Enhancement of lentiviral vectors gene delivery against VEGFR2 expressing cells by co-display of the binding and fusogenic moieties: a two molecules targeting approach

Pseudotyped lentiviral vectors (LVs) were used in cancer therapy as gene delivery systems for inhibiting tumor angiogenesis by targeting cells overexpressing "vascular endothelial growth factor receptor-2 (VEGFR2)". Herein, we report that switching from chimeric sindbis virus glycoprotein (SVG) harboring VEGFR2-specific nanobody (VEGFR2-Nb) to Two-Molecules Targeting Approach (TMTA: independent co-display of binding and fusogenic moieties) highly enhanced the transduction efficiency (TE) of the targeted LVs. Several LVs co-displaying either the VEGFR2-Nb or the natural ligand (VEGF121) as targeting moiety, along with a de-targeted mutant form of SVG (as a binding deficient and fusion competent) fusogenic moiety were produced. LVs were constructed via various backbones and linkers (platelet-derived growth factor receptor "PDGFR" and CD28 as transmembrane domains, and HL and Fc as spacer domains). Expression and incorporation of the VEGFR2-Nb and SVG onto lentiviral particles were confirmed by flowcytometry and Western blotting while their co-display was demonstrated by virus-capture ELISA and virus-cell binding assays. LVs co-enveloped with fusogen and either VEGFR2-Nb or VEGF121 showed higher TEs in VEGFR2-expressing cells (72% and 91%, respectively) over LVs pseudotyped with chimeric fusogen containing the same nanobody (30%). In silico analyses indicated a direct correlation for the TE and the distance between the nanobody and the lipid bilayer. Compared to the chimeric strategy, the two-molecule targeting approach of LVs, due to its flexible and modular nature provides higher TE and thus great potentials for targeted gene delivery.