Hypothesis

Intermittent elevation of NAD+ levels during the off‑days of pulsed OSK expression prevents the re‑accumulation of age‑associated DNA methylation and maintains histone acetylation, thereby locking in a youthful epigenome after reprogramming stops.

Rationale

Partial reprogramming with OSK resets epigenetic clocks but the effect is transient because cellular NAD+ declines between pulses, reducing SIRT1 activity and allowing histone deacetylases to re‑establish repressive marks. Prior work shows that NAD+ boosters improve SIRT1‑mediated deacetylation and enhance DNA repair, which synergizes with OSK‑driven chromatin opening. Senolytic combinations already improve healthspan in flies, suggesting that metabolic support can prolong reprogramming benefits.

Prediction

Mice receiving pulsed OSK (2 days on/5 days off) plus a NAD+ precursor (e.g., nicotinamide riboside) administered only during the off‑days will show:

• Persistent reduction of epigenetic age in blood and liver beyond 8 weeks after the last OSK pulse,
• Sustained improvement in frailty scores and tissue function,
• No increase in tumorigenesis compared with OSK alone.

Experimental Design

1. Groups (n=15 per group, 124‑week‑old C57BL/6):
   • Vehicle control
   • Pulsed OSK only (AAV‑OSK, doxycycline 2 days on/5 days off)
   • NAD+ booster only (nicotinamide riboside 400 mg/kg daily, off‑days only)
   • Combined OSK + NAD+ booster (same schedule)
2. Readouts (baseline, 4 wks, 8 wks post‑last OSK):
   • DNA methylation age (Horvath mouse clock)
   • Histone H3K27ac ChIP‑seq in liver and muscle
   • Frailty index, grip strength, echocardiogram
   • Tumor surveillance (MRI, histology)
3. Analysis: Compare slope of epigenetic age change; test interaction effect.

Potential Outcomes

• If NAD+ supplementation maintains low epigenetic age and high H3K27ac after OSK cessation, the hypothesis is supported.
• If epigenetic age rebounds to control levels despite NAD+, the hypothesis is falsified, indicating that NAD+/SIRT1 is not the limiting factor for durability.

Falsifiability

The study provides a clear, quantitative endpoint (epigenetic age trajectory) that can confirm or refute the predicted stabilizing effect of NAD+ during off‑cycles.