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Abstract

Plasminogen activator inhibitor 1 (PAI-1) is an inhibitor of fibrinolysis, thereby promoting blood clot stabilization. Circulating PAI-1 increases with age, and contributes to thrombosis, diet-induced obesity, and age-associated diseases. In contrast, partial PAI-1 deficiency protects patients from cardiovascular morbidity and extends lifespan. Decreasing circulating PAI-1 levels has both experimental and therapeutic value. However, previously developed PAI-1 inhibitors have stalled in clinical trials. RNA-based therapeutics offer an alternative, as they can regulate expression of proteins not amenable to traditional therapies. We developed a therapeutic approach to induce long-lasting PAI-1 knockdown in vivo with siRNA-lipid nanoparticles (siPAI-1). siPAI-1 decreased thrombus weight in both young and aged mouse models of venous thrombosis, normalized supraphysiologic PAI-1 levels in aged and obese mice, and prolonged lifespan in a fast-aging mouse model. Thus, siPAI-1 represents a long-term anti-thrombotic approach to limit pathologic impact of PAI-1 in aging and in age-related diseases. Conversely, complete PAI-1 deficiency is a rare genetic disorder caused by mutations in the SERPINE1 gene, characterized by absent circulating PAI-1 protein, moderate to severe bleeding, and cardiac fibrosis. No effective therapy currently exists for replacing PAI-1. We developed an mRNA therapy (mPAI-1) encapsulated in lipid nanoparticles to increase circulating PAI-1 levels. Following intravenous administration, mPAI-1 induced PAI-1 production in the liver, and resulted in supraphysiologic circulating PAI-1 levels and demonstrated functional activity by inhibiting fibrinolysis ex vivo. Expression of PAI-1 using mRNA-based approaches has the potential to be a preventative therapy for bleeding and cardiac fibrosis for PAI-1 deficient patients. To advance the translational relevance of these complementary approaches, we established a workflow to evaluate both siRNA- and mRNA- LNP in large animal models. Swine were selected given their cardiovascular, coagulation, and fibrinolytic systems closely resemble humans. As proof of concept, we employed plasminogen as a representative protein for siRNA knockdown and nanoluciferase as a reporter for mRNA delivery, chosen for their ease of detection and quantification. This platform enabled us to assess delivery, expression, and therapeutic modulation in vivo in a clinically relevant system.