Theses (PHD)

Corneal neovascularization significantly contributes to global blindness statistics, affecting approximately 1.4 million individuals annually, with 12% experiencing total blindness. Conventional anti-angiogenic therapies often require repetitive doses and later develop drug resistance. Recent advancements in genetically engineered mesenchymal stem cells (MSCs) using viral vectors present a promising strategy to enhance the efficacy of anti-angiogenic therapies. Adeno-associated virus (AAV) has been utilized in clinical settings to deliver therapeutic genes to the cornea due to its characteristic of transient integration into the genome. This study aims to genetically engineer MSCs overexpressed sFlt1 (MSCs.hsFlt1) using AAV to treat corneal neovascularization. Small and large-scale MSCs.hsFtl1 were produced and validated with gene and protein expressions. The functional in vitro study, based on tube formation assay, showed significant growth inhibition in human umbilical vein endothelial cells (HUVECs) at a low dose of MSCs.hsFlt1 and demonstrated a comparable anti-angiogenesis effect to bevacizumab. The toxicity and efficacy in vivo study involved 24 New Zealand white rabbits with mechanical wound injuries inflicted on the cornea. All rabbits were randomly divided into 3 groups and were treated subconjunctivally with normal saline, MSCs and MSCs.hsFlt1. Neovessels were measured and graded clinically. On day 91 post-treatment, neovascular lengths in MSCs.hsFlt1 decreased significantly and exhibited better anti-angiogenic effects than control and MSCs. Subconjunctival injection of genetically engineered MSCs.hsFlt1 effectively inhibits corneal neovascularization in both in vitro and in vivo models. These findings demonstrated safety and efficacy in managing neovascular conditions, especially the cornea, paving the way for further advancements in cellular gene therapy in a clinical setting.