal / mol) comparable to that of vitamin A, and that this occurs at a different binding site than RBP4 (FIGS 6A and B). To confirm delivery of ZA to the RPE, Alexa647-labeled ZA intraperitoneally to mice was administered (50 μg / kg, three times) and imaged RPE flatmounts by super-resolution imaging. Significant ZA signal in the RPE was observed (FIGS.6C and D), predominantly near the apical surface, suggesting, without being limited by theory, that ZA likely binds RPE melanosomes, which act as a sink or depot. These data suggest that ZA can function as a targeted delivery vehicle to the RPE. Example 6. Zoledronic Acid is transported to the RPE after bindoing to transthyretin (TTR) in the serum.

[0071] In silico modeling of ZA binding to TTR and / or RBP4 showed that ZA binds TTR with a strong binding affinity (-6.7 kCal / mol) comparable to that of vitamin A, and that this occurs at a different binding site than RBP4 (FIG.7A and 7B). Super-resolution live imaging (FIG.7C) and fixed imaging (FIG.7D) of Alexa647-labeled ZA (Red) in mouse RPE flatmounts after intraperitoneal administration (50 μg / kg, three times) showed significant ZA signal in the RPE, predominantly near the apical surface, suggesting, without being limited by theory, that ZA likely binds RPE melanosomes, which act as a sink or depot. Experimental Methods Mice

[0072] Wild-type (Jackson Labs; 129S1 / SvlmJ) and Abca4- / -mice (Jackson Labs; Abca4tm1Ght / J), both on an Rpe65 Leu450 background, were maintained on a 12-hour light / dark cycle with a standard diet. Mice were euthanized at specified times and eyecups processed for immunohistochemistry, live imaging, transcriptomics, or biochemical studies. All animal procedures were approved by the Institutional Animal Care and Use Committee at the University of California, San Francisco. In vivo drug treatments

[0073] Pyrvinium pamoate salt hydrate (Sigma-Aldrich P0027-10MG), was dissolved in DMSO, and then diluted in PBS to a final DMSO concentration of 0.1%. Abca4- / -mice were administered 100 μl of either vehicle or pyrvinium at a final dose of 0.5 mg / kg via intraperitoneal (IP) injection, three times per week for 3 weeks.

[0074] Zoledronic acid disodium salt tetrahydrate (ZA, Toronto Research Chemicals 165800-07-7) was dissolved in PBS.16-mo Abca4- / - mice were administered 100 μl of either vehicle or 100 μl of a solution containing ZA (final dose 5 g / kg) and pyrvinium (final dose 0.3 mg / kg) via intraperitoneal (IP) injection, three times per week for 4 weeks. Immunostaining of mouse RPE flatmounts

[0075] Mouse eyes were collected at specified times after light onset. The anterior segments of the eyes, including the lens, were removed. Four relaxing cuts were made on the remaining eyecups, which were then fixed in 4% paraformaldehyde (PFA) for 30 minutes at room temperature. Following three 5-minute washes with 1% BSA in PBS, the retinas were clipped and removed, and additional relaxing cuts were made. Each RPE flatmount was then blocked for 1 hour at room temperature in 300 L of 1% BSA in PBS with 0.1% Triton-X. Flatmounts were incubated for 3 days at 4°C with the indicated primary antibodies diluted in 1% BSA in PBS. The flatmounts were then washed three times for 10 minutes each with 1% BSA in PBS and gentle shaking, followed by overnight incubation at 4°C with AlexaFluor secondary antibodies (1:500, ThermoFisher Scientific, Waltham, MA) diluted in 1% BSA. After secondary antibody staining, the flatmounts were washed as above and stained with DAPI (Sigma-Aldrich, St. Louis, MO; D9542, 1:200 in PBS) for 15 minutes at room temperature. Finally, after three additional washes, the RPE flatmounts were mounted and sealed on clean slides with PBS and Vectashield (Vector Labs, Peterborough, UK). Flatmounts were imaged using a Nikon dual camera spinning disk confocal microscopy system equipped with a 100× / 1.49 NA oil objective (see below). Consistent laser power and exposure times were maintained for each antibody within a set of experiments. Live imaging of mouse RPE flatmounts

