Submitted November 25, 2008
Accepted March 1, 2009
Efficient construction of producer cell lines for a SIN lentiviral vector for SCID-X1 gene therapy by concatemeric array transfection
Robert E. Throm, Annastasia A. Ouma, Sheng Zhou, Anantharaman Chandrasekaran, Timothy Lockey, Michael Greene, Suk See De Ravin, Morvarid Moayeri, Harry L. Malech, Brian P. Sorrentino, and John T. Gray*
Division of Experimental Hematology, Department of Hematology, St. Jude Children's Research Hospital, Memphis, TN, United States
Department of Therapeutics Production and Quality, St. Jude Children's Research Hospital, Memphis, TN, United States
Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, United States
* Corresponding author; email: john.gray{at}stjude.org.
Retroviral vectors containing internal promoters, chromatin insulators, and self-inactivating (SIN) 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 GFP, which when grown in a bioreactor generated over 20 liters of supernatant with titers above 107 tu/ml. Further refinement of our technique enabled the rapid generation of whole populations of stably transformed cells which produce similar titers. Finally, we describe the construction of an insulated, SIN lentiviral vector encoding the human IL2 receptor common 
-chain (IL2RG) gene and the efficient derivation of cloned producer cells that generate supernatants with titers above 5 x 107 tu/ml, and which are suitable for use in a clinical trial for X-linked SCID (SCID-X1).
