IF a dual-modality lysosomal repair intervention—comprising (1) subretinal or intravitreal AAV9-mediated overexpression of the V-ATPase subunit ATP6V0D1 to restore lysosomal acidification, co-administered with (2) lysosome-targeted PLGA acidic nanoparticles (aNPs) encapsulating a thermostable bacterial bisretinoid-oxidizing enzyme (specifically a carotenoid-cleaving dioxygenase, CCD, from Novosphingobium sp. or structurally analogous genus, engineered with a mannose-6-phosphate (M6P) lysosomal-targeting signal and an acidic pH activity optimum) — is delivered to aged female C57BL/6J mice (22–24 months) and/or ABCA4−/− mice (12–18 months) with established bisretinoid/lipofuscin burden in the RPE,

THEN a measurable reduction in RPE autofluorescent aggregate burden (≥30% decrease in fundus autofluorescence intensity and ≥25% reduction in HPLC-quantified A2E/bis-A2E levels within 12 weeks), accompanied by ≥0.35 unit decrease in lysosomal pH and ≥40% restoration of Cathepsin D proteolytic activity relative to aged vehicle-injected controls, will be observed,

BECAUSE the following mechanistic chain operates:

1. Age-associated lysosomal alkalinization (~pH 5.5–6.5 in aged RPE vs. ~4.5–5.0 in young RPE) directly drives accumulation of autofluorescent granules, as demonstrated by the finding that pharmacologically elevating lysosomal pH to >6.0 in RPE cells is sufficient to induce lipofuscin-like aggregate formation, and that restoration of acidification with PLGA/m-tartaric acid aNPs within 24 hours rescues Cathepsin D activity and reduces granule formation (impaired lysosomal acidification drives autofluorescent granule biogenesis in RPE)[https://doi.org/10.1167/iovs.62.9.39].

2. ATP6V0D1 overexpression via AAV increases V-ATPase proton pump density at the lysosomal membrane, counteracting the age-related decline in V-ATPase assembly that underlies lysosomal alkalinization; this shift from pathological pH (>5.5) to physiological pH (4.5–5.0) restores Cathepsin D proteolytic efficiency, which loses 60–80% activity as pH rises from 4.5 to 6.0 (Cathepsin D is exquisitely pH-sensitive, losing 60–80% activity at pH 6.0 relative to pH 4.5)[https://doi.org/10.1167/iovs.62.9.39].

3. Re-acidification alone is necessary but insufficient for clearing pre-existing cross-linked, oxidized lipofuscin: evidence explicitly demonstrates that advanced lipofuscin species contain covalently cross-linked bisretinoid adducts that resist Cathepsin D-mediated hydrolysis even after full pH normalization, limiting autofluorescence clearance to ~40–50% and predominantly achieving removal of non-crosslinked precursors only (cross-linked oxidized lipofuscin resists degradation even after acidic pH is restored)[https://doi.org/10.1167/iovs.62.9.39].

4. The co-encapsulated thermostable CCD enzyme, delivered lysosome-directionally via the M6P receptor pathway, introduces a non-mammalian oxidative cleavage activity capable of cleaving the polyene back...

SENS category: GlycoSENS

Key references: • doi.org/10.1167/iovs.62.9.39]. • doi.org/10.1167/iovs.62.9.39],