Hypothesis

Engineered AAV capsids that combine liver detargeting, muscle tropism, and a protease‑cleavable linker can achieve sex‑neutral longevity benefits by restricting Klotho and follistatin expression to senescent skeletal muscle while avoiding hepatic exposure and neutralizing antibody epitopes.

Mechanistic Rationale

• Liver detargeting via surface tyrosine mutations (as in AAV2‑THGTPAD/NLPGSGD) reduces hepatocyte transduction and episomal dilution [https://pubmed.ncbi.nlm.nih.gov/35810332/].
• Muscle tropism is enhanced by inserting a myotrophin‑derived peptide into the VP3 hypervariable region, a strategy shown to improve infectivity in skeletal muscle [https://www.insights.bio/cell-and-gene-therapy-insights/journal/article/3367/generation-of-novel-aav-serotypes-with-enhanced-infectivity-specificity-and-lower-toxicity-via-aav-capsid-engineering-platform].
• Protease activation: Senescent muscle exhibits elevated MMP‑9 activity. A cleavable GG‑PK↓L sequence placed between the capsid and the transgene cassette (flanked by self‑cleaving 2A peptide) remains inert in circulation but releases Klotho‑FST cargo upon MMP‑9 cleavage, limiting systemic exposure.
• Sex‑specific toxicity mitigation: By confining expression to muscle, circulating Klotho levels stay within physiological range, preventing the ulcerogenic overexpression observed in females with liver‑dominant AAV‑Klotho [https://lifespan.io/news/a-klotho-gene-therapy-extends-life-in-male-mice/].
• Immune evasion: Surface grafting of a TLR2‑inhibitory peptide (derived from HSV‑gC) reduces innate activation and masks epitopes targeted by pre‑existing neutralizing antibodies [https://www.dovepress.com/adeno-associated-virus-engineering-and-load-strategy-for-tropism-modif-peer-reviewed-fulltext-article-IJN].

Testable Predictions

1. In aged (18‑month) female mice, a single intravenous dose of the protease‑activated, liver‑detargeted AAV (AAV‑MMP9‑Klotho/FST) will produce:
   • Skeletal muscle Klotho and follistatin protein levels ≥2‑fold over baseline, with serum Klotho unchanged (ELISA).
   • No gastric ulcers or histologic liver toxicity (H&E, ALT/AST) at 6 months post‑dose.
   • Median survival increase of ≥15 % relative to saline‑treated controls, matching the benefit seen in males with conventional AAV‑Klotho [https://lifespan.io/news/a-klotho-gene-therapy-extends-life-in-male-mice/].
2. In male mice, the same vector will not diminish the lifespan extension already observed with liver‑detargeted AAV‑Klotho, confirming non‑inferiority.
3. Serum neutralizing antibody titers against the vector will be ≤20 % of those measured after standard AAV9‑Klotho administration, reflecting epitope masking [https://www.dovepress.com/adeno-associated-virus-engineering-and-load-strategy-for-tropism-modif-peer-reviewed-fulltext-article-IJN].
4. In vitro, incubation of the vector with recombinant MMP‑9 will trigger transgene expression in C2C12 myotubes but not in HepG2 hepatocytes, confirming protease dependence.

Experimental Outline

• Vector production: Triple‑plasmid transfection in HEK293F, purification by iodixanol gradient.
• In vitro assay: MMP‑9 cleavage Western blot of capsid; luciferase reporter assay in muscle vs liver cell lines ± MMP‑9 inhibitor.
• In vivo study: Randomized cohorts (n=15/sex/group) receive IV 1×10¹³ vg/kg of test vector, AAV9‑Klotho (positive control), or saline. Monitor survival, clinical chemistry, ulcer scoring, muscle histology, and serum biomarkers every 3 months.
• Statistical analysis: Kaplan‑Meier with log‑rank test; two‑way ANOVA for longitudinal endpoints.

If the protease‑activated, liver‑detargeted AAV fulfills these predictions, it would demonstrate a mechanistic route to disentangle longevity benefits from sex‑specific toxicities, thereby expanding the therapeutic window for systemic AAV‑based anti‑aging interventions.