title: HSV-1 amplicon vector-mediated expression of ATM cDNA and correction of the ataxia-telangiectasia cellular phenotype.
abstract: Ataxia-telangiectasia (A-T) is an autosomal recessive disorder characterized by neurodegeneration, immunodeficiency, cancer predisposition, genome instability, and radiation sensitivity. Previous research has shown that it is possible to correct the hereditary deficiency A-T by DNA transfection in cell culture, but the large size of the ATM cDNA (9 kb) limits the use of many vector types for gene replacement. HSV-1 amplicon vectors provide a means to deliver large genes to cells efficiently and without toxicity. In this study, the FLAG-tagged cDNA for human ATM was inserted into an HSV-1 amplicon under control of the CMV promoter (designated as HGC-ATM). FLAG-ATM expression was confirmed in 293T/17 cells and human A-T fibroblasts (GM9607) after transduction, by immunoprecipitation, Western analysis, and immunocytochemistry. Functional recovery was assessed by two independent assays. First, in vitro kinase assay showed that vector-derived ATM in GM9607 cells could successfully phosphorylate wt p53 using recombinant GST-p53(1-101). Second, in A-T cells infected with the HGC-ATM vector, the extent of accumulation in G2/M phase at 24 h postirradiation was similar to that observed in cells with wild-type endogenous ATM and lower than that observed in A-T cells infected with a control vector. Thus, these vectors provide a tool to test the feasibility of HSV-amplicons as gene therapy vectors for A-T.
authors.affiliation: Molecular Neurogenetics Unit, Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA 02129, USA.
authors.forename: M L
authors.initials: ML
authors.lastname: Cortés
authors.forename: C J
authors.initials: CJ
authors.lastname: Bakkenist
authors.forename: M V
authors.initials: MV
authors.lastname: Di Maria
authors.forename: M B
authors.initials: MB
authors.lastname: Kastan
authors.forename: X O
authors.initials: XO
authors.lastname: Breakefield
mesh_terms: D001260:Ataxia Telangiectasia / Q000628:therapy*; D064007:Ataxia Telangiectasia Mutated Proteins; D018797:Cell Cycle Proteins; D002460:Cell Line; D002478:Cells, Cultured; D004268:DNA-Binding Proteins; D005347:Fibroblasts / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000008:administration & dosage*; D018259:Herpesvirus 1, Human / Q000235:genetics*; D006801:Humans; D017346:Protein Serine-Threonine Kinases / Q000032:analysis / Q000235:genetics*; D014161:Transduction, Genetic / Q000379:methods*; D025521:Tumor Suppressor Proteins
chemical_list: D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D025521:Tumor Suppressor Proteins; C576324:ATM protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; D017346:Protein Serine-Threonine Kinases