Diphtheria Toxin A-Resistant Cell Lines Enable Robust Production and Evaluation of DTA-Encoding Lentiviruses

Abstract

Suicide genes have been widely investigated for their utility as therapeutic agents and as tools for in vitro negative selection strategies. Several methods for delivery of suicide genes have been explored. Two important considerations for delivery are the quantity of delivered cargo and the ability to target the cargo to specific cells. Delivery using a lentiviral vector is particularly attractive due to the ability to encode the gene within the viral genome, as well as the ability to limit off-target effects by using cell type-specific glycoproteins. Here, we present the design and validation of a diphtheria toxin A (DTA)-encoding lentiviral vector expressing DTA under the control of a constituitive promoter to allow for expression of DTA in a variety of cell types, with specificity provided via selection of glycoproteins for pseudotyping of the lentiviral particles. DTA exerts its toxic activity through inhibition of eukaryotic translation elongation factor 2 (eEF2) via adenosine diphosphate (ADP)-ribosylation of a modified histidine residue, diphthamide, at His715, which blocks protein translation and leads to cell death. Thus, we also detail development of DTA-resistant cell lines, engineered through CRISPR/Cas9-mediated knockout of the diphthamide 1 (DPH1) gene, which enable both robust virus production by transfection and evaluation of DTA-expressing virus infectivity.

Figure 1

A proviral plasmid encoding DTA blocks reporter EGFP translation in 293FT and TZM-GFP cells. (a) Schematic representation of the DTA-encoding lentiviral vector, pHIV-CMV-DTA. (b) Fluorescent images at 40x magnification of 293FT (top row) or TZM-GFP (bottom row) cells transfected with a combination of pCMV-EGFP and filler DNA (left column) or pCMV-EGFP and pHIV-CMV-DTA (right column). (c) Quantification of EGFP fluorescence in 293FT cells transfected as in (b) by flow cytometry. Experiments were performed at least three times in triplicate. A representative experiment is shown. Error bars represent standard deviation of triplicate transfections.

Figure 2

Engineering DTA-resistant cell lines using CRISPR/Cas9. Schematic representation of the lentiCRISPRv2 vector, guide RNA (gRNA) sequence specific to the DPH1 gene, and workflow for DPH1 knockout cell line generation. Black bars represent  long terminal repeats (LTRs). Psi = packaging signal, RRE = Rev response element, cPPT = central polypyrimidine tract, EFS = elongation factor short promoter, P2A = cleavage peptide, Puro = puromycin resistance cassette, WPRE = Woodchuck Hepatitis Virus posttranscriptional regulatory element.

Figure 3

Knockout of DPH1 prevents the DTA-mediated block to EGFP protein translation in 293FT and TZM-GFP cells. (a) Fluorescent images at 40x magnification of 293FT (top left), 293FT-DPH1KO (top right), TZM-GFP (bottom left), and TZM-GFP-DPH1KO (bottom right) cells transfected with a combination of pCMV-EGFP and pHIV-CMV-EGFP. (b,c) Quantification of EGFP fluorescence in 293FT (b), 293FT-DPH1KO (b), TZM-GFP (c), and TZM-GFP-DPH1KO (d) transfected with either pCMV-EGFP and filler DNA (black bars) or pCMV-EGFP and pHIV-CMV-DTA (gray bars) by flow cytometry. Raw EGFP fluorescence values were normalized to the pCMV-EGFP + filler DNA transfection controls, and data is shown as transfection efficiency relative to the controls. Experiments were performed at least three times in triplicate. A representative experiment is shown. Error bars represent standard deviation of triplicate transfections.

Figure 4

Knockout of DPH1 allows for robust production of DTA-encoding lentiviral particles. (a) Fluorescent images at 100x magnification of TZM-GFP (top row) and TZM-GFP-DPH1KO (bottom row) cells 48 hours post-transduction with VSV-G-pseudotyped HIV-CMV-DTA produced in 293FT cells (left column) or 293FT-DPH1KO cells (right column) transfected with a combination of pHIV-CMV-DTA (1000 ng) and pVSV-G (100 ng). (b) Quantification of EGFP fluorescence 48 hours post-transduction in TZM-GFP and TZM-GFP-DPH1KO cells. Virus was produced in either 293FT cells (black bars) or 293FT-DPH1KO cells (gray bars). Experiments were performed at least three times in triplicate. One representative experiment is shown. Error bars represent standard deviation of transductions from triplicate transfections.

Figure 5

Knockout of DPH1 yields enhanced DTA resistance (a) and infectious virus production (b) as compared to mutation of EF2. (a) Quantification of EGFP fluorescence in 293FT, 293FT-DPH1KO, and 293T-5H7 cells 48 hours post-transfection with either pCMV-EGFP and filler DNA or pCMV-EGFP and pHIV-CMV-DTA by flow cytometry. Raw EGFP fluorescence values were normalized to the pCMV-EGFP + filler DNA transfection controls, and data is shown as transfection efficiency relative to the controls. Experiments were performed twice in triplicate. A representative experiment is shown. Error bars represent standard deviation of triplicate transfections. (b) Fluorescent images comparing transduction efficiency of TZM-GFP-DPH1KO cells transduced with parental virus, HIV-CMV-EGFP, produced in 293FT-DPH1KO or 293T-5H7 cells (left column) and HIV-CMV-DTA virus produced in 293FT-DPH1KO or 293T-5H7 cells (right column).

Figure 6

Lentiviruses encoding DTA induce cell death in wildtype cells, but not in DPH1 knockout cells. (a) Crystal violet staining of TZM-GFP and TZM-GFP-DPH1KO cells transduced in triplicate with HIV-CMV-DTA at an MOI > 1. Non-transduced controls are also shown. Crystal violet staining was performed 96 hours post-transduction. (b) Bright field images at 100x magnification of TZM-GFP (left) and TZM-GFP-DPH1KO (right) cells 96 hours post-transduction with HIV-CMV-DTA at an MOI > 1.

Figure 7

HIV-CMV-DTA-mediated, glycoprotein-targeted killing of 293 mCAT-1/Cherry cells in a mixed culture with 293FT cells. 293 mCAT-1 cells were transfected with a plasmid for expression of mCherry under the CMV promoter. 48 hours post-transfection, 293FT and 293 mCAT-1/Cherry cells were plated in a mixed culture at a 1:1 ratio by seeding 10,000 cells per well in 96-well plates. 293 mCAT-1/Cherry cells were also plated in a single culture at 10,000 cells per well in 96-well plates. Cells were infected or not infected with HIV-CMV-DTA pseudotyped with MLV Env (a,b) or VSV-G (b, bottom panel) at an MOI > 1. After 72 hours, cells were re-plated to allow detachment of dead cells and re-adherence of live cells. Bright field and fluorescent images at 100x magnification were taken 96 hours post-infection using an Olympus XI81 microscope. Remaining mCAT-1 cells expressing mCherry were also quantified by Flow Cytometry (c).