Sequential sub-retinal AAV-ABCA4 gene augmentation followed by intravitreal co-delivery of remofuscin and hydroxyprop...

IF a sequential, dual-route protocol consisting of:

1. Sub-retinal delivery of AAV-ABCA4 (dual-vector hybrid system, ~1×10¹¹ vg/eye) at Week 0, and
2. Intravitreal co-injection of remofuscin (soraprazan, ~50–100 µM estimated vitreal concentration) plus hydroxypropyl-β-cyclodextrin (HP-βCD, ~1–5 mM) at Week 6–8 post-AAV transduction

is administered to aged (12–18 month) male and female Abca4⁻/⁻ mice (a genetically and temporally established model of heavy bisretinoid loading),

THEN quantitative autofluorescence (qAF) imaging will show ≥40% reduction in RPE lipofuscin fluorescence within 12 weeks of remofuscin/HP-βCD delivery, with preservation of photoreceptor outer nuclear layer (ONL) thickness (≤10% thinning vs. young Abca4⁻/⁻ controls) and no recurrence of A2E accumulation at 6 months post-treatment, compared to either remofuscin alone, AAV-ABCA4 alone, or vehicle controls,

BECAUSE the following mechanistic chain operates:

1. Loss-of-function ABCA4 in photoreceptor disc membranes permits N-retinylidene-phosphatidylethanolamine to condense with a second all-trans-retinal molecule, generating A2E precursors that are phagocytosed daily by the RPE and accumulate as undegradable lysosomal lipofuscin — this is the established root pathology driving both the Stargardt phenotype and accelerated AMD-like atrophy in aged mice (described in evidence set: Molday et al., Journal of Biological Chemistry; Sparrow et al., Progress in Retinal and Eye Research).

2. AAV-ABCA4 sub-retinal gene augmentation restores functional ABCA4 flippase activity in photoreceptors, halting new bisretinoid synthesis — the 6–8 week lag before remofuscin delivery is required to allow transgene expression to reach therapeutic levels and establish a "sealed source," so that any A2E mobilized by subsequent pharmacotherapy is not immediately replenished (NCT01367444; SAR422459 preclinical data referenced in evidence set).

3. Remofuscin (soraprazan), a lysosomotropic potassium-competitive acid blocker, accumulates in the acidic lysosomal compartment of loaded RPE cells and triggers lysosomal exocytosis, releasing the stored bisretinoid cargo basally toward Bruch's membrane and the choroidal circulation for systemic disposal (Julien & Schraermeyer, Neurobiology of Aging, cited in evidence set; NCT03951737).

4. [SPECULATIVE — NOVEL MECHANISTIC LINK] The exocytosed A2E, now resident in the subretinal/interphotoreceptor space and at the RPE–Bruch's membrane interface, represents an acute extracellular toxicity hazard that current remofuscin-alone protocols do not address: free A2E is amphiphilic and membrane-destabilizing; once extracellular, it can be re-internalized by neighbouring RPE cells or photoreceptors, activate extracellular complement via Factor H inhibition, and stimulate NLRP3 inflammasome assembly in bystander cells, potentially worsening the local inflammatory milieu during the very window of intended clearance. This "exocytosis trap" is ...

SENS category: LysoSENS

Key references: • doi.org/10.1073/pnas.1400530111]. • doi.org/10.1371/journal.pone.0067263], • doi.org/10.1371/journal.pone.0067263]. • doi.org/10.1073/pnas.1400530111],