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

Gene Therapy volume 10, pages 1321–1327 (2003)Cite this article

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-p531–101. 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.

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Acknowledgements

We thank Douglas Olson and Joanne Yetz-Aldape who performed the FACS analysis. We thank Dr Yossi Shiloh for helpful suggestions and Dr Yoshinaga Saeki for advice and reagents. This work was supported by the A-T Children's Project, by NIH Grants CA71387 and CA21765 (to MBK), and by the American Lebanese Syrian Associated Charities of the St Jude Children's Research Hospital.

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Authors and Affiliations

  1. Department of Neurology, Molecular Neurogenetics Unit, Massachusetts General Hospital, Harvard Medical School, Charlestown, 02129, MA, USA

    M L Cortés, M V Di Maria & X O Breakefield

  2. Department of Hematology-Oncology, St Jude Children's Research Hospital, Memphis, 38105, TN, USA

    C J Bakkenist & M B Kastan

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Cortés, M., Bakkenist, C., Di Maria, M. et al. HSV-1 amplicon vector-mediated expression of ATM cDNA and correction of the ataxia–telangiectasia cellular phenotype. Gene Ther 10, 1321–1327 (2003). https://doi.org/10.1038/sj.gt.3301996

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