Sex‑Specific Senolytic‑Reprogramming Synergy Targets Endothelial Senescence via Transient OSK Expression

Hypothesis: Combining intermittent senolytic dosing with transient, lipid‑nanoparticle‑delivered OSK (Oct4, Sox2, Klf4) mRNA reprogramming will preferentially clear senescent endothelial cells and reverse vascular aging in a sex‑dependent manner, overcoming the proinflammatory limitation of quercetin observed in females.

Mechanistic Rationale: Quercetin reduces senescence in male vascular cells that harbor a higher baseline senescent load, but in females—whose endothelium is comparatively healthy—it raises CRP and triggers pro‑inflammatory pathways, likely by interfering with Nrf2‑mediated antioxidant responses in non‑senescent cells (NAD.com trial). Transient OSK expression resets chromatin to a more youthful state, dampening SASP production and restoring Nrf2 activity without inducing pluripotency, thereby creating a permissive environment where senolytics can act selectively on truly senescent cells. This two‑step approach addresses the critical gap of integrating senolytics with epigenetic reprogramming to achieve robust rejuvenation rather than incremental improvement.

Experimental Design: Use aged (20‑month) male and female C57BL/6 mice. Group 1 receives vehicle; Group 2 receives intermittent dasatinib + quercetin (D+Q) as per published regimen (PMID: PMC12190739); Group 3 receives OSK‑mRNA LNPs (dose calibrated to achieve transient expression <48 h, based on recent mRNA‑LNP work); Group 4 receives the sequential combination—OSK‑mRNA LNPs administered 24 h before each D+Q cycle. Treatment spans 3 months with monthly dosing cycles. Primary endpoints: (1) endothelial senescence measured by SA‑β‑gal and p16^Ink4a^ flow cytometry in aortic isolations; (2) vascular function via ex vivo aortic ring vasoreactivity; (3) systemic inflammation (plasma CRP, IL‑6); (4) epigenetic age using the Horvath skin‑&‑blood clock on blood DNA. Secondary endpoints include exercise tolerance and cardiac echocardiography.

Predicted Outcomes: In males, D+Q alone will reduce senescent endothelial cells ~30 % (consistent with prior vasculature data). In females, D+Q alone will show minimal senolysis and a rise in CRP. The OSK‑primed group (Group 3) is expected to lower baseline SASP markers and shift the epigenetic clock backward by ~1.5 years across sexes. The combination group (Group 4) should achieve synergistic senolysis (>50 % reduction in p16^+ endothelial cells) in both sexes, normalize CRP, improve vasodilatory response to acetylcholine by >25 %, and reverse epigenetic age by ~2.5 years, with no signs of teratoma formation or off‑target pluripotency markers.

Potential Pitfalls and Mitigations: Transient OSK expression risks inadvertent reprogramming of non‑senescent cells; mitigating by using mRNA LNPs with endothelial‑specific ligands (e.g., anti‑PECAM‑1 antibodies) and confirming limited duration via single‑cell RNA‑seq. Sex‑specific pharmacokinetics of quercetin may confound results; measuring plasma quercetin levels will allow dose adjustment. Finally, epigenetic clock variability in mice can be addressed by using multiple clocks (Horvath, Hannum) and longitudinal sampling.

Falsifiability: If the combination fails to surpass senolytic monotherapy in senescent cell clearance, epigenetic age reversal, or functional improvement in either sex—or if it exacerbates inflammation—the hypothesis is refuted. Conversely, meeting the predicted outcomes supports the mechanistic claim that transient epigenetic reprogramming creates a sex‑neutral senolytic‑sensitive state, enabling robust vascular rejuvenation.