Designing, Construction and Functional Analysis of Sfn0011 As A Possible Next Generation Antiangiogenic Molecule.

Background and aim: age-related macular degeneration (amd) is the most common cause of irreversible blindness among the elderly population. the current treatment options for amd include intravitreal injection of anti-vascular endothelial growth factor (anti-vegf). however, clinical experiences demonstrate that the efficacy of such therapies is limited due to overlapping and compensatory alternative angiogenic pathways which culminate to escape mechanisms. sflt01 is a novel fusion protein that consists of the vegf/plgf (placental growth factor) binding domains of human vegfr1/flt-1 (hvegfr1) which are fused to the fc portion of human igg (1) through a polyglycine linker. methods: we investigated sflt01 molecule structural components via bioinformatics tools and achieved to its amino acid and nucleotide sequences. we augmented the nucleotide sequence of sflt01ʹs by another genetic syntax, against to a nominated antigenic factor. so, we analyzed the secondary and tertiary structures of the cognate tri-specific molecule with swissmodel and i-tasser. the best models were applied in protein-protein docking analysis with cluspro. the cloning process of the construct was performed in the aav2 vector and the result was confirmed by conventional pcr and restriction enzyme digestion. rna extraction and culture condition media collection were performed following the transfection of hek293t cell line by aav2-sfn0011. expression of the gene of interest and its protein output was evaluated by pcr and western blotting respectively. to confirm the functional anti-angiogenic potency of the protein, tube formation assay, phospho-tie2 assay, and ang2-tie2 interaction elisa was performed.results: we designed, constructed, and produced a sflt01-based novel tri-specific molecule (sfn0011) that targets vegfa, plgf, and a third party angiogenic factor that could efficiently inhibit tube formation, tie2 phosphorylation and ang2-tie2 interaction in vitro.conclusion: we propose that targeting various angiogenic pathways by sfn0011 may be a fundamental approach in development of next generation antiangiogenic therapeutic drugs for age-related macular degeneration and other related diseases.