An obligate heterodimeric mitoARCUS delivered in a single AAV9 vector exploits the distance-enforced specificity of t...

IF a single recombinant AAV9 vector encoding an obligate heterodimeric mitoARCUS (HD-mitoARCUS) — comprising two non-identical, engineered I-CreI monomers (designated mitoARCUS-8470 and mitoARCUS-13447) each fused to the COX8A mitochondrial targeting sequence (MTS) and co-expressed from a bicistronic cassette using a P2A self-cleaving peptide under a CKMT2 (cardiac) or SYNP (neuronal) tissue-specific promoter — is delivered via intravenous tail-vein injection (2×10¹¹ vg, single dose) to aged C57BL/6J mice (≥20 months, both sexes), in which Δ4977bp-equivalent common deletion heteroplasmy has been confirmed in heart, skeletal muscle, and substantia nigra,

THEN selective elimination of deletion-bearing mtDNA genomes will reduce Δ4977bp heteroplasmy by ≥50% in cardiac and skeletal muscle tissue within 8 weeks post-injection, accompanied by restoration of mitochondrial respiratory chain Complex I and Complex IV enzymatic activity (≥30% recovery toward young-mouse reference levels), measurable by droplet digital PCR (ddPCR) heteroplasmy quantification, Blue Native-PAGE in-gel enzyme activity assays, and Seahorse XF respirometry on freshly isolated tissue homogenates,

BECAUSE the following step-by-step causal chain applies:

1. The Δ4977bp deletion removes a 4,977 bp segment between positions 8470 and 13447, generating a unique chimeric neo-junction sequence (the fusion of the 3′ end of the upstream direct repeat to the 5′ end of the downstream direct repeat) that is entirely absent from full-length wild-type mtDNA — this junction is the sole recognition target (mitoARCUS meganuclease platform demonstrated to recognize and cleave pathogenic mtDNA targets with high specificity via the I-CreI scaffold) (mitoARCUS selectively eliminates mutant mtDNA in vivo)

2. A standard homodimeric ARCUS targeting only the neo-junction risks partial recognition of the native 13 bp direct repeat sequences (5′-ACCTCCCTCACCA-3′) present in wild-type mtDNA. An obligate heterodimeric architecture, in which mitoARCUS-8470 binds only the half-site 5′ of the breakpoint (positions ~8450–8470) and mitoARCUS-13447 binds only the half-site 3′ of the breakpoint (positions ~13447–13467), enforces a distance-dependent specificity constraint: a productive DSB can only occur when both monomers bind simultaneously at adjacent positions — a configuration geometrically impossible on wild-type mtDNA, where the two half-sites are 4,977 bp apart [SPECULATIVE — heterodimeric ARCUS format for common deletion has not been directly validated; rationale derived from obligate heterodimer logic validated for mitoTALENs, as described in the evidence synthesis literature review]

3. The two monomers are encoded in a single ORF separated by a P2A self-cleaving peptide, with the entire construct (2× MTS + 2× ~1.1 kb ARCUS monomer + P2A linker + regulatory elements) estimated at ~4.2–4.5 kb — within the ~4.7 kb packaging limit of AAV —...

SENS category: RepleniSENS

Key references: • doi.org/10.1038/s41467-021-23561-7