Enhanced vsvg variants for binder‑dependent and high‑specificity transduction of primary T cells

Abstract Although certain canonical mutation combination of VSVG effectively reduces the affinity between VSVG and LDLR—thereby improving transduction specificity of VSVG-pseudotyped lentiviral vectors—it impairs VSVG fusion activity when co-expressed with cell-targeting binders, resulting in reduced transduction efficiency relative to wild type VSVG. Additionally, this combination also lowers viral production titers when applied for LV pseudotyping. To overcome these limitations, we employed a rational and AI-assisted design strategy to disrupt electrostatic interactions and side-chain conformational stability within and around the VSVG–LDLR binding interface. A series of novel amino acid substitutions at both canonical and non-canonical positions were systematically generated and evaluated through transduction assays in T-lineage cells, with or without the co-application of T cell–specific surface binders, to assess each mutation's ability to disrupt LDLR binding and enable binder-mediated, cell-specific entry. This strategy led to the identification of previously unreported substitutions at canonical residues that outperformed traditional mutations in achieving high-specificity transduction of primary T cells. We also uncovered new functional roles for mutations at novel residues, which effectively minimized LDLR affinity and supported binder-dependent T cell targeting. When applied in pseudotyped LVs with T cell–specific binders, these VSV-G variants enabled more specific and efficient T cell transduction than canonical mutations—enhancing the safety profile for in vivo T cell editing. Furthermore, several mutations conferred improved viral resistance to serum complement–mediated neutralization and phagocytic clearance, contributing to enhanced vector persistence in systemic delivery settings. We also evaluated the impact of these mutations on physical LV production titers and observed higher yields relative to canonical VSVG variants under equal plasmid input conditions. In summary, this work establishes a new set of VSV-G mutations that enable high-specificity, binder-dependent transduction while maintaining or improving in-vivo persistence and viral production efficiency. These variants represent valuable tools for targeted LV delivery, particularly in in vivo T cell engineering applications.