Intermittent OSK Pulses Combined with Mitochondrial Uncoupling Amplify Epigenetic Rejuvenation and Reduce Genomic Instability

Hypothesis

Intermittent, low‑dose OSK pulses paired with a mild mitochondrial uncoupler (e.g., BAM15) produce a more stable youthful epigenome than OSK alone, lowering the reprogramming‑induced copy‑number variation (CNV) burden while preserving or enhancing lifespan extension.

Mechanistic Rationale

Partial reprogramming with OSK activates a latent youthful epigenome and boosts mitochondrial oxidative phosphorylation (OXPHOS) [3], 4]. However, the transient OXPHOS surge can raise reactive oxygen species (ROS) enough to provoke DNA damage and CNV [6]. A low dose of a mitochondrial uncoupler mildly dissipates the proton gradient, which:

• Reduces ROS by preventing over‑reduction of the electron transport chain,
• Sustains a modest OXPHOS activation that still drives NAD⁺‑dependent deacetylase activity (e.g., SIRT1) needed for chromatin remodeling,
• Promotes a hormetic mitonuclear signal that reinforces the epigenetic backup without triggering stress‑induced senescence.

Thus, the uncoupler uncouples the pro‑rejuvenation signal from the genotoxic side‑effect, allowing fewer or shorter OSK cycles to achieve the same or greater epigenetic reset.

Testable Predictions

1. Epigenetic age: Mice receiving cyclic OSK + BAM15 will show a greater reduction in DNA methylation age (e.g., −4.5 months) compared with OSK‑only (−2.8 months) after 8 weeks of treatment.
2. Transcriptome: OXPHOS genes will be upregulated to a similar extent as OSK‑only, but ROS‑response genes (e.g., Hmox1, Nrf2 targets) will be significantly lower.
3. Genomic instability: Whole‑genome sequencing will reveal CNV levels comparable to untreated aged controls, whereas OSK‑only will exhibit a 1.8‑fold increase.
4. Lifespan: Median remaining lifespan will increase by ≥130 % over baseline, surpassing the 109 % gain reported for OSK alone [1] .
5. Safety: No teratoma formation or uncontrolled proliferation will be observed in any tissue, matching the safety profile of OSK without c‑MYC [2].

Experimental Design

• Animals: 20‑month‑old C57BL/6J mice, n = 15 per group (OSK‑only, OSK + BAM15, vehicle).
• Intervention: AAV9‑delivered doxycycline‑inducible OSK (no c‑MYC) administered intravenously; doxycycline given 2 days ON / 5 days OFF for 8 weeks. BAM15 formulated in chow at 5 mg kg⁻¹ day⁻¹ (dose shown to be non‑toxic in prior studies).
• Readouts:
  • Epigenetic clock (e.g., Horvath mouse clock) at weeks 0, 4, 8.
  • RNA‑seq of liver, muscle, and brain for OXPHOS and ROS pathways.
  • Low‑pass whole‑genome sequencing for CNV analysis.
  • Histopathology for teratomas and proliferation markers (Ki‑67).
  • Survival monitoring until natural death.

If the OSK + BAM15 group meets predictions 1‑5 while OSK‑only shows the expected intermediate phenotype, the hypothesis is supported. Failure to reduce CNV or improve epigenetic age beyond OSK‑only would falsify the claim that mild uncoupling decouples rejuvenation from genomic risk.