Male mice gain ~20 % lifespan from a single AAV‑Klotho dose, yet females develop severe pathology despite comparable transgene expression [2][3]. This divergence hints at a sex‑dependent downstream effect rather than a simple expression issue.

We hypothesize that endogenous estrogen amplifies hepatic secretion of soluble Klotho, converting a beneficial circulating factor into a pathological hormone excess that disrupts the FGF23‑Klotho phosphate axis and triggers ectopic calcification, inflammation, and hepatic stress.

Estrogen receptors are highly expressed in hepatocytes and can up‑regulate secretory pathway genes, a phenomenon documented for other endocrine proteins [8]. In Klotho‑KO mice, dysregulated microRNAs and tRNA fragments suggest that Klotho loss perturbs RNA‑mediated translational control, hinting that excess Klotho could overwhelm similar regulatory loops in a sex‑specific manner [8].

If hepatic Klotho overproduction drives female toxicity, then redirecting expression away from liver and restricting it to a tissue that does not respond to estrogen‑mediated secretory up‑regulation should abolish the adverse phenotype while preserving longevity benefits.

We will generate an AAV‑PF‑TfR1 capsid (machine‑guided, liver‑detargeted, brain‑penetrant) that carries the Klotho cassette under a muscle‑specific creatine kinase (CK8) promoter coupled to a tetracycline‑responsive element (TRE) for doxycycline‑inducible control [1][4][5][6]. Male and female mice will receive the vector at 12 months of age, with doxycycline administered to achieve physiological Klotho serum levels.

Primary outcomes: survival curves, serum Klotho and FGF23 levels, phosphate balance, histopathology of liver, kidney, and vasculature, plus functional assays of muscle regeneration and cognition. Secondary outcomes: anti‑AAV neutralizing antibody titers and any DRG toxicity signs [7].

If female mice exhibit the same lifespan extension as males without histological signs of ectopic calcification or liver injury, the estrogen‑hepatic secretion hypothesis gains support. Conversely, persistent toxicity despite muscle‑restricted, inducible expression would falsify the mechanism and point to alternative sex‑specific pathways (e.g., estrogen‑dependent immune modulation).

A successful outcome would validate a general strategy for sex‑balanced gene therapy: combine high‑precision capsid engineering, tissue‑specific promoters, and inducible systems to circumvent hormone‑driven off‑target effects, thereby expanding the therapeutic window for longevity interventions such as Klotho, GDF11, or FGF21.