IF a computationally optimized allotopic MT-CYB variant — selected from a 15–20 member library ranked by Rosetta membrane energy scoring, engineered by ProteinMPNN with explicit hydrophobicity penalty constraints targeting the lipid-exposed faces of transmembrane helices B, C, and E of the wild-type apocytochrome b backbone (UniProt P00156), fused to a COX8A mitochondrial targeting sequence (residues 1–29), nuclear codon-optimized (CAI > 0.8), and packaged in AAV9 at MOI 10⁵ — is administered via intramuscular and intracerebroventricular dual-route injection to aged C57BL/6J mice (22–24 months, both sexes) exhibiting measurable age-associated decline in Complex III activity,

THEN a ≥15% restoration of succinate-cytochrome c reductase activity (normalized to citrate synthase) will be observed in cardiac, skeletal muscle, and hippocampal mitochondria at 8 weeks post-delivery, accompanied by >80% cell viability in isolated tissue cybrids, recovery of TMRM-measured mitochondrial membrane potential (ΔΨm), and detectable improvement in grip strength and novel object recognition versus age-matched AAV9-null controls,

BECAUSE the step-by-step causal chain is as follows:

1. Age-accumulated somatic MT-CYB mutations clonally expand in post-mitotic tissues. In neurons, cardiomyocytes, and skeletal muscle fibers, heteroplasmic MT-CYB mutations — including variants at positions corresponding to m.15257G>A and m.15498G>A — accumulate with age via clonal expansion and reach a biochemical threshold that compromises Complex III function, as described in the evidence set's rationale for cybrid model selection and consistent with the known threshold effect in OXPHOS diseases (from Evidence Set: Rossignol et al., 2003, cited therein). This is the accumulated damage state that only allotopic replacement — not prevention — can reverse.

2. Unmodified wild-type allotopic MT-CYB fails because cytosolic hydrophobicity drives aggregation before mitochondrial import. The evidence set explicitly establishes that WT allotopic CYB causes cytoskeletal aggresome formation and cell death (<20% viability) due to its extreme hydrophobicity being incompatible with cytosolic transit (from Evidence Set: described as the reference failure state and consistent with Bannwarth et al., 2012, and Oca-Cossio et al., 2019, cited therein). This is the critical mechanistic failure that all prior allotopic CYB attempts shared.

3. AlphaFold2 structural prediction of apocytochrome b enables precision targeting of lipid-exposed TM helix surfaces. By predicting the folded backbone (Jumper et al., 2021, cited in Evidence Set) prior to redesign, the pipeline identifies which residues on helices B, C, and E face lipid rather than protein partners — distinguishing them from catalytic and heme-binding residues that must remain conserved. [SPECULATIVE: The exact set of permissible substitutions on TM helices B/C/E that preserve assembly without disrupting the Qi/Qo sites has not been exper...

SENS category: LysoSENS