[0076] Mouse RPE flatmounts were labeled with Mitotracker (200 nM, 15 min) and Biotracker ATPRed (10 μM, 15 min) in recording media (1 x HBSS, 4.5 g / L glucose, 0.01 M HEPES), rinsed, and mounted onto a Warner chamber as previously described (11). Images were acquired at 37°C in Okolab humidified environmental chamber using CFI60 Apochromat TIRF 100x oil immersion objective (1.49 NA). Live imaging was performed onthe Nikon spinning disk confocal microscope equipped with: Yokogawa CSU-X1 confocal spinning disk head, Nikon Eclipse Ti2-E inverted microscope, Live-SR super-resolution module, Andor iXon Ultra 888 EMCCD camera, TI2-S-SE-E motorized stage with piezo-Z for rapid Z-stack acquisition, and a laser combiner with four solid-state lasers at 405, 488, 561, and 640 nm and the corresponding band-pass emission filter sets (Chroma) loaded on a FLI high speed filter wheel. The Live-SR is a super-resolution module that increases x-y resolution to ~ 120-140 nm, making it comparable to the resolution achieved by structured illumination microscopy (SIM). During image acquisition, care was taken to maintain the same laser power, exposure and electron-multiplying gain settings. Movies were acquired at the following rates: Movie 1 (~2 s intervals for 2 min), Movie 2 (~15 s intervals for 2 min), Movie 3 (5 s intervals for 1 min), Movie 4 (no delay (~3.7 s intervals for a z-stack) for 5 min), Movie 5 (15 s intervals for 5 min), Movie 9 (10 s intervals for 3 min). Image analysis

[0077] Images were subjected to Gaussian filtering and background subtraction prior to analysis using the same thresholds for all images. Imaris 9.9 (Bitplane) was used for surface rendering using the “Surfaces” module to determine parameters such as mean intensity, object volumes, and number of discrete objects. For quantification of mitochondrial volumes, thresholds for surface creation were guided by automatic thresholding. For timelapse analyses, tracking was enabled to track intensity changes with time, and intensity values were exported from the statistics tab. For ATP analysis, surfaces were created for the ATP channel as detailed above. The number of ATP puncta was exported from the statistics tab and normalized to Mitotracker intensity. NIS Elements (Nikon) was used for automatic deconvolution of images, generation of kymographs, and line intensity profiles. Insets for kymographs and line intensity profiles were exported from NIS Elements using the “Create View Snapshot” function. All exported data were compiled on Microsoft Excel and plotted on Prism 8 (GraphPad). RNA sequencing and analysis

[0078] Mice were sacrificed at the indicated times after light onset, and enucleated eyes were processed as detailed above. Eyecups were placed in pre-chilled RNase-free microcentrifuge tubes and kept on ice at all times. Disposable plastic pestles were used to dissociate RPE from the eyecups into sterile 1X PBS, with gentle twisting motion applied to the RPE side. RPE suspensions from both eyes of the same mouse were pooled, and lysisbuffer from the RNAqueous-Micro Total RNA Isolation Kit (Thermo Fisher, AM1931) was added immediately. Total RNA was isolated according to the manufacturer’s protocol. Cell lysates were vortexed at maximum speed for 20 seconds, followed by the addition of half- volume molecular-grade ethanol and brief vortexing. Lysates were passed through the Micro filter cartridge with 14,000xg centrifugation, followed by three washes using the provided wash solutions. Finally, RNA was eluted in the elution buffer and subjected to DNase I treatment. Samples were submitted to Novogene (Sacramento, CA) for bulk RNA sequencing using the Illumina NovaSeq6000 platform. All samples were confirmed to have RIN > 7, assessed using the RNA Nano 6000 Kit on the Bioanalyzer 2100 system (Agilent Technologies, CA). FASTQ files were processed through fastp, and paired-end clean reads were aligned to the reference genome using the Spliced Transcripts Alignment to a Reference (STAR) software. Gene-level differential expression analysis was performed using DESeq2 with the Wald test, and p-values were adjusted using the Benjamini & Hochberg (BH) procedure, which was conducted using R Studio software. Gene ontology analysis was conducted on selected genes using DAVID Bioinformatics Resources, and enrichment was determined based on the adjusted p-value and gene ratio. Data visualization was performed using the hiplot database. Mitochondrial bioenergetics

