IF AAV9-TBG-PINK1 (1×10¹² viral genome particles, single retro-orbital injection, liver-specific thyroxine-binding globulin promoter) is administered to 22-month-old male and female C57BL/6J mice two weeks prior to two intermittent cycles of dasatinib (5 mg/kg) plus quercetin (50 mg/kg) oral gavage (days 1–3 of weeks 3 and 7), with senolytic timing gated by confirmed mitophagy flux restoration (mt-Keima red/green ratio ≥3-fold above aged vehicle baseline) rather than administered on a fixed calendar schedule,

THEN at week 16 post-injection, hepatic p16^Ink4a^-positive cell burden will be reduced by ≥50% compared to D+Q alone, SA-β-gal-positive hepatocytes per 1,000 cells will decrease by ≥40%, and SASP factors IL-6 and MCP-1 in liver lysate will be suppressed by ≥60% relative to D+Q-alone controls, with the mt-Keima red/green ratio sustained ≥3-fold above aged baseline through week 16, measured by confocal ratiometric imaging in ≥200 hepatocytes per animal,

BECAUSE the following mechanistic chain connects mitophagy restoration to durable suppression of re-senescence:

1. Aged hepatocytes exhibit an approximately 40% deficit in basal PINK1/Parkin-mediated mitophagy flux relative to young controls, quantifiable by mt-Keima ratiometric imaging, indicating that depolarized mitochondria fail to be delivered to the acidic lysosomal compartment and instead accumulate as a standing pool of damaged organelles (Sun et al., Molecular Cell, 2015 — cited in Evidence Set as describing the ~40% flux deficit in aged hepatocytes).

2. This failure of mitophagic clearance permits outer mitochondrial membrane permeabilization and cytosolic leakage of mitochondrial DNA (mtDNA), which activates the cGAS-STING innate immune pathway; STING-driven NF-κB and IRF3 signaling sustains the SASP and promotes paracrine induction of senescence arrest in neighboring hepatocytes, creating a feed-forward re-senescence loop that senolytics alone cannot interrupt (Sliter et al., Nature, 2018 — cited in Evidence Set as demonstrating PINK1 overexpression blunts IL-6 and MCP-1 via mitophagy restoration).

3. AAV9 vectors employing the liver-specific TBG promoter achieve >90–95% hepatocyte transduction efficiency following tail-vein or retro-orbital injection, providing durable, parenchyma-restricted PINK1 overexpression that avoids ectopic mitochondrial depolarization in cardiac or skeletal muscle tissue where ubiquitous promoters (CMV/CAG) cause off-target effects (Bell et al., Human Gene Therapy, 2011 — cited in Evidence Set).

4. Forced PINK1 overexpression via AAV9-TBG at doses of 1×10¹¹–5×10¹¹ vg/mouse restores sufficient PINK1 kinase activity to phosphorylate ubiquitin and Parkin at Ser65, re-initiating autophagic engulfment of defective mitochondria; at 1×10¹² vg (the proposed dose, one log-unit above the published lower effective bound), this should produce supraphysiological but hepatocyte-confined flux restoration sufficient to overcome the high proteostatic stress lo...

SENS category: GlycoSENS