Hypothesis

Combining transient OSK expression with periodic NAD+ precursor supplementation yields longer-lasting epigenetic age reversal than OSK alone by sustaining SIRT1/6 activity and preventing NAD+ depletion-driven feedback inhibition.

Mechanistic Rationale

Partial reprogramming with OSK opens chromatin at loci regulating NAD+ biosynthesis, notably increasing NAMPT transcription [1][2]. Elevated NAMPT raises intracellular NAD+, which activates SIRT1 and SIRT6 deacetylases. These enzymes remove acetyl groups from histones and transcription factors, stabilizing a youthful epigenome [3]. However, heightened PARP activity—triggered by OSK‑induced DNA‑damage signaling—consumes NAD+, creating a negative feedback loop that attenuates SIRT signaling over weeks [4]. Providing exogenous NAD+ precursors (e.g., nicotinamide riboside) during OSK pulses replenishes the pool, tipping the balance toward sustained deacetylase activity and prolonging the rejuvenated state.

Testable Predictions

• Prediction 1: In 24‑month‑old mice, systemic AAV‑OSK plus bi‑weekly nicotinamide riboside (NR) will maintain epigenetic age reduction (measured by Horvath‑mouse clock) for >6 months after the final OSK dose, whereas OSK alone will show reversal decay to baseline by 3 months.
• Prediction 2: The OSK+NR group will exhibit higher hepatic NAD+ levels and increased SIRT1/6 target deacetylation (e.g., PGC‑1α, H3K9ac) at 1 month and 4 months post‑treatment compared with OSK‑only.
• Prediction 3: No increase in tumorigenic incidence will be observed in the OSK+NR cohort relative to OSK‑only, confirming that NAD+ boosting does not compromise the safety profile of transient OSK.

Experimental Design

1. Groups (n=15 per group): (a) AAV‑OSK + NR (200 mg/kg oral, every 3 days for 4 weeks), (b) AAV‑OSK + vehicle, (c) AAV‑GFP + NR, (d) AAV‑GFP + vehicle.
2. OSK delivery: Single intravenous AAV9‑OSK dose (1×10¹² vg) with doxycycline‑inducible system active for 10 days.
3. Readouts: Blood NAD+ quantification, liver SIRT1/6 activity assays, whole‑blood epigenetic clock, frailty index, grip strength, and histopathology for tumors at 1, 3, and 6 months post‑induction.
4. Statistical analysis: Two‑way ANOVA with factors treatment and time; significance set at p<0.05.

Falsifiability

If OSK+NR fails to prolong epigenetic age reversal beyond OSK alone, or if NAD+ levels do not correlate with sustained SIRT activity, the hypothesis is refuted. Conversely, demonstration of extended rejuvenation without added risk supports the mechanistic link between NAD+ metabolism and OSK‑driven epigenetic remodeling.