We hypothesize that coupling brief OSK expression cycles with timed senolytic interventions will prevent senescence rebound and lock in youthful DNA methylation states, thereby sustaining lifespan extension beyond factor withdrawal.

Recent work shows that systemic OSK gene therapy extends median remaining lifespan in old mice by 109% and reverses epigenetic age without teratoma formation [https://pmc.ncbi.nlm.nih.gov/articles/PMC10909732/]. However, rejuvenation effects decay once OSK expression stops, with methylation age rebounding after treatment ends [https://www.fightingaging.org/archives/2025/10/a-review-of-the-present-state-of-epigenetic-reprogramming-to-treat-aging/]. This durability loss coincides with a senescence rebound that occurs when OSKM exposure persists beyond day 13‑15, triggering a p16^INK4a‑dependent secretory phenotype that undermines the epigenetically youthful state [https://pmc.ncbi.nlm.nih.gov/articles/PMC6351826/].

We propose that the senescence burst induced by transient OSK is not merely a toxic side‑effect but a signal that can be harnessed. OSK activates a stress response that upregulates p16^INK4a and SASP factors in a subset of cells; removing those senescent cells shortly after each OSK pulse should interrupt the inflammatory feedback loop that drives remethylation of aging‑associated loci. Senolytic agents such as dasatinib plus quercetin efficiently clear p16^INK4a‑positive cells and have been shown to improve healthspan in aged mice [https://doi.org/10.1016/j.cell.2015.05.031]. By administering senolytics 24 h after each 3‑day OSK ON period, we aim to eliminate the senescence rebound before it can remodel the chromatin landscape, allowing the youthful methylation pattern established during the OSK window to persist.

Mechanistically, senolysis may reduce NF‑κB‑driven HDAC recruitment and preserve TET‑mediated demethylation at key developmental promoters, maintaining an open chromatin configuration that resists de novo methylation by DNMT1. This would explain why epigenetic age stays low even after OSK withdrawal, as the cellular milieu no longer reinforces the aging methylome.

To test this, we will assign aged (24‑month) mice to four groups: (1) OSK pulses alone (3 days ON/4 days OFF), (2) senolytic pulses alone matched to OSK timing, (3) combined OSK + senolytic as described, and (4) vehicle control. All groups receive AAV‑mediated OSK delivery where applicable. Primary outcomes are epigenetic age (via Illumina EPIC mouse array) in liver, brain, and skin at 4‑week intervals, frailty index, and median lifespan. Secondary outcomes include tumor incidence, senescence marker (p16^INK4a) burden, and SASP cytokine levels.

If the combined group shows a statistically significant delay in methylation age rebound, sustained frailty improvement, and extended median lifespan compared to OSK‑only, while senolytic‑only shows no effect, the hypothesis is supported. Conversely, if epigenetic age rebounds at the same rate as OSK‑only despite senolytic treatment, or if no lifespan gain is observed, the hypothesis is falsified. This experiment directly tests whether clearing OSK‑induced senescence can convert a transient reprogramming stimulus into a durable anti‑aging intervention.