Cytotoxicity associated with artemis overexpression after lentiviral vector-mediated gene transfer.
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.
Multhaup
Megan
M
Gene Therapy Program, Institute of Human Genetics, Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis, MN 55455, USA.
Karlen
Andrea D
AD
Swanson
Debra L
DL
Wilber
Andrew
A
Somia
Nikunj V
NV
Cowan
Morton J
MJ
McIvor
R Scott
RS
D000818:Animals; D017209:Apoptosis; D001483:Base Sequence; D015153:Blotting, Western; D002470:Cell Survival / Q000502:physiology*; D002478:Cells, Cultured; D004268:DNA-Binding Proteins; D004720:Endonucleases; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D051379:Mice; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism*
D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; C426344:DCLRE1C protein, human; D004720:Endonucleases