sFLT01-anti-ANG2: a Novel Potent Inhibitor as a Next-generation Anti-angiogenic Molecule

Abstract

Purpose : Current treatment for neovascular age-related macular degeneration includes intravitreal injection of anti-vascular endothelial growth factors (anti-VEGFs). However, clinical experience demonstrates that the efficacy of such therapies is restricted due to the overlapping and compensatory alternative angiogenic pathways. Here, we introduce a new anti-angiogenic molecule (sFLT01-anti-ANG2) that neutralizes VEGF, Placental Growth Factor, and Angiopoietin2 simultaneously and reduces the initiation of associated signaling pathways.

Methods : We investigated sFLT01 molecule components via bioinformatics tools and got access to its sequences. We augmented the nucleotide sequence of sFLT01 by another genetic syntax, against Angiopoietin2. Therefore, we analyzed the tertiary structures of the molecule with MODELLER and Nanome. The best models were applied in docking analysis with ClusPro. The cloning process of the construct in the AAV2 vector was performed and the result was confirmed by PCR and restriction enzyme digestion. RNA extraction and condition media collection were performed following the transfection of HEK293T cell line by AAV2-sFLT01-anti-ANG2. Expression of the gene of interest and its protein output was evaluated by real-time PCR and western blotting respectively. To confirm the functional anti-angiogenic potency of the protein, tube formation assay, phospho-Tie2 assay, and ligand-receptor interaction ELISA were performed

Results : The sFLT01-anti-ANG2 molecule was designed by bioinformatics tools and cloned into the pAAV-MCS vector.HEK293T cells were successfully transduced. RT-qPCR represented that the gene was expressed 4000 fold in HEK293T cell culture. Western blot technique confirmed the presence of this protein in the condition media collected from transfected HEK293T cell culture. The decrease in vascular network observed in tube formation assay showed that the molecule was functional. Also, the reduced amount of absorption observed from phospho-Tie2 assay (0.172.5/1.034,1.0455) and Ang2-Tie2 interaction ELISA assay (0.558/0.995,0.868) demonstrated effective function of this molecule.

Conclusions : We propose that targeting various angiogenic pathways by sFLT01-anti-ANG2 may be a fundamental approach in development of the next generation antiangiogenic therapeutic drugs.

This is a 2021 ARVO Annual Meeting abstract.