4. Developing an Engineered Nipah Virus Glycoprotein Based Lentiviral Vector System Retargeted To Cell Surface Receptors of Choice

Abstract

Delivery of genes selectively into specific cell types is a major bottleneck in successful gene therapy. Receptor-targeted lentiviral vectors (RT-LVs) can be an effective tool to achieve selective transfer of genes to cell types of choice, ex vivo and in vivo. So far, many different target receptors specific for cell types present in many different tissue types have been successfully addressed. RT-LVs based on engineered measles virus (MV) glycoproteins are most selective but can only be produced at moderate vector titers and suffer from susceptibility towards neutralising antibodies induced by vaccination against measles. In this study, LVs have been pseudotyped with modified Nipah virus (NiV) glycoproteins in order to generate LVs with high specificity for target cell populations and enhanced titers. The glycoproteins were engineered to make them deficient for the use of EphrinB2/3, the natural NiV receptor, and truncated in their cytoplasmic tails to allow efficient incorporation into LV particles. Designed ankyrin repeat proteins (DARPins) or single chain antibodies (scFv) specific for the cell surface receptor of choice were fused to the G glycoprotein to mediate receptor binding and subsequent cell entry. All G-DARPin/-scFv fusion proteins were efficiently expressed at the cell surface and incorporated into LV particles. The particles exhibited selective entry into their particular target cells and showed stable transgene expression. Importantly, NiV-LVs were at least 250-fold less effectively neutralized than MV glycoprotein pseudotyped LVs if incubated with pooled human intravenous immunoglobulin (Intratect®). So far, EpCAM, CD4, and CD8 served as functional receptors for NiV glycoprotein based RT-LVs. The CD8-targeted vector selectively transduced CD8+ lymphocytes when added to human PBMCs in vitro. Remarkably, these NiV-based RT-LVs could be produced at 8- to 68-fold higher titers compared to the corresponding MV glycoprotein-based RT-LVs. Current efforts focus on the quantification of these observations and identification of the underlying mechanisms. In addition, the capability of NiV-based RT-LVs to transduce target cell populations in vivo is being analyzed.