IF hydroxypropyl-beta-cyclodextrin (HPβCD) is administered via intravitreal injection (1.5 μL, ~500 mM, weekly × 4 doses based on MβCD dosing precedent) to aged Abca4−/− male and female mice (9 months, at peak bisretinoid burden) in a sequential, two-phase protocol — HPβCD clearance first (weeks 1–4), followed by subretinal dual-vector AAV8-ABCA4 gene therapy (1.0–1.5 × 10¹² gc/mL per eye, week 5) —

THEN RPE A2E and total bisretinoid content will be reduced by ≥50% relative to AAV8-ABCA4 monotherapy, ONL thickness will be preserved at ≥80% of young wild-type reference at 6-month post-treatment assessment, and scotopic ERG b-wave amplitudes will be improved ≥30% relative to age-matched vehicle controls,

BECAUSE the causal chain proceeds through the following steps:

1. HPβCD extracts excess free cholesterol from RPE lysosomal membranes, disrupting mTORC1 anchoring at the lysosomal surface and thereby allowing TFEB dephosphorylation and nuclear translocation — a mechanism demonstrated in lysosomal storage disease cells where HPβCD activates TFEB, induces the CLEAR network, and drives autophagic flux (HPβCD promotes TFEB-mediated autophagy activation)[https://doi.org/10.1074/jbc.m113.506246].

2. TFEB nuclear translocation in RPE cells upregulates lysosomal biogenesis and exocytosis genes, causing lysosomes loaded with indigestible A2E and bisretinoids to fuse with the basolateral RPE membrane and expel their contents into the sub-RPE space for choriocapillaris clearance — analogous to how MβCD drove bisretinoid reduction ex vivo and in vivo in Abca4−/− Rdh8−/− retinas (beta-cyclodextrins reduce A2E by >48% and bisretinoids by 25–50% via intravitreal injection)[https://doi.org/10.1073/pnas.1400530111].

3. HPβCD's superior ocular safety profile relative to MβCD — attributable to its hydroxypropyl substitution reducing free-cholesterol stripping from photoreceptor outer segment membranes — permits repeated IVT dosing sufficient for progressive lysosomal unloading over the 4-week clearance phase [SPECULATIVE: direct RPE-specific toxicity comparison at matched IVT doses has not been published; inferred from the known lower membrane-disruptive potency of HPβCD vs. MβCD].

4. Systemic HPβCD penetrates the blood-retinal barrier, as demonstrated by i.p. and subcutaneous administration reducing retinal cholesterol in both wild-type and Cyp27a1−/−Cyp46a1−/− mice (HPβCD crosses the blood-retinal barrier and reduces retinal cholesterol)[https://doi.org/10.1111/bph.15209], suggesting that adjunctive subcutaneous HPβCD co-dosing could supplement IVT delivery to reach the RPE from both the vitreal and choroidal sides [SPECULATIVE: dual-route combined IVT + subcutaneous delivery in Abca4−/− has not been tested].

5. Once existing A2E burden is substantially cleared by HPβCD (weeks 1–4), RPE cells in a reduced-toxicity state are more metabolically competent to process and stably express AAV8-delivered ABCA4 transgene, and t...

SENS category: LysoSENS

Key references: • doi.org/10.1074/jbc.m113.506246], • doi.org/10.1073/pnas.1400530111], • doi.org/10.1074/jbc.m113.506246]. • doi.org/10.1073/pnas.1400530111]. • doi.org/10.1111/bph.15209],