IF a tripartite nuclear-recoded MT-CYB construct — comprising (a) the SOD2 mitochondrial targeting sequence (MTS) N-terminal fusion, (b) the SOD2 3′UTR for mRNA surface localization, and (c) targeted hydrophobicity-reduction mutagenesis of the two most GRAVY-scored transmembrane helices (predicted TM-IV and TM-V) limited to conservative Ile→Val and Leu→Ala substitutions at non-heme-coordinating, non-ubiquinone-contacting residue positions — is delivered first as pseudouridine/5-methylcytosine (Ψ/5mC) modified mRNA and subsequently via AAV9 to MT-CYB-null cybrid cells generated by DdCBE-mediated introduction of a premature stop codon in the endogenous MT-CYB locus,

THEN the allotopically expressed CYB protein will (i) localize to the mitochondrial inner membrane as confirmed by immunogold electron microscopy, (ii) assemble into the dimeric bc1 complex detectable by Blue Native PAGE, (iii) restore Complex III enzymatic activity (cytochrome c reductase assay) to ≥25% of isogenic wild-type levels, and (iv) confer rescue of OXPHOS-dependent proliferation on glucose-free galactose medium within 14 days post-transfection,

BECAUSE the causal chain proceeds as follows:

1. The SOD2 3′UTR directs the recoded MT-CYB mRNA to the mitochondrial outer membrane surface prior to translation, achieving ≥80% mitochondrial mRNA enrichment as demonstrated for ATP6 mRNA by the Corral-Debrinski strategy (ATP6 mRNA localized at 84.6% mitochondrial surface enrichment, enabling full NARP cybrid rescue), thereby co-localizing the ribosome-mRNA-nascent-chain complex with the TOM import channel. (Corral-Debrinski mRNA surface localization strategy validated in Kaltimbacher et al., RNA 2006, as cited in the Literature Task Output and Research Context)

2. Co-translational import eliminates the cytosolic aggregation bottleneck that has prevented all prior CYB allotopic expression attempts. When hydrophobic precursor proteins are translated in the cytosol without surface localization, they misfold, aggregate, and are shunted to proteasomal degradation rather than imported (Oca-Cossio et al., Mitochondrion 2003, as cited in the Literature Task Output). By coupling translation directly to the TOM40 pore at the outer membrane, the nascent CYB chain is threaded into the import pathway before its transmembrane helices can nucleate cytosolic aggregates. (Co-translational import mechanism, Kaltimbacher et al., RNA 2006, and Bonnet et al., Rejuvenation Research 2007, as cited in Literature Task Output)

3. However, CYB's eight transmembrane helices present a hydrophobicity load exceeding the demonstrated import capacity of the SOD2 MTS/3′UTR system alone, as evidenced by the fact that ND6 — with fewer TM domains and lower aggregate GRAVY score — required the full co-translational system to be rescued only in 2023 despite two decades of prior attempts (Liang et al., J Biomed Sci 2023, raising Complex I assembly by 20–23% and ATP by ~53%, as cited ...

SENS category: RepleniSENS