IF an AI-engineered human CYP27A1 variant — redesigned via RFdiffusion All-Atom motif scaffolding to remodel the substrate-access channel and introduce a polar second-shell residue capable of hydrogen-bonding with the C7-keto moiety of 7-ketocholesterol (7KC), followed by ProteinMPNN sequence optimization of second-shell residues (targeting positions beyond the native steric gatekeepers Val367/Val482), validated by AlphaFold2 pLDDT ≥ 85 and Rosetta ΔΔG ≤ −1 kcal/mol) — is packaged as a codon-optimized transgene (≤1.5 kb coding sequence) under the MSR1/SR-A promoter (~800 bp, intrinsically upregulated by oxidized lipids and 7KC itself in foam cells), supplemented with a miR-122-binding site array in the 3′ UTR to suppress hepatocyte off-target expression, and delivered via AAV6 capsid (superior macrophage transduction vs. AAV8) at 1×10¹¹ – 5×10¹¹ vector genomes per mouse via tail-vein injection into 12-week-old male ApoE⁻/⁻ mice that have been fed a Western diet for 8 weeks to establish foam-cell-laden plaques, with the Western diet continued for an additional 8 weeks post-injection (sacrifice at week 24),

THEN aortic plaque-resident macrophages will show:

1. ≥50% reduction in intracellular 7KC concentration (measured by LC-MS/MS after collagenase D digestion + CD11b⁺ magnetic sorting)
2. ≥30% reduction in aortic root lesion area (Oil Red O cross-section morphometry)
3. Reduced macrophage foam-cell content (CD68⁺ area by immunohistochemistry) relative to AAV6-null vector controls,

BECAUSE of the following causal chain:

1. 7KC accumulates irreversibly in foam-cell lysosomes and membranes in ApoE⁻/⁻ plaques during Western diet feeding, and this already-deposited 7KC cannot be cleared by cholesterol efflux transporters (ABCA1/ABCG1), meaning the damage is structural and only enzymatic catabolism can reverse it — wild-type CYP27A1 already processes 7KC at ~4× the rate of cholesterol with a Kd ≈ 0.07 μM, indicating native active-site pre-organization for 7KC, but the absolute kcat is rate-limiting for clearance under foam-cell lipid loading conditions (Pikuleva reconstituted system data)[https://pmc.ncbi.nlm.nih.gov/articles/PMC3090233/][https://pubmed.ncbi.nlm.nih.gov/21411718/]

2. The AI-engineering strategy targets a specific structural opportunity: the C7-ketone of 7KC (absent in cholesterol) presents a hydrogen-bond acceptor that the native CYP27A1 active site does not exploit — RFdiffusion All-Atom, which explicitly models non-protein components including heme cofactors and small molecules during diffusion, can condition backbone generation on the presence of 7KC to sculpt a binding pocket that stereochemically favors the oxysterol and introduces a new polar contact (e.g., a Thr or Asn at a second-shell position ~5–6 Å from C7) without displacing the native heme-thiolate coordination geometry (Evidence Set, Section 7 — RFdiffusion All-Atom description, Krishna et al., Science 2024 as cited in Evidence Set)

3. **...

SENS category: LysoSENS