Muscle-Directed AAV Delivery of Mitochondrial-Targeted Senolytics Extends Healthspan via Mitokine-Mediated Systemic Rejuvenation

Hypothesis

Engineered AAV capsids with strong skeletal muscle tropism and minimal liver uptake can deliver a mitochondrially targeted senolytic peptide (e.g., a BCL‑XL inhibitor fused to a mitochondrial targeting sequence) to post‑mitotic myonuclei, achieving long‑term expression that reduces senescent cell burden and mitochondrial ROS in muscle. The resulting improvement in muscle mitochondrial function triggers secretion of mitokines such as FGF21 and GDF15, which act systemically to improve metabolic health, reduce inflammation, and extend lifespan in aging mice.

Rationale

• Recent advances show AAV9 crosses the BBB and AAVrh.74 leads in cardiac transduction, while AAV4 exhibits pan‑endothelial tropism via sialic acid binding independent of AAVR [1] [2].
• AI‑driven platforms like AAVGen can now simultaneously optimize capsid features for kidney tropism, thermostability, and manufacturability [3].
• Despite these capabilities, longevity applications remain unexplored; no clinical trials have tested AAV delivery of telomerase, Yamanaka factors, senolytics, or mitochondrial genes [1] [4].
• Skeletal muscle is a post‑mitotic, long‑lived tissue that can act as a secretory depot; myotropic AAV transduction yields stable transgene expression without the risk of insertional mutagenesis [5].
• Targeting the senolytic to mitochondria concentrates activity where ROS are produced, augmenting antioxidant defenses and reducing the SASP that drives systemic aging [6].

Experimental Plan

1. Capsid selection – Use directed evolution or AI design to generate a muscle‑biased AAV variant (e.g., AAVrh.74‑derived) with validated low liver transduction (<5% of total vector genomes) and high skeletal muscle uptake (>60% of injected dose) in mice.
2. Transgene construct – Clone a senolytic peptide (e.g., navitoclax‑derived BCL‑XL inhibitor) fused to a mitochondrial targeting signal (MTS) under a muscle‑specific promoter (CK8 or MHCI).
3. Animal cohorts – (a) Muscle‑targeted AAV‑senolytic‑MTS; (b) Liver‑targeted AAV‑senolytic‑MTS (AAV8 control); (c) Empty vector control; (d) Wild‑type aged mice. All groups receive a single intravenous dose at 18 months of age.
4. Readouts – At 1, 3, and 6 months post‑dose: quantify vector genome copy number in muscle vs liver (qPCR), assess senescence markers (p16^INK4a, SA‑β‑gal) and mitochondrial ROS (MitoSOX) in muscle biopsies, measure circulating FGF21 and GDF15 (ELISA), evaluate frailty index, grip strength, and glucose tolerance. Survival monitored up to 30 months.
5. Falsifiable outcomes – If muscle‑targeted AAV does not reduce muscle senescence or mitochondrial ROS relative to liver‑targeted or control groups, or if circulating mitokines and functional improvements are absent, the hypothesis is refuted. Conversely, a significant reduction in senescence/ROS, increased mitokines, and extended median lifespan would support the hypothesis.

Mechanistic Insight

The hypothesis extends current capsid engineering by proposing that the location of transgene expression—specifically, post‑mitotic muscle—creates a durable, low‑immunogenic secretory platform. Unlike hepatocytes, which turnover and can present capsid antigens, myonuclei persist, providing continuous senolytic expression without repeated dosing. Mitochondrial localization ensures the senolytic acts at the source of ROS, lowering oxidative damage and inhibiting the SASP. The resulting mitokine surge reprograms distant tissues (adipose, liver, brain) to a more youthful metabolic state, mirroring the endocrine effects observed with exercise‑induced myokines. This mechanism bridges the gap between advanced AAV delivery and longevity biology by exploiting muscle’s natural role as a systemic signaling organ.