Introduction:: Lentivirus-based gene delivery is a commonly used technology in modern laboratories and clinical therapies due to their safety and stable integration of genes into human cell genomes. The human Low-Density Lipoprotein Receptor (LDLR) has recently been identified as the natural target of VSV-G and serves as the entry receptor for VSV-G pseudotyped lentiviral vectors. However, many cell types have poor transducibility (gene delivery) by the most widely used VSV-G-pseudotyped lentiviral vectors, mostly due to the lack of expression of the proper lentiviral entry receptor, LDLR. These include murine immune cell types, such as T cells, canine immune cells, and also human naïve T cells, certain epithelial cells, and more. We aim to overcome this limitation in these cell types with the design of a broadly applicable method to increase the efficiency of transduction. Here we show that in cells without LDLR expression, chemical labeling restored the recognition of target cells by lentiviruses with a paired pseudotyping viral envelope directed toward this chemical group, resulting in the restoration of lentiviral transduction ex-vivo (Figure 1A). We envision that this method will be a valuable and flexible tool in both academic and industrial settings.
Materials and Methods:: The LDLR KO K562 cells were chemically labeled with NHS-FITC at 1 μM and transduced with a pseudotyped lentivirus expressing a blue fluorescent protein (BFP). NHS-FITC is a fluorescein molecule conjugated to N-hydroxysuccinimide at the 5’ carbon, allowing it to covalently form amide bonds with free primary amines. Lentivirus particles containing a chimeric protein anti-Fluorescein (αFITC) envelope protein, were produced by transfection of HEK293T cells, using a 1:1:0.25:0.25 plasmid mass ratio of sPAX:BFP:MD2G: αFITC. NHS-FAM labeled cells were then mixed with lentiviruses at 1 million per ml and transduced via a standard spin infection protocol at 2000g for 90min at 35℃. Transduction efficiency was monitored by flow cytometry analysis of BFP expression.
Results, Conclusions, and Discussions:: We developed a K562 cell line with LDLR knocked out via CRISPR-Cas9 to model various cell types with low LDLR expression (Figure 1B). The transduction efficiency of LDLR KO K562 cells was evaluated using BFP-expressing VSV-G pseudotyped lentivirus, and the transduction efficiency was only ~20% in LDLR KO cells, compared to a typical transduction efficiency of about >85% in WT K562 cells (Figure 1C). This shows that lack of LDLR results in lower transduction and transduction efficiency correlates with the presence of the LDLR at least in K562 cells. Using this model LDLR KO K562 cell line, we developed a simple two-step approach to harness active membrane protein internalization to promote pseudotyped lentivirus entry into target cells (Figure 1A). This process involves 1) labeling target cell surface proteins rapidly and unbiasedly with a small hapten molecule (e.g., FITC), 2) pseudotyped lentivirus with a hapten-targeting moiety (e.g., anti-FITC scFV). The unbiased labeling of cell surface proteins with the hapten molecule essentially repurposed all surface proteins as lentivirus entry receptors and prepared the cells for lentivirus docking and cell entry. Towards this end, the LDLR KO K562 cells could be efficiently labeled with various concentrations of NHS-FITC (Figure 1D). The labeled LDLR KO K562 cells could be transduced with BFP-expressing lentivirus pseudotyped with an αFITC envelope, resulting in a marked increase of transduction efficiency from 20% to over 70% (Figure 1E). We tested the ability of this approach to improve the lentiviral transduction of canine T cells which are known to be refractory to lentivirus transduction and showed a successful boost of the transduction efficiency from < 5% to up to 40% (data not shown). Finally, to demonstrate the modularity of this platform and the potential application for targeted gene delivery, we labeled LDLR KO K562 cells with a FITC-conjugated anti-CD71 antibody. This CD71-specific surface labeling significantly restored the transduction of αFITC-pseudotyped lentivirus from less than 10% to over 70% (data not shown). These results highlight the potential of this surface engineering approach (non-specific or specific) to improve gene delivery to cell lines and primary cells ex vivo.
