Abolishing Retro-Transduction of Producer Cells in Lentiviral Vector Manufacturing

Abstract

Transduction of producer cells during lentiviral vector (LVV) production causes the loss of 70-90% of viable particles. This process is called retro-transduction and it is a consequence of the interaction between the LVV envelope protein, VSV-G, and the LDL receptor located on the producer cell membrane, allowing lentiviral vector transduction. Avoiding retro-transduction in LVV manufacturing is crucial to improve net production and, therefore, the efficiency of the production process. Here, we describe a method for quantifying the transduction of producer cells and three different strategies that, focused on the interaction between VSV-G and the LDLR, aim to reduce retro-transduction.

Keywords: LDL receptor; VSV-G pseudotyping; gene therapy; lentiviral vector; lentiviral vector manufacturing; retro-transduction.

Figure 1

Retro-transduction during LVV production mainly occurs via VSV-G envelope protein. (A) Comparison of retro-transduction events analysis (TIPs) in VSV-G-LVV and LVV missing VSV-G envelope. Statistical analysis was performed using an unpaired t-test (** = p < 0.01). (B) LVV transduction efficiency in the presence of increasing concentrations of a VSV-G antibody. (C) Functional (bars) and physical (circles) titres were obtained for LVV with and without VSV-G. Each experiment is the average of two biological replicates. Error bars indicate standard deviation.

Figure 2

sLDLR producer cell line production and analysis. (A) Evaluation of sLDLR gene expression level in sLDLR+ in comparison to HEK293T cells. (B) LVV transduction efficiency in the presence of sLDLR+ supernatant at different production times of sLDLR+ cells. (C) LVV transduction in sLDLR+ and HEK293T producer cells (TIPs). Statistical analysis was performed using an unpaired t-test (* = p < 0.05). (D) Physical and (E) infective titers of LVV produced in sLDLR+ and HEK293T cell lines. Each experiment is the average of two biological replicates. Error bars indicate standard deviation.

Figure 3

Addition of CR3 to reduce retro-transduction. (A) Transduction efficiency assay in the presence of increasing concentrations of CR3 peptide. Each experiment is the average of two biological replicates. Error bars indicate standard deviation. (B) Retro-transduction events analysis in LVV production in the presence of CR3 peptide.

Figure 4

Co-expression of RAP increases LVV production and reduces retro-transduction. (A) Different RAP constructs were used in this study. (B) LVV production in control cells (i.e., co-transfected with an empty plasmid), cells expressing RAP ΔHNEL, cells expressing RAP ΔHNEL_mut, cells expressing GLUCsp-RAP ΔHNEL, cells expressing GLUCsp-RAP ΔHNEL_mut. (C) TIPs of the producer cells are described in (B). Each experiment is the average of two biological replicates. Error bars indicate standard deviation. Statistical analysis was performed for experimental conditions compared to control (HEK293T co-transfected with an empty plasmid, pCDNA3) using one-way ANOVA and Dunnet test (ns = not significant; * = p < 0.05; ** = p < 0.01; *** = p < 0.001; **** = p < 0.0001).

Figure 5

LVV production in HEK293T wild-type and LDLR Knock Out (KO) cells. (A) Infective titre of LVV produced in HEK293T wild-type and the three HEK293T LDLR KO Cell clones previously selected. (B) Physical titre, obtained by p24 ELISA, of the LVV produced in HEK293T wild-type and LDLR KO cells. (C) LVV transduction of producer cells during production (TIPs) in the mentioned cell clones. Statistical analysis was performed for experimental conditions compared to control (HEK293T WT versus LDLR KO cell lines) using one-way ANOVA and the Dunnet test (*** = p < 0.001). Each experiment is the average of two biological replicates. Error bars indicate standard deviation.

Figure 6

LVV production in HEK293T wild-type (WT) and LDLR knock-out (KO) cells with and without cholesterol supplementation. (A) Infective titre obtained in HEK293T WT and LDLR KO clone 1 (KO1) with (+Chol) or without (−Chol) cholesterol supplementation. (B) Physical titre, obtained by p24 ELISA, of the LVV produced in HEK293T wild-type and LDLR KO clone 1 (KO1) with (+Chol) or without (−Chol) cholesterol supplementation. (C) LVV retro-transduction of producer cells during production (TIPs) in the mentioned cell clones. Statistical analysis was performed for experimental conditions compared to control (HEK293T WT–Chol versus the other three groups) using one-way ANOVA and Dunnet test (ns = not significant, * = p < 0.05). Each experiment is the average of two biological replicates. Error bars indicate standard deviation.

Figure 7

Cholesterol content in LVV producer and non-producer HEK293T and LDLR KO1 cells. (A) Cholesterol content in HEK293T wild-type cells without (filled bar) or with (patterned bar) cholesterol supplementation. Cholesterol content was measured in LVV producer (+LVV, black) and non-producer (−LVV grey) cells. (B) Cholesterol content in HEK293T LDLR KO1 cells without (filled bar) or with (patterned bar) cholesterol supplementation. Cholesterol content was measured in LVV producer (+LVV, dark green) and non-producer (−LVV, light green) cells. Each experiment is the average of two biological replicates. Error bars indicate standard deviation.