IF a sequential two-phase intervention consisting of (Phase A) systemically administered TFSA (trifluoromethylsulfonyl-functionalized) pH-buffering polymeric nanoparticles (10–25 mg/kg i.p., 2×/week, 4 weeks) to chemically re-acidify RPE lysosomes, followed by (Phase B) subretinal injection of a self-complementary AAV9 (scAAV9) vector encoding human ATP6V1H (V-ATPase V1 H-subunit) under the VMD2/BEST1 RPE-specific promoter with a C-terminal HA epitope tag, is administered to aged (18–22 month) male and female C57BL/6 mice with established RPE lysosomal dysfunction,

THEN LysoSensor Yellow/Blue DND-160 ratiometric imaging of RPE flat mounts will show measurable re-acidification of RPE lysosomes (pH shift from ~5.5–5.8 baseline in aged RPE toward the youthful ~4.7–5.0 range), accompanied by a ≥30% reduction in RPE lipofuscin/A2E autofluorescence, structural preservation of the photoreceptor-RPE interface on OCT, and maintained ERG b-wave amplitude relative to vehicle-treated aged controls,

BECAUSE:

1. In aged RPE, V-ATPase assembly efficiency declines, causing lysosomal alkalinization that impairs proteolytic degradation of photoreceptor outer segment (POS) material, leading to progressive accumulation of bisretinoid lipofuscin (A2E) — the defining LysoSENS damage in AMD pathogenesis. (Evidence Set: Biodistribution and BRB Crossing section; In Vivo Ratiometric Imaging section)
2. TFSA/PBFSU-class pH-buffering polymeric nanoparticles are engineered to internalize via endocytosis, buffer the luminal pH of late endosomes and lysosomes through a proton-sponge or hydrolytic acid-releasing mechanism, acutely restoring lysosomal acidity in target cells. (Evidence Set: Introduction — Systemic Delivery; Specific pH-Buffering Platforms section)
3. Systemically administered nanoparticles (<200 nm) can cross the fenestrated choriocapillaris to access the basolateral RPE surface, and optimized surface chemistry enables transcytosis or endocytic uptake across the tight-junction-forming RPE, allowing luminal lysosomal access. (Evidence Set: BRB crossing section — "optimized polymeric systems achieving sufficient posterior segment accumulation")
4. Chemical acidification via TFSA nanoparticles is transient and does not address the root cause (deficient V-ATPase subunit availability); therefore, Phase B genetic supplementation of ATP6V1H via scAAV9-VMD2 provides durable restoration of the V1 catalytic sector assembly, sustaining proton-pumping activity independent of continued nanoparticle dosing. (Evidence Set: Existing Gene Therapy Constructs section; Vector Genome Configuration section) [SPECULATIVE — the sequential "priming then genetic consolidation" concept has not been tested; however each component has independent mechanistic precedent]
5. scAAV9 delivered subretinally achieves superior RPE transduction compared to intravitreal routes, and the VMD2 promoter restricts expression to RPE cells, preventing ectopic photoreceptor or Müller glia V...

SENS category: LysoSENS

Key references: • doi.org/10.1038/s41586-020-2975-4] • doi.org/10.1101/2025.04.28.651096] • doi.org/10.1038/s41586-020-2975-4],