Gene therapy for the treatment of ocular neovascularization has reached clinical trial phases. The AAV2-sFLT01 construct was already evaluated in a phase 1 open-label trial administered intravitreally to patients with advanced neovascular age-related macular degeneration. SFLT01 protein functions by binding to VEGF and PlGF molecules and inhibiting their activities simultaneously. It consists of human VEGFR1/Flt-1 (hVEGFR1), a polyglycine linker, and the Fc region of human IgG1. The IgG1 upper hinge region of the sFLT01 molecule makes it vulnerable to radical attacks and prone to causing immune reactions. This study pursued two goals: (i) minimizing the immunogenicity and vulnerability of the molecule by designing a truncated molecule called htsFLT01 (hinge truncated sFLT01) that lacked the IgG1 upper hinge and lacked 2 amino acids from the core hinge region; and (ii) investigating the structural and functional properties of the aforesaid chimeric molecule at different levels (in silico, in vitro, and in vivo). Molecular dynamics simulations and molecular mechanics energies combined with Poisson–Boltzmann and surface area continuum solvation calculations revealed comparable free energy of binding and binding affinity for sFLT01 and htsFLT01 to their cognate ligands. Conditioned media from human retinal pigment epithelial (hRPE) cells that expressed htsFLT01 significantly reduced tube formation in HUVECs. The AAV2-htsFLT01 virus suppressed vascular development in the eyes of newborn mice. The htsFLT01 gene construct is a novel anti-angiogenic tool with promising improvements compared to existing treatments.
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This work was supported by the National Institute of Genetic Engineering and Biotechnology (NIGEB) through grant no.649. Our gratitude thanks to the INSF, Iran National Science Foundation, for the Doctoral Research Support Grant to perform this study (No. 96004188).
The authors declare no competing interests.
Ethics approval and consent to participate
Animal experiments were performed under the ARVO (Association for Research in Vision and Ophthalmology) protocol for the use of animals and protocols and were approved by the ethical committee of the “Ophthalmic Research Center, Shahid Beheshti University of Medical Sciences” and the “National Institute of Genetic Engineering and Biotechnology”.
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Zakeri, F., Latifi-Navid, H., Soheili, ZS. et al. Design, construction and in vivo functional assessment of a hinge truncated sFLT01. Gene Ther (2022). https://doi.org/10.1038/s41434-022-00362-1