Hypothesis

Transient expression of OSK combined with periodic senolytic clearance yields a durable epigenetic age reset that outlasts factor withdrawal, because senescent cells secrete SASP factors that drive re‑methylation and chromatin compaction after reprogramming.

Rationale

Partial reprogramming with OSK reverses mesenchymal drift and resets DNA methylation clocks, but the effect fades once factors are removed [1,2]. One reason for rebound is the persistence of a senescent microenvironment: SASP cytokines (IL‑6, TGF‑β) activate DNMTs and HDACs in neighboring cells, pushing the epigenome back toward an aged state [5]. Senolytics such as dasatinib + quercetin eliminate these cells, reducing SASP burden and potentially locking in the youthful chromatin configuration achieved by OSK.

Experimental Design

Model: Naturally aged C57BL/6 mice (24 mo). Groups: (1) Vehicle control; (2) Cyclic OSK AAV‑inducible (2 d on/5 d off) × 4 cycles; (3) Senolytic regimen (dasatinib 5 mg/kg + quercetin 50 mg/kg, i.p., once weekly) × 4 cycles; (4) Combined OSK + senolytic on the same schedule. Readouts:

• Epigenetic age measured by multi‑tissue Horvath clock at baseline, post‑treatment, and every 4 weeks for 20 weeks after cessation.
• Tissue‑specific methylation arrays (liver, kidney, brain) to assess durability.
• Functional assays: grip strength, treadmill endurance, frailty index.
• Senescence burden (p16^Ink4a^‑positive cells, SASP ELISA).
• Safety: tumor incidence, karyotype. Duration: 20 weeks post‑intervention observation window.

Predicted Outcomes

• Primary: Group 4 will show a statistically significant slower rebound of epigenetic age compared with groups 2 and 3, maintaining a youthful methylation profile for ≥12 weeks after the last OSK cycle.
• Secondary: Improved functional endpoints in group 4 relative to OSK alone, correlating with lower residual SASP levels.
• Safety: No increase in tumor formation versus OSK alone, confirming that senolytic addition does not compromise the safety of transient reprogramming.

Potential Pitfalls & Mitigations

• Senolytic toxicity: Intermittent dosing reduces off‑target effects; monitor liver enzymes.
• Variable OSK expression: Use a tightly regulated doxycycline‑inducible AAV to ensure uniform transient expression.
• Compensatory proliferation: Include BrdU labeling to detect hyperplastic responses; adjust senolytic schedule if needed.

If the combined approach fails to extend the reset duration, the hypothesis would be falsified, indicating that SASP‑driven re‑methylation is not a major barrier to durability and prompting investigation of alternative maintenance mechanisms (e.g., NAD+ boosters or histone chaperone modulation).