An AI-scaffolded, lysosome-targeted peroxidase engineered by RFdiffusion and ProteinMPNN with a substrate-optimised A...

IF an AI-engineered chimeric peroxidase — designated AcidoHRP-v1 — constructed by using RFdiffusion to scaffold the HRP catalytic triad (proximal His170, distal His42/Arg38, heme-coordination geometry) onto a de novo acid-stable backbone, with ProteinMPNN sequence optimization for maximal folding probability and net-negative surface charge at pH 5.0–5.5, further fused at the C-terminus to the LAMP1 lysosomal targeting domain (YXXΦ motif: GYQTI) via an Igκ signal peptide leader sequence, and packaged into AAV5 driven by the RPE-specific VMD2 promoter at 1×10^10 viral genomes per eye delivered by subretinal injection,

is administered to 12-month-old male and female Abca4-/- mice (C57BL/6J background, both sexes included to detect sex-dependent lipofuscin differences), a species and age at which lysosomal A2E burden is established and progressive,

THEN at 4 weeks post-injection, RPE tissue from treated eyes will exhibit a ≥30% reduction in A2E concentration (measured by HPLC-MS/MS, femtomole sensitivity, CV <15%, normalized to RPE protein content), compared to eyes receiving either wild-type HRP-AAV5 or empty AAV5 vector, with ERG a/b-wave amplitudes maintained at ≥80% of pre-injection baseline and outer nuclear layer thickness maintained at ≥90% of baseline by OCT; confocal autofluorescence imaging at 488 nm excitation / 560 nm emission will show a ≥25% reduction in mean granule fluorescence intensity in treated RPE flat-mounts targeting F1 lipofuscin granules,

BECAUSE the following mechanistic chain is operative:

1. Wild-type HRP exhibits substantial loss of catalytic turnover (kcat) against hydrophobic polyene substrates at pH ≤5.0, primarily due to protonation of the distal His42 which is required for heterolytic peroxide bond cleavage; this renders the native enzyme non-functional in the RPE lysosomal lumen (pH 4.5–5.5), as described in the evidence synthesis referencing Veitch 2004 and structural peroxide-cleavage mechanisms.

2. RFdiffusion's motif-scaffolding mode fixes the precise Cartesian coordinates of the heme-binding pocket and catalytic dyad while generating entirely novel protein backbones around this core, thereby decoupling catalytic geometry preservation from the acid-labile secondary structure elements of the native plant HRP scaffold — a capability demonstrated for oxidoreductase active-site transplantation by (Watson et al., Nature, 2023, as cited in the Evidence Set).

3. ProteinMPNN solves the inverse-folding problem to design sequences that maximise thermodynamic packing density and surface polarity of the RFdiffusion-generated backbone, parameters strongly correlated with resistance to acid-induced unfolding, while conditioning toward net-negative surface residues that remain stable and soluble at lysosomal ionic strength — as described in the evidence synthesis referencing Dauparas et al., Science, 2022.

4. [SPECULATIVE] An additional ProteinMPNN design pass, constrained by docking the A2E ...

SENS category: LysoSENS