Efficient Downregulation of Flt-1 Mediated by Splice Switching ASO in Murine Endothelial Cells

Abstract

Impaired angiogenesis is a common feature of several pathological conditions, including neuromuscular disorders. Such vascular defects not only contribute to disease progression but also may compromise the efficacy of systemically delivered therapies such as antisense oligonucleotides (ASOs) and adeno-associated virus vectors. Enhancing muscle vascularization is therefore an attractive strategy to improve both therapeutic delivery and tissue regeneration. Vascular endothelial growth factor A (VEGF-A) is the principal driver of angiogenesis, but its bioavailability is negatively regulated by VEGFR1/Flt-1, a high-affinity decoy receptor. Here, we investigated a splice-switching ASO (SSO) approach to downregulate Flt-1 expression in murine endothelial cells. We designed ASOs to induce skipping of an out-of-frame exon in the Flt1 transcript, triggering nonsense-mediated decay and reducing protein expression. Screening in C166 endothelial cells identified a lead SSO that efficiently skipped exon 5, resulting in robust Flt-1 downregulation, similar to levels achieved with a control siRNA. Functionally, Flt-1 knockdown enhanced endothelial cell proliferation, survival, and migration upon VEGF-A stimulation. These results provide proof-of-concept for targeting Flt-1 via exon skipping to promote angiogenesis, with potential applications in degenerative or ischemic contexts where vascularization is impaired.