IF a dual-regulatory allotopic ND1 construct — encoding nuclear-codon-recoded ND1 (CAI ≈ 0.70) fused to the Su9-DHFR mitochondrial targeting sequence (MTS), placed under a ROS-responsive ARE/NRF2-driven minimal promoter (rather than constitutive CMV) with an attenuated Kozak sequence, delivered by lentiviral transduction (MOI = 5, single administration)

is stably integrated into aged (24-month-old) C57BL/6J mice via subretinal AAV9 co-delivery (for retinal ganglion cell [RGC] targeting) and systemically by intravitreal injection, in animals harboring accumulated somatic mtDNA mutations in RGC-enriched retinal tissue (confirmed by long-range PCR heteroplasmy quantification),

THEN at 12 weeks post-injection: (i) RGC-layer Complex I–linked oxygen consumption rate (Seahorse XF, rotenone-sensitive fraction, ex vivo retinal explants) will recover ≥60% relative to aged vehicle-injected controls; (ii) precursor ND1 protein import rate (pulse-chase ³⁵S-Met/Cys, 30-min chase, immunoprecipitation of mature vs. precursor band) will exceed that of a matched CAI ≈ 0.95 construct by ≥1.5-fold in age-matched cybrid lines; (iii) cytosolic ND1 precursor aggregation (filter trap assay, anti-ND1, cytosolic fraction) will be ≤20% of total ND1 signal — versus ≥50% aggregate fraction predicted for the CAI ≈ 0.95 control in aged cells where TIM23 capacity is further diminished,

BECAUSE:

1. Maximally codon-optimized (CAI ≈ 0.95) constructs drive burst-rate ribosomal translation that generates precursor protein flux exceeding TIM23 translocase stoichiometric throughput, causing cytosolic stalling and aggregation rather than productive mitochondrial import — as established by kinetic import saturation data (TIM23 import stalling under high precursor flux)[https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7729113/].
2. Aged tissues experience a compounded bottleneck: TIM23 subunit stoichiometry declines with organismal age due to proteostatic impairment and accumulation of mis-imported precursor "traffic jams" in the import channel [SPECULATIVE — direct TIM23 subunit quantification in aged murine retina is an evidence gap], meaning the optimal CAI set-point for productive import in old tissue is shifted further downward from the CAI ≈ 0.70 established in young cybrid models.
3. A constitutive CMV promoter cannot self-adjust to this age-variable TIM23 capacity; a ROS/NRF2-responsive promoter creates a negative-feedback loop: elevated mitochondrial ROS (the primary pathophysiological signal of Complex I dysfunction in both LHON cybrid models and aged RGCs) transcriptionally activates the allotopic construct precisely when import demand is highest — and simultaneously, ROS-induced NRF2 activation upregulates mitochondrial chaperones (HSP70/HSPA9, DNAJC19) that facilitate TIM23-assisted import, transiently increasing import capacity in synchrony with precursor delivery (self-matching flux-to-capacity) [SPECULATIVE — direct co-activation of NRF2 and mitochondrial chapero...

SENS category: LysoSENS