Hypothesis

We hypothesize that a liver‑detargeted, muscle‑tropic AAV capsid delivering a secreted klotho protein fused to an albumin‑binding domain (ABD) will achieve sustained systemic klotho levels, efficiently cross the blood‑brain barrier via megalin‑mediated transcytosis, and extend lifespan in both male and female mice without relying on hepatocyte turnover or direct brain injection.

Mechanistic Rationale

1. Muscle as a secretory depot – Skeletal muscle is post‑mitotic, providing a stable niche for long‑term transgene expression, circumventing the dilution problem observed in hepatocytes (see discussion on liver‑detargeted AAVs)[https://pmc.ncbi.nlm.nih.gov/articles/PMC10000783/]. Muscle‑derived secreted proteins can enter the circulation and reach distant tissues.

2. Albumin‑binding domain extends half‑life – Fusion of klotho to an ABD (derived from streptococcal protein G or albumin‑binding motifs) exploits the neonatal Fc receptor (FcRn) recycling pathway, increasing plasma half‑life from hours to days, similar to albumin‑fusion therapeutics.

3. Klotho crosses the BBB – Soluble klotho can bind megalin (LRP2) on brain endothelial cells, facilitating receptor‑mediated transcytosis; this pathway is functional in adult mice and is upregulated under oxidative stress, a hallmark of aging.

4. Liver‑detargeted capsid reduces off‑target transduction and immune clearance – Engineered capsids with mutated hepatocyte‑binding residues (e.g., AAV9‑Y731F) lower liver uptake while preserving muscle tropism, decreasing anti‑AAV antibody generation and preserving transgene expression.

Predictions

• Biochemical: Mice receiving the ABD‑klotho AAV will show ≥2‑fold higher plasma klotho concentrations at 4, 8, and 12 weeks post‑injection compared with AAV‑klotho lacking ABD.
• Neurobiological: Brain klotho levels (measured by ELISA) will rise ≥1.5‑fold in cortex and hippocampus, correlating with reduced microglial activation (Iba1 staining) and improved synaptic markers (PSD‑95).
• Physiological: Treated mice will exhibit improved grip strength, enhanced rotarod performance, and preserved bone mineral density relative to controls.
• Longevity: Median lifespan will increase by ≥15% in both sexes when treatment is initiated at 12 months of age, surpassing the 19.7% increase reported for intracerebral klotho AAV in males only.

Experimental Design (testable & falsifiable)

1. Vector construction: Generate AAV9‑mut (Y731F) carrying a cassette: muscle‑specific CK8 promoter → secreted klotho‑ABD (HA‑tagged). Control vectors: AAV9‑WT‑klotho (no ABD) and AAV9‑mut‑klotho (no ABD).
2. Animal cohort: Male and female C57BL/6J mice, n=20 per group, treated at 12 months via tail‑vein injection (1e11 vg). Monitor for 12 months.
3. Readouts: Plasma klotho (ELISA) every 4 weeks; brain klotho via Western blot; immunohistochemistry for microglial activation; functional tests (grip strength, rotarod); bone density (DEXA); survival analysis.
4. Statistical plan: Power analysis to detect 15% lifespan increase (α=0.05, power=0.8). Use log‑rank test for survival; two‑way ANOVA for longitudinal biomarkers.

Falsifiability

If plasma klotho does not exceed control levels by ≥1.5‑fold, or brain klotho fails to rise despite elevated plasma levels, the hypothesis that ABD extends half‑life and enables BBB translocation is falsified. Likewise, if lifespan extension is not observed in either sex despite biochemical success, the claim that muscle‑derived secreted klotho drives systemic longevity effects is refuted.

This integrated approach leverages post‑mitotic muscle secretion, albumin‑mediated pharmacokinetic enhancement, and receptor‑mediated BBB transport to overcome the current limits of AAV‑klotho longevity therapy.