IF a dual rAAV6 vector system co-delivering two complementary mitoTALEN half-constructs (each half encoding one TALE DNA-binding arm fused to a monomeric FokI nuclease domain, with each arm designed to recognize a unique sequence spanning the breakpoint junction of the murine 3867-bp common mtDNA deletion rather than a flanking wild-type sequence) is administered via bilateral intramuscular injection at a combined dose of ~1×10¹¹ vector genomes per limb under a muscle-specific desmin promoter is administered to 22–24-month-old male C57BL/6 mice exhibiting established sarcopenia and confirmed tibialis anterior / gastrocnemius accumulation of the 3867-bp common mtDNA deletion at heteroplasmy levels ≥60%,

THEN a ≥60% reduction in deletion-bearing mtDNA heteroplasmy within treated myofibers (quantified by deletion-junction-specific droplet digital PCR and long-range PCR across the deletion breakpoint), accompanied by ≥40% restoration of complex IV enzymatic activity in SDH/COX dual-stain histochemistry and a measurable improvement in ex vivo high-resolution respirometry (state 3 respiration, complex IV-linked oxygen flux), and a ≥15% increase in fiber cross-sectional area over a 12-week treatment window, will be observed,

BECAUSE the following causal chain is operative:

1. Large-scale mtDNA deletions create a unique chimeric breakpoint junction sequence — the precise nucleotide sequence spanning the deletion border — that is entirely absent from wild-type mtDNA. Designing the two TALE DNA-binding domains against this junction sequence (rather than sequences flanking the deletion in wild-type mtDNA, as in standard restriction enzyme approaches) confers absolute molecular discrimination: the FokI heterodimer can only assemble and cleave when both half-sites are present on the same molecule, and both half-sites coexist exclusively on deleted genomes. This represents a structural improvement over mitochondrially targeted restriction enzymes (mitoREs), which require a fortuitous restriction site near the deletion and cannot inherently distinguish breakpoint junctions. (Allele-specific mitoTALEN activity with full specificity controls was demonstrated in patient-derived heteroplasmic cells)[https://doi.org/10.1038/nm.3261]

2. AAV9-delivered mitoTALENs targeting the m.5024C>T point mutation achieved ~75% reduction of mutant mtDNA in cardiac and skeletal muscle in vivo, restoring tRNA^Ala steady-state levels without depleting total mtDNA copy number, demonstrating that selective degradation of targeted genomes triggers compensatory repopulation by wild-type molecules. (AAV9-mitoTALEN selectively reduced mutant mtDNA fraction and restored tRNA^Ala in vivo)[https://doi.org/10.1038/s41591-018-0166-8] This repopulation principle is mechanistically critical: because mitochondrial nucleoids in post-mitotic fibers are not fixed in copy number, selective elimination of deleted genomes creates a replicative advantage for intact wild-type genomes, which expand to n...

SENS category: RepleniSENS

Key references: • doi.org/10.1038/nm.3261] • doi.org/10.1038/s41591-018-0166-8] • doi.org/10.1038/s41591-018-0166-8 • doi.org/10.1038/s41591-018-0166-8].