Hypothesis

Transient OSK expression followed by NAD+ replenishment during the post‑expression recovery phase will extend and stabilize the epigenetic youthfulness induced by partial reprogramming beyond the 4‑week window observed with OSK alone, without compromising cellular identity or increasing tumorigenic risk.

Rationale

1. OSK‑driven demethylation is TET‑dependent – OSK pulses activate TET1/2, leading to active DNA demethylation at aging‑altered loci [3]. TET enzymes require α‑ketoglutarate (α‑KG) as a co‑substrate and are inhibited by succinate and fumarate, metabolites that rise when NAD⁺ is low.
2. NAD⁺ fuels both sirtuins and TET activity – Elevated NAD⁺ increases α‑KG production via enhanced TCA cycle flux and activates SIRT1, which deacetylates and stabilizes TET2 [4]. NAD⁺ also supports PARP‑mediated DNA repair, reducing oxidative stress that can impede demethylation.
3. Recovery phase is a permissive window – After OSK withdrawal, cells undergo a chromatin remodeling phase where demethylation peaks and epigenetic age continues to decline [4]. Providing NAD⁺ during this window should amplify TET activity and lock in a youthful methylome.
4. Safety is preserved – OSK alone avoids pluripotency and teratoma formation [2]. NAD⁺ boosters such as nicotinamide riboside (NR) or nicotinamide mononucleotide (NMN) have extensive safety data in mammals and do not induce proliferation when administered intermittently.

Experimental Design

• Animal model: Naturally aged C57BL/6 mice (24 months old).
• Groups (n = 15 per group):
  1. Vehicle control.
  2. Transient OSK (AAV‑OSK, 2‑day on/5‑day off, 4 cycles).
  3. NAD+ booster alone (NR 400 mg/kg/day, administered during the same recovery windows as group 2).
  4. Combined OSK + NAD+ (OSK pulses as in group 2, NR given daily only during the 5‑day off periods).
• Readouts (collected at baseline, 2 weeks, 4 weeks, and 8 weeks post‑final OSK pulse):
  • Epigenetic clocks (DNAm PhenoAge, GrimAge) from blood and liver.
  • TET activity (5‑hmC levels) and α‑KG/succinate ratios via LC‑MS.
  • Transcriptomic signatures of youthfulness (PATHWAY analysis).
  • Functional assays: grip strength, frailty index, retinal electrophysiology.
  • Safety monitoring: karyotype, pluripotency marker (OCT4, NANOG) expression, tumorigenic surveillance via ultrasound and histology.

Predicted Outcomes

• Group 4 will show a significantly greater reduction in epigenetic age (−2.5 years vs. −1.2 years in group 2) that persists to week 8, whereas group 2’s effect wanes after week 4.
• Elevated 5‑hmC and α‑KG/succinate ratios will be detected specifically in group 4 during recovery phases, linking NAD⁺ to enhanced TET activity.
• Functional improvements (strength, frailty, vision) will be additive, exceeding those of either intervention alone.
• No increase in pluripotency markers, chromosomal aberrations, or tumor incidence will be observed in any group.

Falsifiability

If group 4 does not demonstrate a statistically significant extension of epigenetic youthfulness beyond group 2 at any time point, or if NAD⁺ administration during recovery fails to boost TET activity or α‑KG levels, the hypothesis is refuted. Conversely, a clear synergistic effect would support the model that metabolic priming of the recovery window stabilizes and amplifies the benefits of transient OSK expression.