Designing, construction and functional analysis of SFN۰۰۱۱ as a possible next generation antiangiogenic molecule.
- سال انتشار: 1399
- محل انتشار: چهارمین کنگره بین المللی و شانزدهمین کنگره ملی ژنتیک
- کد COI اختصاصی: CIGS16_054
- زبان مقاله: انگلیسی
- تعداد مشاهده: 307
نویسندگان
Department of Molecular Medicine, National Institute of Genetic Engineering and Biotechnology, Tehran, Iran
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
Molecular Biomarkers Nano-Imaging Laboratory, Brigham and Women's Hospital, Boston, MA, USA
Department of Molecular Medicine, National Institute of Genetic Engineering and Biotechnology, Tehran, Iran
چکیده
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. sFLT۰۱ is a novel fusion protein that consists of the VEGF/PlGF (placental growth factor) binding domains of human VEGFR۱/Flt-۱ (hVEGFR۱) which are fused to the Fc portion of human IgG (۱) through a polyglycine linker.Methods: We investigated sFLT۰۱ molecule structural components via bioinformatics tools and achieved to its amino acid and nucleotide sequences. We augmented the nucleotide sequence of sFLT۰۱ʹs 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 AAV۲ 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 HEK۲۹۳T cell line by AAV۲-SFN۰۰۱۱. 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-Tie۲ assay, and Ang۲-Tie۲ interaction ELISA was performed.Results: We designed, constructed, and produced a sFLT۰۱-based novel tri-specific molecule (SFN۰۰۱۱) that targets VEGFA, PLGF, and a third party angiogenic factor that could efficiently inhibit tube formation, Tie۲ phosphorylation and Ang۲-Tie۲ interaction in vitro.Conclusion: We propose that targeting various angiogenic pathways by SFN۰۰۱۱ may be a fundamental approach in development of next generation antiangiogenic therapeutic drugs for age-related macular degeneration and other related diseases.کلیدواژه ها
Age-related macular degeneration , Angiogenesis, sFLT۰۱, SFN۰۰۱۱, VEGF, Resistanceاطلاعات بیشتر در مورد COI
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