AAV8-delivered lysosome-targeted BgaD-D amplifies the senescent hepatocyte lysosomal compartment's prodrug-activating...

IF AAV8 vectors encoding lysosome-targeted Bacillus circulans BgaD-D (fused to the N-terminal GNPTAB lysosomal-targeting signal peptide sequence) under the hepatocyte-specific TBG promoter are delivered by tail-vein injection (1.0 × 10¹¹ vg) to aged (20–22 month) C57BL/6J male mice, followed 4 weeks later by systemic administration of galacto-navitoclax (PZ-15227) at a sub-therapeutic sub-senolytic dose (estimated 1–2 mg/kg, representing ≤30% of the dose used in RMR1 daily-dosing regimens),

THEN senescent hepatocyte burden—quantified as p16INK4a⁺ cells/HPF, SA-β-gal⁺ area percentage, and p21⁺ hepatocyte frequency—will decrease by ≥40% relative to aged AAV8-GFP controls at the 8-week post-injection timepoint, while systemic navitoclax-associated hematologic toxicity (thrombocytopenia, platelet counts) will be significantly attenuated compared to full-dose galacto-navitoclax monotherapy,

BECAUSE the following causal chain operates:

1. AAV8 serotype demonstrates superior hepatocyte tropism and achieves robust transduction (>90% of hepatocytes) following tail-vein administration, and this efficiency is maintained in aged C57BL/6J livers despite inflammaging-associated polyploidy, making TBG-driven transgene expression feasible across the hepatocyte population (Nathwani et al., AAV8 hepatocyte transduction cited in Evidence Set as NEJM 2011; Borel et al., Molecular Therapy 2014, cited in Evidence Set).

2. The GNPTAB lysosomal-targeting signal directs the expressed BgaD-D fusion protein into the mannose-6-phosphate receptor pathway, routing it to the lysosomal lumen, where it augments total intralysosomal β-galactosidase enzymatic activity—a mechanistic precedent firmly established in enzyme replacement gene therapy for lysosomal storage diseases (Evidence Set, Section 5: microbial enzyme gene therapy precedent for lysosomal function modulation in hepatocytes).

3. In senescent hepatocytes specifically, the lysosomal compartment is massively expanded compared to non-senescent neighboring cells—SA-β-gal (GLB1) is detectable at pH 6.0 precisely because lysosomes are enlarged, more numerous, and contain greater total GLB1 protein per cell (Dimri et al., 1995, PNAS, cited in Evidence Set)—meaning AAV8-delivered lysosome-targeted BgaD-D accumulates to disproportionately higher molar concentrations within the expanded lysosomal network of senescent hepatocytes relative to non-senescent hepatocytes, even when driven by a constitutive hepatocyte promoter. [SPECULATIVE: the magnitude of this differential accumulation due to lysosomal volume expansion in senescent vs. non-senescent hepatocytes is not yet quantified for an exogenous lysosome-targeted enzyme; however, the principle that expanded lysosomal compartments accumulate more lysosome-targeted protein per cell is consistent with enzyme replacement therapy uptake kinetics.]

4. Galacto-navitoclax (PZ-15227) is a prodrug in which navitoclax is capped by a galactose moiety; its cytotox...

SENS category: GlycoSENS