Design, Construction and in-vivo Functional Assessment of SFN0011: a Novel Potent Inhibitor Developed as a Next-generation Anti-angiogenic Molecule
Hamid Latifi-Navid1 , Zahra-Soheila Soheili1 *, Mehdi Sadeghi2 , Shahram Samiei3 , Narges Zolfaghari1 , Sepideh Taghizadeh1 , Seyed Shahriar Arab4 , Hamid Ahmadieh5 , Mozhgan Rezaei Kanavi6 , Ramin Nourinia5
- Department of Molecular Medicine, National Institute of Genetic Engineering and Biotechnology, Tehran, Iran
- Department of Medical Genetics, National Institute for Genetic Engineering and Biotechnology, Tehran, Iran
- Blood Transfusion Research Center, High Institute for Research and Education in Transfusion Medicine, Tehran, Iran
- Department of Biophysics, School of Biological Sciences, Tarbiat Modares University, Tehran
- Ophthalmic Research Center, Research Institute for Ophthalmology and Vision Science, Shahid Beheshti University of Medical Sciences, Tehran, Iran
- Ocular Tissue Engineering Research Center, Research Institute for Ophthalmology and Vision Science, Shahid Beheshti University of Medical Sciences, Tehran, Iran
Abstract: Intravitreal injection of anti-VEGF agents plays a principal role in the treatment of age-related macular degeneration. However, clinical experience indicates that the efficacy of such therapies is restricted due to overlapping and compensatory alternative angiogenic pathways leading to escape mechanisms. Therefore, we designed a new anti-angiogenic molecule (SFN0011) –based on sFLT01 chimeric protein - to neutralize both vascular endothelial growth actor A (VEGF-A) and Angiopoietin 2 (ANG2) simultaneously and reduce the initiation of their associated signaling pathways.
Methods: We investigated sFLT01 molecule structural components via bioinformatics tools and accessed to its amino acid and nucleotide sequences. We augmented the nucleotide sequence of sFLT01ʹs by another genetic syntax, against to a nominated antigenic factor. Then, we analyzed the secondary and tertiary structures of the cognate tri-specific molecule with modeller and nanome. The best models were applied in 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 real-time PCR and western blotting respectively. To study in vivo functional assessment of SFN0011 in both physiological and pathological angiogenesis, we used intravitreal AAV2-SFN0011 injection in new-born and laser-induced choroidal neovascularization mice models, respectively.
Results: The SFN0011 molecule was designed by bioinformatics tools and cloned into the pAAV-MCS vector. HEK293T cells were successfully transduced. RT-qPCR represented a 4000-fold gene expression 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 was observed from phospho-Tie2 assay (0.172.5/1.034,1.0455) and Ang2-Tie2 interaction ELISA assay (0.558/0.995,0.868) demonstrating effective function of this molecule.
Conclusion: We propose that targeting various angiogenic pathways by SFN0011 may be a fundamental approach in development of next generation anti-angiogenic drugs for AMD and other neovascular ocular diseases.
