ABSTRACT
Artemis is an endonucleolytic enzyme involved in nonhomologous double-strand break repair and V(D)J recombination. Deficiency of Artemis results in a B- T- radiosensitive severe combined immunodeficiency, which may potentially be treatable by Artemis gene transfer into hematopoietic stem cells. However, we recently found that overexpression of Artemis after lentiviral transduction resulted in global DNA damage and increased apoptosis. These results imply the necessity of effecting natural levels of Artemis expression, so we isolated a 1 kilobase DNA sequence upstream of the human Artemis gene to recover and characterize the Artemis promoter (APro). The sequence includes numerous potential transcription factor-binding sites, and several transcriptional start sites were mapped by 5' rapid amplification of cDNA ends. APro and deletion constructs conferred significant reporter gene expression in vitro that was markedly reduced in comparison to expression regulated by the human elongation factor 1-α promoter. Ex vivo lentiviral transduction of an APro-regulated green fluorescent protein (GFP) construct in mouse marrow supported GFP expression throughout hematopoeitic lineages in primary transplant recipients and was sustained in secondary recipients. The human Artemis promoter thus provides sustained and moderate levels of gene expression that will be of significant utility for therapeutic gene transfer into hematopoeitic stem cells.
Subject(s)
5' Untranslated Regions , Bone Marrow Transplantation , Bone Marrow/metabolism , Gene Expression , Green Fluorescent Proteins/metabolism , Molecular Targeted Therapy/methods , Nuclear Proteins , Promoter Regions, Genetic , Severe Combined Immunodeficiency/therapy , Animals , Base Sequence , Binding Sites , Cell Line , DNA Repair/genetics , DNA-Binding Proteins , Endonucleases , Genes, Reporter , Green Fluorescent Proteins/genetics , Hematopoietic Stem Cells/cytology , Hematopoietic Stem Cells/metabolism , Humans , Lentivirus , Mice , Mice, Inbred C57BL , Molecular Sequence Data , Nuclear Proteins/chemistry , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Peptide Elongation Factor 1/genetics , Peptide Elongation Factor 1/metabolism , Protein Binding , Severe Combined Immunodeficiency/genetics , Severe Combined Immunodeficiency/immunology , Severe Combined Immunodeficiency/metabolism , Transcription Factors/metabolism , Transduction, GeneticABSTRACT
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.
