Abstract Body (Do not enter title and authors here): Introduction:
Messenger RNA (mRNA) technology has the potential to revolutionize current therapeutic strategies for heart failure. However, mRNA-based therapies require high dosing for a robust therapeutic effect. Self-amplifying RNA (saRNA) bypasses this by enabling intracellular replication which significantly enhances protein expression and substantially reduces the initial RNA dose required. However, the comparative efficiency of saRNA versus conventional mRNA in the heart remains largely unexamined. Here, we aim to investigate this using healthy and heart failure murine model.

Materials and Methods:
We administered 0.5 µg of Luciferase (Luc) saRNA or Luc mRNA into the left ventricular (LV) myocardium of mice and monitored serial Luc expression over 14 days using an in vivo imaging system (IVIS). To evaluate off-target expression, ex vivo analysis was performed 24 hours after administration. In a myocardial infarction-induced heart failure mouse model, 0.5 µg of VEGF saRNA, VEGF mRNA, Luc saRNA, or PBS was injected into the infarct border area. Cardiac function was evaluated by echocardiography at two and four weeks after administration, followed by histological analysis.

Results:
IVIS analysis showed that Luc expression in the heart peaked on days 1–2 for both RNAs, with saRNA showing a 31.5±4.7-fold higher signal than mRNA (P<0.0001), sustained through day 14 (P=0.0001). Ex vivo analysis revealed no detectable expression in off-target organs. Mice treated with VEGF saRNA exhibited significantly reduced LV dilation (diastolic diameter (Dd): 4.26±0.08 mm; systolic (Ds): 3.34±0.04 mm) and improved ejection fraction (EF: 45.17±1.92%) compared to the VEGF mRNA (Dd: 4.66±0.06 mm, Ds: 3.99±0.07 mm, EF: 35.83±2.11%), Luc saRNA (Dd: 4.89±0.07 mm, Ds: 4.26±0.06 mm, EF: 29.33±1.87%), and PBS (Dd: 4.96±0.06 mm, Ds: 4.28±0.07 mm, EF: 31.01±1.63%) groups (all P<0.0001). Picro-Sirius Red staining revealed reduced fibrosis rate in the VEGF saRNA group (16.42±1.41 %) compared to the other groups (25.16±1.66 % vs. 27.93±1.98 % vs. 26.96±1.72 %, P=0.0006), and the number of CD31-stained endothelial cells in the border area was higher in the VEGF saRNA-treated group (3752.4±127.3/mm²) than in the other groups (2879.1±164.9/mm² vs. 2604.9±103.1/mm² vs. 2427.1±152.6/mm², P<0.0001).

Conclusions:
VEGF saRNA administration significantly enhanced protein expression in the heart, and improved cardiac function in a myocardial infarction-induced heart failure mouse model.