IF a secreted glucosepanase engineered as a collagen-binding domain (CBD) fusion protein — encoded in an AAV9 vector under a liver-specific TBG (thyroxine-binding globulin) promoter at a dose of 5×10¹¹ viral genomes administered via tail-vein injection — is delivered to 20-month-old male and female C57BL/6J mice rendered hypergly­cemic by low-dose streptozotocin (STZ; 50 mg/kg × 5 days i.p.) at 12 months of age to accelerate glucosepane accumulation,

THEN aortic pulse wave velocity (PWV) will be reduced by ≥20% relative to AAV9-TBG-GFP vehicle controls at 12 weeks post-injection, accompanied by a ≥40% reduction in aortic glucosepane concentration measured by isotope-dilution LC-MS/MS and a ≥15% decrease in ex vivo aortic stiffness by pressure myography,

BECAUSE the following causal chain is activated:

1. Chronic hyperglycemia in STZ-conditioned aged mice amplifies glucosepane crosslink density in aortic collagen beyond wild-type aged levels, overcoming the fundamental translational gap in which standard C57BL/6J mice accumulate glucosepane at concentrations orders of magnitude lower than aging humans, thereby creating a substrate-rich target tissue sufficient to detect therapeutic effect. [SPECULATIVE — magnitude of STZ-driven amplification in aged aorta is inferred from the literature review's recommendation to use STZ models; direct quantitative data in aged aortic tissue is a critical gap identified in Section 5 of the Evidence Set]

2. AAV9 administered systemically at 5×10¹¹ vg transduces hepatocytes broadly and enables sustained secretion of the glucosepanase-CBD fusion protein into systemic circulation, exploiting the liver as a biofactory for long-term enzyme production without requiring direct vascular transduction; (AAV9-CAG vectors effectively transduce multiple tissues including aorta in mice at doses like 5×10¹¹ vg, enabling systemic enzyme expression confirmed via immunohistochemistry)[https://pmc.ncbi.nlm.nih.gov/articles/PMC10543735/]. Use of a TBG promoter further restricts expression to hepatocytes, reducing the risk of off-target immunogenic presentation in non-hepatic antigen-presenting niches.

3. The collagen-binding domain fusion confers preferential retention of the secreted enzyme within the aortic ECM, addressing the critical barrier identified in the literature review — that diffusion of an unmodified secreted enzyme through the protease-rich, densely crosslinked medial layer of aged aorta would be insufficient to achieve catalytic concentrations at substrate (Evidence Set, Section 8: "therapeutic efficacy depends on the enzyme's stability in the protease-rich extracellular environment and its ability to access deep medial layers of the aorta"). The CBD, derived from CNA35 (collagen-binding adhesin from Staphylococcus aureus or analogous fibronectin-type-III collagen-binding repeats), binds fibrillar collagen types I and III with Kd ~1 µM, physically anchoring the enzyme to its macromolecular substrate ...

SENS category: GlycoSENS