A monomeric mitoTev-TALE targeting the unique breakpoint junction of somatic age-accumulated mtDNA deletions, deliver...

IF a monomeric mitoTev-TALE nuclease engineered to recognize the unique breakpoint junction sequence of the age-accumulated somatic mitochondrial common deletion (ΔmtDNA; in humans Δ4977, spanning the 13-bp direct-repeat junction that exists exclusively in deleted but not wild-type mtDNA genomes) is packaged as a single AAV-PHP.B vector carrying a skeletal-muscle-tropic serotype capsid and systemically administered (tail vein, 5×10¹¹ vg/mouse) to aged C57BL/6 mice (22–24 months, both sexes) carrying a validated heteroplasmic large-deletion mtDNA model,

THEN a ≥50% reduction in the ratio of deleted-to-total mtDNA molecules in skeletal muscle and cortical neurons (measured by droplet digital PCR with junction-spanning and total-mtDNA primer sets) will be observed within 12 weeks, accompanied by a ≥20% improvement in ex vivo mitochondrial respiratory capacity (Complex I–linked OCR, Seahorse XFe96), reduction in the proportion of cytochrome c oxidase (COX)-negative muscle fibers (serial section immunohistochemistry), and attenuation of grip-strength decline versus AAV-null controls,

BECAUSE the following mechanistic chain is engaged:

1. The mitoTev-TALE architecture fuses the I-TevI GIY-YIG endonuclease domain to a programmable TALE DNA-binding array, generating a monomeric enzyme that fits within a single AAV's ~4.7 kb cargo limit—unlike the obligate heterodimeric mitoTALENs or mtZFNs that require dual-vector co-transduction—enabling high single-cell transduction efficiency in post-mitotic tissue (mitoTev-TALE reduced MERRF m.8344A>G mutant load and improved OXPHOS in heteroplasmic cells as a monomer)[https://doi.org/10.15252/emmm.201708084].

2. The Δ4977 common deletion (and its murine equivalent, identifiable by long-range PCR) generates a neo-junction sequence at the deletion breakpoint that is entirely absent from wild-type mtDNA; TALE arrays designed against this junction sequence achieve inherent allelic selectivity with zero risk of cleaving the WT genome—a selectivity advantage that surpasses SNP-discriminating mitoTALENs that must distinguish a single-base mismatch under competitive hybridization conditions [SPECULATIVE for the junction-targeting design specifically; the monomeric cleavage principle is established][https://doi.org/10.15252/emmm.201708084].

3. Double-strand breaks introduced into deleted mtDNA molecules are processed by rapid degradative mechanisms in the mitochondrial matrix (Pol γ exonuclease, MGME1 exonuclease) rather than by non-homologous end joining, causing selective elimination of the linearized deleted genome (mitoTALEN-induced DSBs eliminated mutant mtDNA without depletion of total copy number in vivo)[https://doi.org/10.1038/s41591-018-0166-8].

4. Following selective clearance of deleted mtDNA molecules, organellar copy-number homeostatic mechanisms upregulate replication of the surviving wild-type mtDNA pool, repopulating mitochondria with fully encoded genomes and restoring complete OXPHOS complex ...

SENS category: LysoSENS

Key references: • doi.org/10.15252/emmm.201708084]. • doi.org/10.1038/s41591-018-0165-9], • doi.org/10.1038/s41591-018-0166-8]. • doi.org/10.1038/s41591-018-0165-9]. • doi.org/10.15252/emmm.201708084