IF a single AAV9 vector encoding a triple-engineered human ND1 transgene — comprising (1) a codon-harmonized (not simply codon-maximized) nuclear recoding of all 7 non-universal mitochondrial codons with strategic synonymous slow-codon insertions at each of ND1's hydrophobic transmembrane domain boundaries, (2) the COX8A mitochondrial targeting sequence (MTS) at the N-terminus, and (3) the 3′ UTR of the nuclear-encoded mitochondrial gene AKAP1 appended to the mRNA to anchor ND1 transcripts to the outer mitochondrial membrane (OMM) for pseudo-co-translational import — is delivered via AAV9 at MOI 10⁵ vg/cell to LHON patient-derived fibroblasts (GM3460A, m.3460G>A, cultured in galactose medium),

THEN at day 14 post-transduction, mitochondrial colocalization of allotopic ND1 protein (measured by immunofluorescence with anti-ND1 Abcam ab183721 / MitoTracker Deep Red) will exceed ≥70% — compared to the ≤30–40% colocalization typical of standard single-component COX8A-MTS-only constructs — and Complex I-dependent oxygen consumption rate (Seahorse XF, rotenone-sensitive OCR in galactose media) will increase ≥50% versus untreated m.3460G>A fibroblasts, achieving ≥70% of healthy control OCR, with less than 40% deterioration of these OCR gains between days 14 and 28,

BECAUSE the following mechanistic chain addresses the primary failure mode — cytosolic aggregation of hydrophobic ND1 — through two independent and synergistic mechanisms:

1. Cytosolic aggregation is the dominant import bottleneck for allotopic ND1. Standard COX8A MTS-only constructs depend entirely on post-translational import of the fully synthesized ND1 polypeptide. Because ND1 contains eight highly hydrophobic transmembrane segments, cytosolic exposure during and after translation causes irreversible misfolding, ubiquitination, and aggregation before the TOM complex can engage the MTS. This is identified as the primary failure mode for Complex I subunit allotopic expression, distinct from less hydrophobic subunits where allotopic strategies have shown greater success. (Codon optimization as essential parameter for allotopic expression)[https://doi.org/10.1016/j.redox.2020.101429]

2. The AKAP1 3′ UTR localizes ND1 mRNA to the OMM, enabling pseudo-co-translational import that minimizes cytosolic exposure. The 3′ UTR of AKAP1 (A-kinase anchoring protein 1) contains cis-acting RNA localization elements that tether mRNA to the OMM surface. When appended to the allotopic ND1 mRNA, this element recruits ND1 transcripts to OMM-associated ribosomes, allowing the nascent polypeptide chain — while still ribosome-tethered — to be captured by TOM20 and threaded co-translationally into the TOM/TIM23 import channel before full-length hydrophobic exposure. This pseudo-co-translational mechanism mimics the membrane-proximal synthesis used by mitochondrial ribosomes for mtDNA-encoded subunits. The strategy of mRNA localization to the OMM to mitigate hydrophobicity-driven import failure i...

SENS category: LysoSENS

Key references: • doi.org/10.1016/j.redox.2020.101429] • doi.org/10.1016/j.cmet.2016.04.004] • doi.org/10.1016/j.redox.2020.101429], • doi.org/10.1016/j.redox.2020.101429]. • doi.org/10.1016/j.cmet.2016.04.004],