SEARCH:   Advanced

Blood, 21 May 2009, Vol. 113, No. 21, pp. 5104-5110. Prepublished online as a Blood First Edition Paper on March 13, 2009; DOI 10.1182/blood-2008-11-191049. This Article Full Text Full Text (PDF) Supplemental Methods and Figures All Versions of this Article: blood-2008-11-191049v1 113/21/5104    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Rights and Permissions Citing Articles Citing Articles via CrossRef Google Scholar Articles by Throm, R. E. Articles by Gray, J. T. PubMed PubMed Citation Articles by Throm, R. E. Articles by Gray, J. T. Related Collections Gene Therapy Social Bookmarking                   What's this?  Previous Article  |  Table of Contents  |  Next Article  GENE THERAPY Efficient construction of producer cell lines for a SIN lentiviral vector for SCID-X1 gene therapy by concatemeric array transfection Robert E. Throm1, Annastasia A. Ouma1, Sheng Zhou1, Anantharaman Chandrasekaran1, Timothy Lockey2, Michael Greene1, Suk See De Ravin3, Morvarid Moayeri3, Harry L. Malech3, Brian P. Sorrentino1, and John T. Gray1 1 Division of Experimental Hematology, Department of Hematology, and 2 Department of Therapeutics Production and Quality, St Jude Children's Research Hospital, Memphis, TN; and 3 Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD Retroviral vectors containing internal promoters, chromatin insulators, and self-inactivating (SIN) long terminal repeats (LTRs) may have significantly reduced genotoxicity relative to the conventional retroviral vectors used in recent, otherwise successful clinical trials. Large-scale production of such vectors is problematic, however, as the introduction of SIN vectors into packaging cells cannot be accomplished with the traditional method of viral transduction. We have derived a set of packaging cell lines for HIV-based lentiviral vectors and developed a novel concatemeric array transfection technique for the introduction of SIN vector genomes devoid of enhancer and promoter sequences in the LTR. We used this method to derive a producer cell clone for a SIN lentiviral vector expressing green fluorescent protein, which when grown in a bioreactor generated more than 20 L of supernatant with titers above 107 transducing units (TU) per milliliter. Further refinement of our technique enabled the rapid generation of whole populations of stably transformed cells that produced similar titers. Finally, we describe the construction of an insulated, SIN lentiviral vector encoding the human interleukin 2 receptor common chain (IL2RG) gene and the efficient derivation of cloned producer cells that generate supernatants with titers greater than 5 x 107 TU/mL and that are suitable for use in a clinical trial for X-linked severe combined immunodeficiency (SCID-X1). CiteULike    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

	Copyright © 2009 by American Society of Hematology         Online ISSN: 1528-0020