IF a bicistronic nuclear expression cassette co-encoding (i) codon-optimized allotopic MT-CYB fused to the SOD2 mitochondrial targeting sequence (MTS) and the SOD2 3' UTR mRNA-localization element, together with (ii) a co-expressed, matrix-targeted UQCC2 "assembly escort" fragment (residues 1–130, containing the CYB-binding domain but truncated to remove the ribosome-docking domain that is irrelevant outside the mitoribosome context) — delivered via AAV2/9 intravitreally (for optic models) or transfection into MT-CYB-mutant human fibroblasts —

is administered to patient-derived MT-CYB-mutant fibroblasts (Phase 1) and aged (18–22 month) C57BL/6J male mice with confirmed Complex III decline (Phase 2),

THEN a restoration of Complex III enzymatic activity to ≥50% of age-matched wild-type levels (measured by the decylubiquinol spectrophotometric assay of Krähenbühl et al., validated for fibroblasts and isolated mitochondria) (assay validated in fibroblasts and tissues)[https://pubmed.ncbi.nlm.nih.gov/7834868/], accompanied by ≥75% mitochondrial colocalization of allotopic CYB with TOM20 by immunofluorescence, and incorporation of CYB into the ~480 kDa CIII₂ homodimer and I+III₂+IV supercomplexes detectable by Blue-Native PAGE, will be observed —

BECAUSE the following causal chain operates:

1. mRNA surface-localization minimizes cytosolic hydrophobic exposure. The SOD2 3' UTR contains cis-acting elements that recruit RNA-binding proteins on the mitochondrial outer membrane, ensuring allotopic CYB polypeptide emerges from ribosomes in immediate proximity to the TOM complex, dramatically shortening the window during which eight hydrophobic transmembrane helices are exposed to the aqueous cytosol (strategy reviewed in the context of Corral-Debrinski mRNA-targeting, cited in the literature review above). Without this localization, CYB exceeds the hydrophobicity threshold and aggregates before translocation (Claros and Vincens, 1996, as reviewed above; Oca-Cossio et al., 2003, as reviewed above).

2. Post-translational import still bypasses the co-translational UQCC1/UQCC2 chaperone interaction — the identified critical failure mode. In native mitochondrial biogenesis, UQCC1-UQCC2 (orthologs of yeast Cbp3-Cbp6) bind nascent CYB as it exits the mitoribosome, stabilizing it against degradation and guiding membrane insertion before any nuclear-encoded subunits arrive (Tucker et al., Cell Metabolism, 2013, as reviewed above). Allotopically expressed CYB imported post-translationally never encounters this co-translational handshake, resulting in unassembled CYB that is degraded by the mitochondrial quality control protease FTSH/AFG3L2. This assembly failure, distinct from import failure, is the dominant barrier not yet systematically targeted.

3. Co-expressed, matrix-targeted UQCC2 escort fragment reconstitutes the missing chaperone handshake. By independently expressing a truncated UQCC2 construct that retains CYB-binding capacity but l...

SENS category: LysoSENS