IF sequential systemic delivery of AAV9-DdCBE (1×10¹³ vg/kg, retro-orbital injection, week 0) followed by AAV9-mitoTALEN (5×10¹² vg/kg, retro-orbital injection, week 4) is administered to aged male and female C57BL/6J mice (18–20 months) carrying the m.5024C>T tRNA-Ala mutation at 60–70% heteroplasmy,

THEN by week 12, cardiac and skeletal muscle m.5024C>T heteroplasmy will be reduced below 30% of total mtDNA (≥35–40 percentage-point absolute reduction from baseline), complex I activity will be restored by ≥40% relative to vehicle-treated aged controls, ATP production in left ventricular homogenates will increase by ≥35% above baseline, and cardiac RNA-seq at week 8 will uniquely reveal upregulation of PGC-1α, TFAM, NRF1, and NRF2 in the combination arm—surpassing the additive prediction of both monotherapies by ≥20 percentage points of heteroplasmy reduction (i.e., demonstrating synergy)—while total mtDNA copy number remains above 50% of wild-type levels and ALT/AST remain within 3× baseline,

BECAUSE the following mechanistic cascade operates:

1. DdCBE editing (week 0) creates an intra-cellular heteroplasmy gradient across individual mitochondria. AAV9-DdCBE imports a DddA-derived split deaminase fused to TALE arrays targeting the m.5024C strand into the mitochondrial matrix, catalysing C→T conversion on a stochastic, per-organelle basis. Because each mitochondrion contains only a subset of the cell's mtDNA copies, DdCBE editing results in a distribution of per-organelle mutant loads—some mitochondria become predominantly wild-type, others remain predominantly mutant—rather than a uniform shift. [SPECULATIVE: this intra-organelle heterogeneity distribution has not been directly imaged in aged post-mitotic tissue but is consistent with the known stochastic nature of mtDNA replication and DdCBE accessibility.]

2. Per-organelle heteroplasmy gradients generated by DdCBE prime selective BNIP3L/NIX-mediated mitophagy. Mitochondria retaining high mutant burden exhibit reduced membrane potential (ΔΨm) because mutant tRNA-Ala impairs translation of all 13 mtDNA-encoded OXPHOS subunits. Depolarised mitochondria stabilise PINK1 on the outer membrane, recruiting Parkin and activating canonical mitophagy; simultaneously, stress-responsive BNIP3L/NIX upregulation targets mitochondria with the lowest ΔΨm for autophagic capture. This mitophagy selectivity effectively constitutes a third elimination mechanism beyond the two AAV-delivered editing tools. [SPECULATIVE: direct evidence that DdCBE-induced per-organelle heteroplasmy variance specifically amplifies BNIP3L-mediated mitophagy flux has not been published; this is inferred from the known relationship between mutant tRNA burden, ΔΨm, and PINK1/Parkin pathway activation in heteroplasmic cells.]

3. AAV9-mitoTALEN at week 4 eliminates residual high-heteroplasmy mtDNA molecules that survived DdCBE editing and mitophagy. mitoTALEN proteins are imported into the matrix via an N-terminal MTS, where...

SENS category: GlycoSENS