An AI-redesigned CYP27A1 variant, delivered by AAV8 under a foam-cell-responsive MSR1 promoter, creates a self-amplif...

IF an AI-engineered human CYP27A1 variant — redesigned via RFdiffusion backbone remodeling of the B-C and F-G loop substrate-access channels and ProteinMPNN sequence optimization to rigidify the active-site pocket around the 7-keto moiety (targeting residues proximal to Leu369, Val373, and Leu497 identified in PDB: 3M2Q), packaged into AAV8 at 3×10¹¹ vector genomes per mouse under the disease-responsive macrophage scavenger receptor 1 (MSR1/CD204) promoter — is delivered via tail-vein injection to 12-week-old male ApoE⁻/⁻ mice that have been on Western diet for 8 weeks prior to injection (established foam-cell plaque stage), then continued on Western diet for 8 weeks post-injection with sacrifice at week 24,

THEN aortic macrophages isolated by collagenase digestion and CD11b⁺ magnetic sorting will show ≥60% reduction in intracellular 7-ketocholesterol (7KC) by isotope-dilution LC-MS/MS, accompanied by a ≥30% increase in the 7KC-27OH metabolite, a ≥25% reduction in en face aortic Oil Red O surface area, a ≥20% increase in Picrosirius Red-quantified collagen content, and a ≥30% reduction in CD68⁺ plaque macrophage area in aortic root cross-sections relative to AAV8-MSR1-GFP control mice,

BECAUSE the following causal chain operates:

1. Accumulated intracellular 7KC in plaque foam cells is a primary driver of ongoing lysosomal membrane permeabilization, ER stress, and NLRP3 inflammasome activation — not merely a downstream marker of disease — making it a bona fide accumulated damage substrate amenable to enzymatic repair. Wild-type CYP27A1 already processes 7KC with a spectral dissociation constant of ≈0.07 μM and catalytic efficiency approximately 4-fold greater than for cholesterol, establishing proof-of-concept that mitochondrial hydroxylation is a viable intracellular detoxification route. (CYP27A1 kinetics and Kd data)[https://pmc.ncbi.nlm.nih.gov/articles/PMC3090233/]

2. The MSR1 promoter is transcriptionally upregulated specifically in macrophages that are actively engulfing oxidized LDL — the very cells accumulating the most 7KC — creating a disease-severity-proportional transgene expression gradient. This self-amplifying circuit means that the foam cells with the greatest 7KC burden will produce the highest CYP27A1 variant levels, whereas quiescent resident macrophages with low oxLDL uptake will generate minimal off-target expression. This promoter selectivity is functionally superior to constitutive CD68 minimal promoters, which yield 2–5-fold lower macrophage expression than CMV while showing negligible activity in non-myeloid cells (Gough et al., Gene Therapy, 2001, cited in Evidence Set).

3. RFdiffusion-guided remodeling of the B-C loop and F-G loop of CYP27A1, guided by the crystal structure (PDB: 3M2Q, active-site residues Leu369, Val373, Leu497), can sterically narrow the substrate-access channel to preferentially exclude cholesterol's C3-hydroxyl while accommodating 7KC's C7-keto group through engineered polar co...

SENS category: LysoSENS