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Design, construction and in vivo functional assessment of a hinge truncated sFLT01

Gene Therapy volume 30, pages 347–361 (2023)Cite this article

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Abstract

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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Fig. 1: Structure and sequence comparison of htsFLT01 and sFLT01 proteins.
Fig. 2: Protein complex equilibration validation.
Fig. 3: htsFLT01 and sFLT01 3D structures.
Fig. 4: Residual interaction network analysis.
Fig. 5: htsFLT01 western blotting and tube network formation quantification.
Fig. 6: Confocal microscopy of retinal vasculature 2 weeks after AAV2-htsFLT01 intravitreal delivery.

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Acknowledgements

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).

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  1. These authors contributed equally: Fahimeh Zakeri, Hamid Latifi-Navid.

Authors and Affiliations

  1. Department of Molecular Medicine, National Institute of Genetic Engineering and Biotechnology, Tehran, Iran

    Fahimeh Zakeri, Hamid Latifi-Navid, Zahra-Soheila Soheili, Mehdi Sadeghi & Sepideh Taghizadeh

  2. Biophysics Department, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran

    Seyed Shahriar Arab

  3. Blood Transfusion Research Center, High Institute for Research and Education in Transfusion Medicine, Tehran, Iran

    Shahram Samiei

  4. Molecular Biomarkers Nano-Imaging Laboratory, Department of Radiology, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA

    Ehsan Ranaei Pirmardan & Ali Hafezi-Moghadam

  5. Ophthalmic Research Center, Research Institute for Ophthalmology and Vision Science, Shahid Beheshti University of Medical Sciences, Tehran, Iran

    Hamid Ahmadieh

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Contributions

Z-SS designed the research; FZ, SS, ERP, ST, HL-N performed the research; ERP, MS, SSA, HA, AH-M, HL-N analyzed the data. FZ, Z-SS, ERP, SSA; HL-N wrote the paper.

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Correspondence to Zahra-Soheila Soheili.

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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 30, 347–361 (2023). https://doi.org/10.1038/s41434-022-00362-1

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