Novel VSV-G Variants with Enhanced Blinding for Targeted Delivery of Lentiviral Vectors

Targeted in vivo transduction, entailing direct administration of viral vector preparations to patients, is the next big step in gene therapy. To redirect lentiviral vector (LV) particles selectively to desired cell type(s), different glycoproteins have been engineered to alter their tropism, including the glycoprotein G from Vesicular Stomatitis Virus (VSV-G) as the most commonly employed tropism-defining protein for LV particles. For detargeting from its natural receptor, the low-density lipoprotein receptor (LDLR), a VSV-G variant with two blinding substitutions, K47Q and R354A, is commonly used (VSV-G.pub). We provide first evidence for insufficient blinding of VSV-G.pub in human and murine cell lines and primary cells. In silico modeling pointed toward only slightly reduced LDLR binding affinity of VSV-G.pub. To lower the affinity further, we generated three novel VSV-G variants based on charge-reversing substitutions in two, four, or six key residues. These variants achieved a more stringent blinding compared with VSV-G.pub in the tested cell lines and primary cells. Codisplay of a CD4 binder enabled lentiviral particles pseudotyped with the novel variants to selectively transduce CD4-expressing cells. In summary, we present improved VSV-G variants with a better on/off-target ratio as attractive tools for in vivo gene therapy applications.