[0079] Mouse eyes were enucleated post-euthanasia and posterior eyecups containing the RPE-choroid-sclera (RPE-ch-sc) complex were isolated under a dissection microscope. RPE flatmounts were placed RPE side up in Seahorse XF24 Islet Capture Microplates (Agilent) with XF DMEM assay medium (10 mM glucose, 1 mM pyruvate, 2 mM glutamine) and secured using mesh screens. A Seahorse XFe24 sensor cartridge was hydrated overnight in the XF calibrant solution at 37°C in a non-CO2 incubator. On the day of the assay, the cartridge was loaded with the following drugs from the Seahorse XF Cell Mito Stress Test Kit - Port A: oligomycin (final concentration 1.5 μM); Port B: FCCP (final concentration 1.0 μM); Port C: rotenone / antimycin A (final concentration 0.5 μM). Oxygen consumption rates (OCR) were measured using the Seahorse XFe24 Analyzer with a mix (3 min), wait (2 min), and measure (3 min) protocol across sequential drug injections. After the assay, tissues were lysed with HNTG buffer and protein concentrations were quantified using the DC assay (Thermo Fisher Scientific). OCR values were normalized to protein content. Data analysis was performed using Wave Pro software (Agilent) and GraphPad Prism (v10.1.2).Electroretinography

[0080] Stimuli were generated by Celeris rodent electrophysiology system (Diagnosys LLC, Lowell, MA). Mice were dark adapted overnight and anesthetized intraperitoneally with ketamine (80mg / kg) and xylazine (10mg / kg) under dim red illumination. Whiskers were trimmed and a mydriatic solution composed of 5% phenylephrine and 1% tropicamide was applied to the eyes. Generous amounts of 0.3% hypromellose gel were applied to keep the eyes moist. The Celeris system was used with bright stimulators in an active / reference / ground configuration. The grounding electrode needle was placed into the skin of the animal near the tail. The reference electrode needle was placed in the cheeks of the animal and the warmingpad was set to 38 . An 8-step series was taken with 10 sweeps per step with each sweeplasting four seconds with 20 seconds between each sweep. Sweeps were averaged together for a final readout with outlier readouts removed. Brightness was set from 0.01 to 50 cd·s / m². After the recording, atipamezole was administered and mice were allowed to recover in a warm, clean cage. Wave peaks and times were determined by Espion V6 (Diagnosys) software with a-waves starting at 10ms after stimulus with a 30ms range, b-waves had a start time 25ms after stimulus with a 60ms range, and c-waves had a peak start time at 300ms with a 3,000ms range. The peak amplitudes and times from each eye were averaged together and analyzed in Prism using two-way ANOVA with Bonferroni's multiple comparisons test. Statistics

[0081] Data were analyzed using GraphPad Prism 8 (GraphPad Software, Inc., La Jolla, CA, USA) and are expressed as mean ± standard error of the mean (SEM). The normality of the data was assessed using the Shapiro-Wilk test, and homogeneity of variances was evaluated using the Brown-Forsythe test, both in GraphPad Prism 8. For data meeting these assumptions, significant differences were determined using Student’s t-test for two-group comparisons and one-way or two-way analysis of variance (ANOVA) for multigroup comparisons, followed by Tukey’s multiple comparisons test. Statistical significance was set at p < 0.05.

[0082] The embodiments illustrated and discussed in this specification are intended only to teach those skilled in the art the best way known to the inventors to make and use the invention. Nothing in this specification should be considered as limiting the scope of the present invention. All examples presented are representative and non-limiting. The above- described embodiments of the invention may be modified or varied, without departing fromthe invention, as appreciated by those skilled in the art in light of the above teachings. It is therefore to be understood that, within the scope of the claims and their equivalents, the invention may be practiced otherwise than as specifically described. All publications, patents, and patent applications cited in this specification are herein incorporated by reference as if each individual publication, patent, or patent application were specifically and individually indicated to be incorporated by reference.

Claims

WHAT IS CLAIMED IS:

1. A method of treating a retinal condition in a subject, the method comprising administering to the subject a therapeutically effective amount of a nitrogen- containing bisphosphonate (NBP) and a casein kinase 1α (CK1α) agonist.

