Hypothesis: A dynamic, biomarker‑driven dosing regimen for transient OSK expression can keep cells within a rejuvenation window that maximizes epigenetic age reversal while minimizing senescence‑associated stem‑like plasticity and tumorigenic risk.

Rationale: Recent work shows that cyclic partial reprogramming extends lifespan in mice but carries oncogenic hazards when reprogramming exceeds a tissue‑specific threshold [2][3]. Therapy‑induced senescence in cancer cells can paradoxically fuel relapse by inducing stemness, a process that mirrors the plasticity seen during overly aggressive reprogramming [4]. Thus, the therapeutic window is narrow and likely varies across cell types and physiological states.

Mechanistic insight: Epigenetic clocks derived from CpG methylation patterns in circulating cell‑free DNA (cfDNA) reflect the biological age of multiple tissues and can be measured serially with high sensitivity [1]. Simultaneously, senescence‑associated secretory phenotype (SASP) factors such as IL‑6, IL‑8, and MMP‑3 rise when cells approach a plastic, stem‑like state [4]. By integrating cfDNA‑based epigenetic age (e.g., DunedinPACE) with plasma SASP concentrations into a feedback algorithm, we can titrate the dose and frequency of lipid‑nanoparticle‑delivered OSK mRNA to maintain cells in a state where epigenetic age is decreasing but SASP remains below a predefined safety threshold.

Experimental design: 1) Generate a murine model expressing a doxycycline‑inducible OSK cassette alongside a fluorescent senescence reporter (p16‑GFP). 2) Treat aged mice with OSK mRNA LNPs at varying intervals. 3) Serial blood draws yield cfDNA for epigenetic clock analysis and plasma for SASP cytokine measurement via multiplex assay. 4) An algorithm adjusts the next OSK dose: if epigenetic age reduction exceeds 1.5 years per month or SASP IL‑6 rises >2‑fold baseline, the dose is reduced or postponed; if both metrics indicate insufficient rejuvenation (<0.5 year/month epigenetic gain and SASP unchanged), the dose is increased. 5) Endpoints include tissue‑specific epigenetic age, functional assays (grip strength, treadmill endurance), senescence burden (p16‑GFP+, SA‑β‑gal), and tumor incidence over 12 months.

Falsifiable predictions: (a) Mice receiving the closed‑loop regimen will show greater median lifespan extension than fixed‑dose cyclic OSK without increased tumor onset; (b) Exceeding the SASP threshold will correlate histologically with expansion of Keratin‑14+/Sca‑1+ progenitor‑like lesions in epithelial tissues, indicating oncogenic plasticity; (c) Disabling the SASP feedback (e.g., IL‑6 neutralizing antibody) will lead to higher tumor rates despite similar epigenetic age reduction, confirming that SASP serves as a safety readout beyond methylation clocks.

This approach translates static epigenetic biomarkers into a closed‑loop control system, directly addressing the need for tissue‑specific, real‑time titration of reprogramming intensity to maximize benefit while limiting the risk of senescence‑driven tumorigenicity.