Switchable Retargeting of Lentiviral Vectors Through a VSV-G-Binding Adapter Molecule

Selective gene delivery to defined cell populations remains one of the key challenges in lentiviral vector-based gene therapy. The vesicular stomatitis virus glycoprotein (VSV-G) confers high infectivity but lacks cell-type specificity because of the ubiquitous expression of its receptor, LDLR. To enable modular, receptor-specific targeting while retaining the production efficiency of VSV-G-pseudotyped vectors, we designed a bispecific adapter, 929-B6, comprising a VSV-G-binding nanobody and an ERBB2-binding DARPin 9.29. Anti-VSV-G nanobodies were isolated from an alpaca immune library and screened in cell-based pseudoreceptor assays to identify the optimal binder (VSVG-B6). The resulting adapter was evaluated with receptor-ablated (VSV-G_mut) and wild-type VSV-G-pseudotyped LVs across ERBB2-positive and -negative cell lines and in a mouse xenograft model. 929-B6 enabled efficient, receptor-specific transduction of ERBB2-expressing cells without increasing infection of ERBB2-negative controls. Pre-incubation of VSV-G_mut-pseudotyped LVs with 1-2 µg/mL 929-B6 increased transduction up to eight-fold in ERBB2⁺ cells, with similar but smaller effects for VSV-G and VSV-G_mut + 929R pseudotypes. Across breast cancer lines, transduction enhancement correlated with ERBB2 surface density, and co-culture experiments confirmed selective entry into ERBB2⁺ populations. In vivo imaging of ERBB2⁺ tumors revealed a visible tumor-localized luminescent signal following administration of 929-B6-treated vectors. The 929-B6 adapter provides a rapid, scalable means to retarget standard LV stocks toward chosen receptors without re-engineering the envelope or co-packaging pseudoreceptor plasmids. Its modularity suggests a generalizable platform for both gene therapy and oncolytic applications requiring flexible, receptor-defined tropism.