Splice switching of membrane-bound FLT1 into a soluble protein as a treatment for pathological angiogenesis

Abstract Pathological retinal neovascularization is associated with dysregulated vascular endothelial growth factor (VEGF) signaling. Soluble FLT1 (sFLT1), an endogenous VEGF decoy receptor, is generated through alternative 3′-end processing of FLT1 pre-mRNA. We investigated the cis-regulatory elements that control this process and used splice-switching oligonucleotides (SSOs) to increase sFLT1 production. Two overlapping SSO panels were screened in HEK293FT cells using qPCR and ELISA. We validated lead candidates in CRISPRa-engineered, FLT1 -enriched Müller glial cells using qPCR, ELISA, and a VEGF-responsive luminescence assay. Proximal SSOs increased sFLT1 without altering total FLT1 transcript levels, indicating altered RNA processing rather than increased gene expression. Distal SSOs also enhanced sFLT1 by targeting polyadenylation signals. SSO-16 and SSO-19 produced the greatest increases in sFLT1 and significantly attenuated VEGF signaling. These findings identify FLT1 intron 13 regulatory elements as promising therapeutic targets for retinal vascular disease while preserving FLT1 expression and supporting targeted modulation of VEGF signaling.