Circular RNA Telomerase Outperforms mRNA in Reversing Accelerated Aging in Progeria Cells

**Why This Matters** Cardiovascular aging is the leading driver of heart attack and stroke, and Hutchinson-Gilford Progeria Syndrome (HGPS) compresses decades of vascular aging into childhood. Understanding how to reverse endothelial senescence in this extreme model has direct implications for normal aging. Previous work showed that transient transfection with linear mRNA encoding telomerase reverse transcriptase (TERT) could partially rescue HGPS endothelial cells, but mRNA is short-lived, limiting its durability. Circular RNA, lacking the free ends that exonucleases attack, theoretically lasts longer and produces protein over an extended window.

**What Was Studied** The team generated endothelial cells (ECs) from iPSCs derived from HGPS patients and compared them to control ECs from genetically normal parents. They transfected HGPS-ECs once with equimolar amounts of wild-type TERT mRNA, catalytically inactive (CI) TERT mRNA, or TERT circRNA (1 µg/3–4×10⁵ cells), then tracked outcomes from 3 to 28 days post-transfection. Endpoints spanned morphology, angiogenesis, LDL uptake, nitric oxide production, telomere length (q-FISH), SA-β-galactosidase staining, nuclear morphology, gene expression of senescence/inflammatory markers, DNA damage (γH2AX), and mitochondrial health (ROS, membrane potential).

**Key Results** Across every endpoint, TERT circRNA was superior to linear TERT mRNA. Morphologically, circRNA-treated HGPS-ECs were more elongated and smaller in area, resembling healthy controls. SA-β-galactosidase-positive cells were reduced ~3-fold by circRNA vs. a lesser reduction by mRNA, and at 28 days post-transfection, circRNA-treated cells maintained low β-gal levels while mRNA-treated cells had reverted toward untreated HGPS levels. Telomere lengthening, confirmed by q-FISH, was also greater and more durable with circRNA. Senescence genes p16 and p21, the disease-specific protein progerin, and inflammatory cytokines IL-1β, IL-6, IL-8, and MCP-1 were all more substantially reduced by circRNA. Nitric oxide generation, acetylated LDL uptake, and Matrigel network formation (segments, master segments, tube length) were restored to near-normal levels. Mitochondrial ROS was reduced and membrane potential normalized more effectively by circRNA. Catalytically inactive TERT had no benefit, confirming that enzymatic telomerase activity—not mere RNA presence—drives these effects.

**Implications** This study positions TERT circRNA as a potentially transformative RNA therapeutic for vascular aging. A single dose providing weeks of benefit vastly improves on a regimen requiring repeated mRNA injections. Beyond HGPS, the approach could theoretically be applied to normal age-related endothelial dysfunction and cardiovascular disease. This is also the first reported cardiovascular and anti-senescence application of therapeutic circular RNA.

**Caveats** All experiments were conducted in vitro using iPSC-derived cells. Whether circRNA survives in vivo delivery and achieves sufficient tissue concentrations without immune activation remains to be established. The 28-day window, while encouraging, does not address whether a single dose would be sufficient therapeutically or how often re-dosing would be needed.