PROJECT SUMMARY Accumulation of toxic lipid bisretinoids (LBs) in lysosomes (LY) of retinal pigment epithelium (RPE) is a main etiological agent for recessive Stargardt disease (STGD1) and ABCA4-related forms of cone-rod dystrophy (CRD) and retinitis pigmentosa (RP). Furthermore, accumulation of LBs with age is a suspected risk factor for Age-Related Macular Degeneration (AMD). Hence, agents that remove LBs from RPE fulfill an unmet medical need for STGD1, CRD, RP and may help validate LBs as pharmacological targets in AMD. We have recently identified beta cyclodextrins (βCDs), cyclic sugars formed by 7 glucose residues, as promising bioactive compounds capable of removing LB from RPE cultures, from enucleated eyecups obtained from ABCA4/RDH8 double knock out (DKO) mice (a mouse model for accelerated LB deposition) and from DKO mouse eyes after intravitreal (IVT) injection (Nociari et al. PNAS, 2014). βCDs are known for their ability to solubilize free cholesterol (FC) from biological membranes and to clear aberrant storage materials in lysosomal storage disorders, including FC in Niemann-Pick C (NPC), ceroid lipofuscin in Batten Disease as well as α-synuclein (α- syn) in Parkinson and amyloid-β in Alzheimer Disease. In NPC cells, clearance of FC depends on βCDs' ability to form complex with FC. In contrast, their ability to clear ceroid lipofuscin and α-syn, depends on their capacity to modulate transcription factor EB (TFEB), a master regulator of LY, peroxisomal and mitochondrial function and of autophagy. As with FC, βCDs form soluble complexes with LBs; however, the underlying clearance mechanism is unknown. Elucidation of the mechanism mediating LB clearance from RPE will facilitate the therapeutic utilization of βCDs for LB-induced retinal disease. Furthermore, to maximize the safety, potency and retinal biodistribution we propose to optimize nanoparticles (βCD-threaded polyrotaxanes) that have already shown effective βCD delivery into LY of NPC cells. Finally, we will test the efficacy of βCD-based approaches alone or in combination with TFEB-based strategies in ABCA4RDH8-/- mice, an animal model of LB-driven retinal degeneration. Because β- CDs are FDA approved, success in this proposal could be quickly extended into clinical trials in humans.