To the editor:
Vesicular stomatitis virus (VSV) G-protein pseudotyped lentiviral vectors (VSV-G-LVs) signify a major advancement in the gene and immunotherapy field as illustrated by successful clinical trials, for example, for Wiskott Aldrich Syndrome and leukodystrophies.1
Although VSV-G-LVs allow efficient transduction of nondividing cells,2 they do not provide efficient transduction of quiescent T cells, B cells, and hematopoietic stem cells (HSCs), which hampers their application in gene and immune-therapy areas where conservation of cell phenotype is essential. Although these hurdles can be overcome in lymphocytes by LVs pseudotyped with measles virus envelope proteins (MV-LVs3-5 ), the reason as to why VSV-G-LVs were not efficient for gene transfer in these quiescent cells, and in particular in HSCs, remains unclear. Recently, Finkelstein et al revealed a long-kept secret of VSV by identifying its receptor, the low-density lipid receptor (LDL-R), explaining its broad tropism.6 This finding prompted us to evaluate LDL-R levels on unstimulated T, B, and CD34+ cells. Strikingly, we confirmed a very low expression of LDL-R, coinciding with VSV-G-LV–mediated poor transduction in these 3 cell lineages (Figure 1A). Stimulation of T cells through the T-cell receptor or of human CD34+ (hCD34+) cells with “early-acting cytokines” remarkably upregulated the LDL-R surface expression and permitted efficient VSV-G-LV transduction. In contrast, B-cell receptor stimulation augmented LDL-R expression only marginally, in agreement with poor VSV-G-LV transduction levels (Figure 1A and Frecha et al4 ). Binding of the different cell lineages with VSV-G-LVs was detected by incubation with the VSV-G-LVs followed by HIV capsid (p24) detection. VSV-G-LVs bound efficiently to stimulated T and hCD34+ cells but not B cells and barely attached to their resting counterparts (Figure 1B). In contrast, MV-LVs efficiently attached to both stimulated and unstimulated cells (Figure 1B). Next, we used particles formed by VSV-G protein (gesicles7 ) incorporating high levels of green fluorescent protein (GFP) through a farnesylation tag to verify fusion of VSV-G protein with 3 cell lineages (Figure 1A, right panels). Resting T, B, and CD34+ cells showed a poor GFP signal upon contact with GFP-loaded gesicles, while the GFP signal was evident for prestimulated cells, except for B cells (Figure 1A), confirming the presence of VSV and thus the VSV-G-LV receptor, LDL-R. Accordingly, VSV-G-LV transduction of resting T and B cells also resulted in very low levels of reverse-transcribed viral DNA.4
Finally, we confirmed the requirement for VSVG-LV entry and transduction through the LDL-R and its family members using an anti–LDL-R antibody or by competition with soluble LDL-R, resulting in reduction or almost complete inhibition of transduction, respectively (Figure 1C). In contrast, MV-LVs were not sensitive to these LDL-blocking or -competing agents. Interestingly, IL-7–stimulated T-cell VSV-G-LV transduction8 coincided with LDL-R upregulation and was inhibited upon LDL-R blocking. Additionally, low-level transduction in resting cells was lost upon LDL-R blocking (data not shown).
In conclusion, although cellular postentry blocks may still play a role in VSV-G-LV transduction of resting T cells, B cells, and HSCs, we confirmed here that VSV-G-LV entry is compromised by the low expression of the VSV receptor LDL-R and its family members. Therefore, other LV pseudotypes (eg, MV-LVs) are more adapted for gene transfer in these invaluable resting gene-therapy targets.9
The online version of this article contains a data supplement.
Contribution: F.A., C.L., C.C., D.N., and C.X.W. performed and designed experiments; B.E.T. and F.L.C. discussed results; and E.V. coordinated the project, designed and performed the experiments, analyzed the data, discussed results, and wrote the manuscript.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
F.A., C.L., and C.C. contributed equally.