Hypothesis

Transient mRNA-delivered OSK, administered in pulsed cycles matching the dosing of viral OSKM, will achieve comparable tissue‑specific epigenetic rejuvenation and functional improvement as viral OSKM while maintaining a tumor‑free safety profile.

Rationale

• Non‑integrating mRNA eliminates insertional mutagenesis, a key safety advantage over viral vectors 1.
• Viral OSKM, despite integration risk, enables sustained factor expression that can drive robust epigenetic remodeling; however, continuous OSKM expression, especially with c‑Myc, triggers tumorigenesis in multiple organs 23.
• Short, cyclic OSK pulses (2‑4 days on, 5 days off) promote regeneration without tumors in muscle and intestine 45 and OSK alone extends lifespan in progeria/aged mice 6.
• We propose that the missing link between safety and efficacy is the Myc‑independent activation of the AMPK‑PGC‑1α axis. OSK (without Myc) can stimulate AMPK phosphorylation, leading to PGC‑1α‑driven mitochondrial biogenesis and enhanced NAD⁺/SIRT1 activity, which in turn facilitates DNA demethylation and histone turnover independent of HIF‑1α‑mediated hypoxia. This mitochondrial improvement reduces oxidative DNA damage, lowering the oncogenic pressure typically associated with c‑Myc expression.

Testable Predictions

1. Epigenetic parity – Liver and skeletal muscle from mice receiving pulsed mRNA OSK will show comparable reductions in epigenetic age clocks (e.g., Horvath DNAmAge) to those receiving matched‑dose viral OSKM after 8 weeks of treatment.
2. Functional equivalence – Measures of tissue‑specific function (e.g., serum ALT/AST for liver, grip strength for muscle) will not differ significantly between the two delivery modalities.
3. Tumor suppression – Histopathological surveillance over 6 months will reveal zero neoplasms in the mRNA OSK group, whereas a low‑frequency tumor incidence (<5 %) may persist in the viral OSKM group due to residual Myc activity or integration events.
4. Mechanistic validation – Western blot analysis will detect increased p‑AMPK and PGC‑1α protein levels exclusively in the OSK (no Myc) groups, correlating with mitochondrial respiration assays showing elevated OXPHOS capacity.

Experimental Design (brief)

• Groups: (A) viral OSKM (standard dose), (B) mRNA OSK matched total factor exposure, (C) mRNA OSKM (including Myc) as positive control for efficacy, (D) saline control.
• Regimen: 2‑day ON / 5‑day OFF cycles for 8 weeks, total factor dosage calibrated via qPCR of Factor mRNA in plasma.
• Readouts: epigenetic clocks, functional assays, tumor monitoring, mitochondrial assays.

If predictions 1‑2 hold while prediction 3 confirms tumor‑free outcome for mRNA OSK, the hypothesis is supported; failure to achieve epigenetic or functional parity would falsify the claim that mRNA OSK can match viral OSKM efficacy under matched dosing.