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-1a 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 À B À phenotype, crucial for preclinical studies and clinical application of Artemis gene transfer in the treatment of human SCID-A.

Role of Transgene Regulation in Ex Vivo Lentiviral Correction of Artemis Deficiency

“…1B). Similar to our previous report, 18 in a 5-day survival study mouse 3T3 cultures transduced with both EF1a-Puro and PGK-Artemis remained viable at all vector doses ( p > 0.05 at an MOI of 10); however, a dose-dependent decrease in cell survival was observed for cultures transduced with EF1a-Artemis at increasing multiplicities of infection ( p < 0.05 vs. all other groups; Fig. 1B).…”

“…16 We subsequently demonstrated that overexpression of Artemis after lentiviral transduction is associated with cytotoxicity, a halt in cell cycle progression, and fragmentation of genomic DNA ultimately resulting in apoptosis. 18 These results, along with the previous reports demonstrating incomplete immune reconstitution of SCID-A after ex vivo transduction with an exogenous promoter, 16,17 emphasize the importance of providing Artemis expression at a level that is nontoxic and yet sufficient to correct the SCID-A T -B -phenotype. Accordingly, we isolated and characterized the human Artemis promoter (APro) as a sequence extending 1 kilobase upstream from the human Artemis translational start site on human chromosome 10.…”

“…We found that overexpression of Artemis upon lentiviral transduction results in genomic shearing, cell cycle arrest, and apoptosis. 18 Considering the endonucleolytic activity of Artemis 29-32 these results are not surprising, yet they emphasize the importance of providing Artemis expression at a level that is nontoxic and yet sufficient to correct the T -B -phenotype in preclinical studies and in clinical application to human SCID-A. We subsequently reported the isolation and characterization of the endogenous human Artemis promoter as a sequence that effectively mediates gene expression at levels substantially lower than that mediated by the strong EF1a promoter both in vitro and in vivo.…”

“…Lentiviral vector plasmids pCSIIEG, 21 pCSEPuro, 18 pOK/ EF1a-Artemis (including the EF1a intron 1 sequence), and pOK/PGK-Artemis 18 have been previously described. For construction of pOK/APro-Artemis, the EF1a promoter was excised from pOK/EF1a-Artemis with AgeI, yielding pOK/ Artemis.…”

“…18 We subsequently characterized the 1-kb APro segment 19 as a weak promoter both in vitro and in vivo with the intention of employing this endogenous element to regulate Artemis expression in lentiviral vectortransduced cells. To determine the effect of promoter strength on Artemis-mediated cytotoxicity and the potential for correction of SCID-A by lentiviral transduction, an APro-regulated Artemis vector was compared with other vectors previously generated and characterized that contain the Artemis coding sequence regulated by the strong EF1a promoter and the more moderate-strength PGK promoter (Fig.…”