Abstract
Purpose :
Dysregulated angiogenesis is a major pathological factor associated with vasculopathies underpinning conditions linked to vision loss, such as diabetic retinopathy and neovascular age-related macular degeneration. Current treatments are not fully effective in all the patients due to particular issues associated with resistance, response, and side effects. The subcellular localization of RAB13 and NET1 mRNAs critically regulates endothelial cell behaviour and morphogenesis during angiogenesis. Recent studies have demonstrated that antisense oligonucleotides (ASOs) designed to target localization elements within 3‘UTRs can disrupt the correct distribution of these mRNAs. The aim of this study was to investigate the in vitro potential of mRNA mislocalization ASOs as therapeutic tools to control undesired blood vessel formation.
Methods :
The subcellular localization of RAB13, NET1, and GAPDH mRNAs were determined in primary human retinal endothelial cells (HRECs) and human umbilical vein endothelial cells (HUVECs) by single molecule fluorescence in situ hybridization (smFISH), and the polarization indeces were measured to quantify their asymmetric distribution. Fluorescently labelled 2’O-methoxyethyl (2’MOE) modification ASOs were used to determine the efficiency of viability, delivery, and their persistence in endothelial cells. HRECs and HUVECs were treated with 2'MOE mislocalization ASOs targeting RAB13 and NET1 mRNAs for 48 hours. Polarization indeces, protein and RNA levels were examined, and the ability to prevent vessel formation was evaluated using in vitro systems simulating early stages of angiogenesis.
Results :
RAB13 and NET1 is prominently enriched at extending peripheral protrusions in HRECs and HUVECs. Fluorescently labelled ASOs were taken up by virtually all cells and persisted at a concentration above 1 μM, for more than 4 days. smFISH studies after treatment with mislocalization ASOs targeting RAB13 and NET1 mRNAs revealed significant reduction in polarization indeces of these mRNAs, without affecting the protein or RNA levels. The in vitro systems of angiogenesis showed that perturbing the localization of RAB13 and NET1 mRNAs halts endothelial cell sprouting and vessel formation.
Conclusions :
Our results shows that targeting subcellular mRNA localization with mislocalizing ASOs may represent an effective therapy for the management of neovascularization-related retinal disorders.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.