Cytotoxicity associated with artemis overexpression after lentiviral vector-mediated gene transfer

Abstract

Artemis is a hairpin-opening endonuclease involved in nonhomologous end-joining and V(D)J recombination. Deficiency of Artemis results in radiation-sensitive severe combined immunodeficiency (SCID) characterized by complete absence of T and B cells due to an arrest at the receptor recombination stage. We have generated several lentiviral vectors for transduction of the Artemis sequence, intending to complement the deficient phenotype. We found that transduction by a lentiviral vector in which Artemis is regulated by a strong EF-1alpha promoter resulted in a dose-dependent loss of cell viability due to perturbed cell cycle distribution, increased DNA damage, and increased apoptotic cell frequency. This toxic response was not observed in cultures exposed to identical amounts of control vector. Loss of cell viability was also observed in cells transfected with an Artemis expression construct, indicating that toxicity is independent of lentiviral transduction. Reduced toxicity was observed when cells were transduced with a moderate-strength phosphoglycerate kinase promoter to regulate Artemis expression. These results present a novel challenge in the establishment of conditions that support Artemis expression at levels that are nontoxic yet sufficient to correct the T(-)B(-) phenotype, crucial for preclinical studies and clinical application of Artemis gene transfer in the treatment of human SCID-A.

Figures

FIG. 1.

Lentiviral vector constructs. Several lentiviral vectors were constructed for analysis and complementation of Artemis deficiency. (A) Lentiviral vectors engineered to express both Artemis and green fluorescent protein (GFP) cDNA sequences for monitoring transduction. (B) Lentiviral vectors constructed to express either the human or murine Artemis cDNA sequence from either the elongation factor (EF)-1α or phosphoglycerate kinase (PGK) promoter. (C) Control lentiviral vectors that express either the puromycin resistance gene or GFP regulated by EF-1α. CMV, cytomegalovirus early promoter/enhancer region; U3/U5/R, unique 3′/unique 5′/repeat regions of the HIV long terminal repeat; ψ, packaging signal; CPPT, central polypurine tract; WPRE, woodchuck posttranscriptional regulatory element; IRES, internal ribosome entry site; pA, polyadenylation signal; CLP, CpG-less promoter. Arrows indicate sites and direction of transcript initiation.

FIG. 2.

Analysis of Artemis expression in transduced cell populations. (A) Expression of Artemis protein was verified by Western blot analysis (78 kDa) of nuclear extracts derived from tMEFSCIDA wild-type (+/+), Artemis heterozygous (+/−), Artemis-deficient (−/−), and Artemis-deficient MEFs transduced with the ABiG lentiviral vector (−/−t) or the control CSIIEG lentiviral vector (−/−c). (B) Artemis activity was detected in a fluorescence hairpin-opening assay. Nuclear extracts generated from tMEFSCIDA wild-type (+/+), Artemis heterozygous (+/−), and Artemis deficient (−/−) as well as Artemis-deficient MEFs transduced at increasing multiplicity with ABiG lentiviral vector were assayed for hairpin opening activity as described in Materials and Methods. 0, no lysate; −, not transduced. Each value represents the mean of three replicates ± SD.

FIG. 3.

Cell survival response following Artemis transduction. Murine NIH 3T3 cells were transduced at increasing MOI using A) CSIIEG or B) EAIG lentiviral vectors as indicated. Cell survival was assessed over time, plotted as the percentage of cells surviving in control, untreated populations. Each value represents the mean of three replicates ± SD.

FIG. 4.

Growth response after Artemis transfection. 293T cells were transfected with increasing amounts of plasmid EF1α-hArtemis or EF1α-Puromycin, using the DNA-calcium phosphate coprecipitation technique. Cell survival was assessed 5 days later by trypan blue exclusion, expressed here as the percentage of an untreated control cell population. Each value represents the mean of three replicates ± SD.

FIG. 5.

The effect of promoter strength on Artemis toxicity. The effect of promoter strength on Artemis toxicity was assayed by transducing (A) tMEFSCIDA+/+, (B) tMEFSCIDA+/−, and (C) tMEFSCIDA−/− cells at increasing MOI with EF1α-hArtemis, PGK-hArtemis, and the control vector EF1α-Puromycin. To control for integrant frequency, side-by-side titering experiments indicated a consistent copy number between murine NIH 3T3 and tMEFSCIDA cell lines by quantitative PCR (see Materials and Methods) for all three vectors. Cell viability was assayed 5 days later by trypan blue exclusion, presented here as the percentage of an untreated cell population. Each value represents the mean of three replicates ± SD.

FIG. 6.

Comet assay of genomic DNA damage. The comet assay was performed on tMEFSCIDA−/− cells transduced with either CSIIEG or EAIG. Transduced cells were collected by GFP-positive cell sorting and subjected to alkaline comet assay as described in Materials and Methods. Mean tail length ± SD (n = 3) is plotted for Artemis-deficient MEFs transduced at various multiplicities with EAIG and CSIIEG lentiviral vectors.

FIG. 7.

Effect of Artemis overexpression on cell cycle progression. NIH 3T3 cells were transduced with (A) EF1α-Puromycin or (B) EF1α-hArtemis at increasing MOI and then subjected to cell cycle analysis 48 hr later by flow cytometry using propidium iodide (see Materials and Methods). (C) The percentage of cells in G1 of the cell cycle was determined with FlowJo software.

FIG. 8.

Induction of apoptosis in Artemis-transduced cells. NIH 3T3 cells were transduced at increasing MOI with (A) CSIIEG or (B) EAIG lentiviral vectors and then 20 hr later stained with Annexin V and 7-AAD. Transduced GFP-positive cells were gated to determine Annexin V binding and 7-AAD staining in each population, expressed here as the singly and doubly staining cells as a percentage of the whole GFP+ cell population ± SD (n = 3).