Optimisation of lentiviral transduction in B-cells: application of protein A/G-driven transduction

Abstract

Primary B cells are difficult to transduce with lentiviruses and most methods available are not B cell specific. Therefore, we investigated the application of protein AG (pAG) on the lentiviral particle surface to have an antibody-mediated entry system. This should enhance transduction efficiency and allow for cell specific targeting. Our aim was to optimise this system and ultimately transduce BCL6/BCL2L1 in primary B cells, to enhance their survival and mimic germinal centre B cells. The optimisation was done in Raji cells, an established cell line that resemble B cells. Enhanced Green Fluorescent Protein (eGFP) was used as the gene of interest initially, because of its simplicity. This allowed the focus to be on pAG to determine if the mechanism worked as intended. The cells were analysed using flow cytometry. Transduction in Raji cells was poor, <10% of eGFP-positive cells were found. No antibody-dependent entry was observed. To investigate potential causes, the lentiviral particles were tested in HEK293T cells. This revealed that high, antibody-independent transduction was possible. These results confirmed that the lentiviral particles were functional and successful in causing eGFP expression in cells. The production of the lentiviral particles was therefore deemed satisfactory. Further research is needed to clarify why the antibody-mediated cell entry was unsuccessful. A switch to other lentiviral envelope glycoproteins, such as measles virus glycoproteins or baboon endogenous retrovirus glycoproteins, can be considered to target B cells.