Hypothesis

Hepatocyte-targeted self-spreading CRISPR-mediated ANGPTL3 knockout lowers circulating triglycerides and reduces lipotoxic stress, which when paired with intermittent senolytic administration decreases senocyte burden and mitochondrial ROS, leading to improved healthspan in aged mice.

Mechanistic Rationale

• ANGPTL3 inhibition enhances lipoprotein lipase activity, accelerating triglyceride clearance 2. Lower lipid accumulation in hepatocytes diminishes ceramide-induced mitochondrial permeability transition, curbing ROS production.
• Self-spreading CRISPR platforms achieve >3‑fold editing efficiency in liver parenchyma after a single dose, enabling sustained ANGPTL3 suppression without repeated vector administration 7.
• Intermittent senolytic dosing (e.g., dasatinib + quercetin) selectively induces apoptosis in p16^high^ senescent cells, reducing SASP‑driven oxidative inflammation 8.
• Combined, reduced lipid stress and senocyte clearance synergistically lower systemic oxidative damage, preserving stem‑cell function and tissue homeostasis.

Experimental Design

1. Animal model: C57BL/6 mice, 20 months old, n=15 per group.
2. Groups:
   • G1: Vehicle control.
   • G2: Self-spreading CRISPR ANGPTL3 knockout (single IV injection of lipid‑nanoparticle‑ packaged SaCas9‑gRNA).
   • G3: Intermittent senolytic (dasatinib 5 mg/kg + quercetin 50 mg/kg, i.p., once weekly for 4 weeks, then 2‑week rest, repeat three cycles).
   • G4: Combined CRISPR ANGPTL3 knockout + senolytic regimen (same schedules as G2 and G3).
3. Intervention timeline: Treatments start at baseline; assessments at 0, 3, 6, and 9 months.
4. Readouts:
   • Plasma triglycerides, HDL‑C, LDL‑C measured via enzymatic assays.
   • Hepatic ANGPTL3 mRNA and protein levels quantified by qPCR and Western blot.
   • Senocyte burden assessed through p16^Ink4a^ immunostaining and flow cytometry for p21^high^ cells.
   • Mitochondrial ROS measured using MitoSOX fluorescence in isolated hepatocytes.
   • Functional healthspan evaluated by grip strength, treadmill endurance, and a frailty index incorporating grip, gait, and activity metrics.
   • Serum ALT and AST monitored to detect hepatotoxicity.
   • Off‑target editing assessed by amplicon deep sequencing of predicted homologous sites.

Predicted Outcomes

• G2 will show approximately a 40 % reduction in plasma triglycerides relative to G1, accompanied by a modest (~15 %) decline in hepatic mitochondrial ROS.
• G3 is expected to exhibit roughly a 30 % decrease in senocyte frequency, with an intermediate (~20 %) reduction in ROS.
• G4 should achieve greater than 60 % triglyceride lowering, greater than 50 % senocyte clearance, and the most pronounced ROS suppression (≈45 % vs G1).
• Functional readouts predict a ≥25 % improvement in grip strength, a ≥30 % increase in treadmill endurance, and a significant reduction in frailty score for G4 compared with all other groups.
• Safety metrics (ALT/AST) are anticipated to remain within normal ranges, and off‑target indels are projected to stay below 0.1 % of total alleles.

Falsifiability

If G4 fails to demonstrate a statistically significant (p<0.05) improvement in at least two of the three functional healthspan metrics (grip strength, treadmill endurance, frailty index) relative to both G2 and G3, the hypothesis is falsified. Likewise, if oxidative stress markers (MitoSOX, plasma 8‑iso‑PGF2α) show no meaningful change across groups, or if deep sequencing reveals persistent off‑target edits exceeding 0.1 % in liver DNA, the safety and mechanistic premises of the approach would be invalidated.