Optimization of the VSV-G backbone for amino terminal fusion with nanobodies allowing its specific retargeting to HER2 receptors

Vesicular stomatitis virus (VSV) is a promising oncolytic virus. Additionally, its glycoprotein G (VSV-G) is the most commonly used envelope glycoprotein to pseudotype lentiviral vectors for gene therapy. However, VSV receptors (low-density lipoprotein receptor [LDL-R] family members) are ubiquitous and expressed at the surface of non-target cells, precluding <i>in vivo</i> gene therapy. Recently, we identified VSV-G mutants that no longer bind to LDL-R but retain their fusion activity. This created the opportunity to specifically retarget the glycoprotein to the desired receptors. Here, we engineered chimeric glycoproteins by fusing nanobodies to the amino terminus of VSV-G. Using experimental evolution, we identified two mutations in VSV-G improving the folding and functionality of chimeric glycoproteins, regardless of the nanobody inserted at the amino terminus. We then generated chimeric glycoproteins using several nanobodies targeting HER2 receptor, and into these chimeras, we introduced additional mutations that abolish recognition of LDL-R. VSV and lentiviruses pseudotyped with these glycoproteins specifically infect cells expressing HER2. We have, therefore, identified VSV-G mutations that optimize its backbone to tolerate amino-terminal insertion of a nanobody and establish proof of concept that this approach can be used to confer a new tropism on VSV-G. This paves the way for the development of targeted <i>in vivo</i> therapies.