HSV-1 amplicon vector-mediated expression of ATM cDNA and correction of the ataxia-telangiectasia cellular phenotype

被引:12
作者
Cortés, ML
Bakkenist, CJ
Di Maria, MV
Kastan, MB
Breakefield, XO
机构
[1] Harvard Univ, Sch Med, Massachusetts Gen Hosp, Dept Neurol,Mol Neurogenet Unit, Charlestown, MA 02129 USA
[2] St Jude Childrens Res Hosp, Dept Hematol Oncol, Memphis, TN 38105 USA
关键词
HSV-1; amplicon; ataxia-telangiectasia; irradiation; ATM;
D O I
10.1038/sj.gt.3301996
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 G(2)/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.
引用
收藏
页码:1321 / 1327
页数:7
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