2. The method of claim 1, wherein the NBP is zoledronic acid and / or the CK1α agonist is pyrvinium.

3. The method of claim 1 or 2, wherein the retinal condition is Stargardt macular dystrophy.

4. The method of claim 1 or 2, wherein the retinal condition is dry age- related macular degeneration.

5. The method of claim 1 or 2, wherein the retinal condition is selected from the group consisting of Batten's Disease, Bietti’s crystalline dystrophy, Niemann-Pick disease Type C, Doyne’s honeycomb dystrophy, Farber disease, and Best vitelliform macular dystrophy.

6. The method of any one of claims 1-5, wherein the NBP and CK1α agonist are administered sequentially in either order.

7. The method of any one of claims 1-5, wherein the NBP and CK1α agonist are administered concurrently.

8. The method of claim 7, wherein NBP and CK1α agonist are administered as a pharmaceutical composition that comprises the therapeutically effective amounts of NBP and CK1α agonist.

9. The method of claim 8, wherein the pharmaceutical composition is formulated for parenteral delivery or ocular delivery.

10. The method of claim 8, wherein the pharmaceutical composition is formulated for intramuscular injection, intravenous injection, or subcutaneous delivery.

11. The method of any one of claims 8-10, wherein the pharmaceutical composition is contained in a syringe for injection.

12. The method of claim 8, wherein the pharmaceutical composition is formulated for topical delivery to the eye.

13. The method of claim 8 or 10 wherein the pharmaceutical composition is incorporated within an implant; drug-eluting device, structure, or material; polymeric drug- eluting wafer; injectable hydrogel; or implantable hydrogel scaffold.

14. The method of claim 7, wherein the therapeutically effective amount of NBP is administered in a first pharmaceutical composition and the therapeutically effective amount of CK1α agonist is administered in a second pharmaceutical composition.

15. The method of any of claims 1-14, wherein the NBP is administered at a dose of about 100 ng / kg body weight to about 100 μg per kg body weight and the CK1α agonist is administered at a dose of about 100 ng / kg to about 500 μg per kg body weight.

16. The method of any of claims 1-15, wherein the CK1α agonist and NBP are administered at a frequency once per year, once per month, twice per month, weekly, twice weekly, every other day, or daily.

17. A pharmaceutical composition comprising a therapeutically effect amount of an NBP and a therapeutically effective amount of a CK1α agonist.

18. The pharmaceutical composition of claim 17, wherein the BP inhibitor is zoledronic acid and the CK1α agonist is pyrvinium.

19. The pharmaceutical composition of claim 17 or 18, comprising a CK1α agonist at a concentration of about 100 ng / kg to about 2.0 mg / kg body weight and an NBP at a concentration of about 100 ng / kg to about 100 μg per kg body weight.

20. A syringe containing a composition comprising a CK1α agonist at a concentration of about 100 ng / kg to about 2.0 mg / kg body weight and a dose of NBP at a concentration of about 100 ng / kg to about 100 μg per kg body weight.

21. The syringe of claim 20, wherein the CK1α agonist is pyrvinium and / or the NBP is zoledronic acid.

22. An intravitreal or suprachoroidal implant loaded with a pharmaceutical composition comprising a CK1α agonist in amount of about 0.0001 to about 0.3 mg and an NBP in an amount of about 0.0001 to about 0.2 mg.

23. The intravitreal or suprachoroidal implant of claim 22, wherein the CK1α agonist is pyrvinium and / or the NBP is zoledronic acid.

24. A topical ophthalmic preparation comprising a CK1α agonist in an amount of about 0.001 to about 0.3 mg / dose and an NBP in an amount of about 0.001 to about 0.2 mg / dose.

25. The topical ophthalmic preparation of claim 24, wherein the CK1α agonist is pyrvinium and / or the NBP is zoledronic acid.

26. A method of treating a retinal condition in a subject, the method comprising administering to the subject a therapeutically effective amount of zoledronic acid and a therapeutically effective amount of pyrvinium, wherein zoledronic acid and pyrvinium are administered concurrently.

27. The method of claim 26, wherein the zoledronic acid and pyrvinium are administered in the same pharmaceutical composition.

28. A method of treating a retinal condition in a subject, the method comprising administering to the subject a therapeutically effective amount of a casein kinase 1α (CK1α) agonist.

29. The method of claim 28, wherein the CK1α agonist is pyrvinium.

30. The method of claim 28 or 29, wherein the retinal condition is Stargardt macular dystrophy.

31. The method of claim 28 or 29, wherein the retinal condition is dry age- related macular degeneration.

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