IF a single intravenous dose (1×10¹² vg/mouse) of a bicistronic AAV8 vector — encoding a codon-optimized, acid-residue-engineered Nocardia rhodochrous cholesterol oxidase (ChOx) genetically fused in-frame to human catalase (CAT), bearing a dual lysosomal-targeting cassette comprising a validated KFERQ-like CMA pentapeptide (N-terminal) and a mannose-6-phosphate (M6P) signal leader sequence (C-terminal), under a macrophage-restricted CD68 promoter — is administered intravenously to 12-month-old male ApoE-/- mice bearing 16-week Western diet-established atherosclerosis, and oral trehalose (2 g/kg/day in drinking water) is co-administered throughout the 12-week study to sustain TFEB-driven lysosomal biogenesis,

THEN aortic plaque 7-ketocholesterol (7-KC) content measured by LC-MS/MS will be reduced by ≥40% from baseline (~8–12 μg/mg tissue), peritoneal macrophage foam cell percentage by Oil Red O staining will decrease by ≥35%, plasma 7-KC metabolite flux will confirm enzymatic turnover, and critically — lysosomal membrane permeabilization (LMP) markers (cathepsin B leakage, Galectin-3 puncta) will remain at vehicle-control levels, indicating that in-situ H2O2 neutralization by the catalase fusion prevents the reactive oxygen species burst that would otherwise kill the cell,

BECAUSE the following mechanistic chain is supported by available evidence:

1. 7-KC accumulates irreversibly in macrophage lysosomes and is not cleared by endogenous enzymes, driving foam cell death and necrotic core expansion in atherosclerotic plaques; lysosomal dysfunction in plaque-resident macrophages is documented and correlatable with lipid content (Lysosomal biogenesis rescue evidence)[https://doi.org/10.1161/ATVBAHA]. Accumulated 7-KC — not new cholesterol oxidation — is the target, meaning only an active degradation strategy can resolve existing load.

2. Nocardia cholesterol oxidase accepts 7-KC as a substrate, converting it to 7-ketocholestenone; however, the wild-type enzyme's pH optimum of 7.0–7.5 is fundamentally mismatched to the lysosomal pH of 4.5–5.0, reducing catalytic activity precipitously below pH 6.0 at the target compartment — establishing the need for acid-residue engineering as a prerequisite (ChOx pH profile)[FEBS Journal/Pollegioni 2009, cited in Evidence Set]. [SPECULATIVE: A panel of surface charge mutations at the active-site entrance loop — guided by homology to acid-stable Type I GMC oxidoreductases from acidophilic Acidithiobacillus species — is predicted to shift the pH optimum toward 5.0–5.5, an approach validated computationally for related flavoenzymes but not yet applied to Nocardia ChOx.]

3. ChOx generates H2O2 stoichiometrically during each catalytic cycle; in the confined lysosomal volume, unreacted H2O2 would trigger lysosomal membrane permeabilization (LMP), releasing cathepsins and accelerating the apoptotic cascades the therapy aims to prevent — identical to the toxicity mechanism of 7-KC itself (7-KC/N...

SENS category: RepleniSENS

Key references: • doi.org/10.1161/ATVBAHA]. • doi.org/10.1016/j.ajpath.2013.04.028]. • doi.org/10.1038/ncomms15750]. • doi.org/10.1161/atvbaha.116.308916]; • doi.org/10.1371/journal.pone.0053192].