Efficient construction of producer cell lines for a SIN lentiviral vector for SCID-X1 gene therapy by concatemeric array transfection.

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 10(7) 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 gamma chain (IL2RG) gene and the efficient derivation of cloned producer cells that generate supernatants with titers greater than 5 x 10(7) TU/mL and that are suitable for use in a clinical trial for X-linked severe combined immunodeficiency (SCID-X1).

[1]  Christine Kinnon,et al.  Insertional mutagenesis combined with acquired somatic mutations causes leukemogenesis following gene therapy of SCID-X1 patients. , 2008, The Journal of clinical investigation.

[2]  F. Bushman,et al.  Insertional oncogenesis in 4 patients after retrovirus-mediated gene therapy of SCID-X1. , 2008, The Journal of clinical investigation.

[3]  A. Schambach,et al.  Physiological promoters reduce the genotoxic risk of integrating gene vectors. , 2008, Molecular therapy : the journal of the American Society of Gene Therapy.

[4]  Y. Durocher,et al.  Inducible packaging cells for large-scale production of lentiviral vectors in serum-free suspension culture. , 2008, Molecular therapy : the journal of the American Society of Gene Therapy.

[5]  A. Nienhuis,et al.  An experimental system for the evaluation of retroviral vector design to diminish the risk for proto-oncogene activation. , 2008, Blood.

[6]  C. Lengner,et al.  Sequential expression of pluripotency markers during direct reprogramming of mouse somatic cells. , 2008, Cell stem cell.

[7]  A. Nienhuis,et al.  A chromatin insulator blocks interactions between globin regulatory elements and cellular promoters in erythroid cells. , 2007, Blood cells, molecules & diseases.

[8]  A. Schambach,et al.  Lentiviral vectors containing an enhancer-less ubiquitously acting chromatin opening element (UCOE) provide highly reproducible and stable transgene expression in hematopoietic cells , 2007, Blood.

[9]  G. Stamatoyannopoulos,et al.  Extended core sequences from the cHS4 insulator are necessary for protecting retroviral vectors from silencing position effects. , 2007, Human gene therapy.

[10]  Christof von Kalle,et al.  and insertional genotoxicity Cell culture assays reveal the importance of retroviral vector design for , 2006 .

[11]  E. Simons.  Gene Therapy of X-Linked Severe Combined Immunodeficiency by Use of a Pseudotyped Gammaretroviral Vector , 2006, Pediatrics.

[12]  T. Kafri,et al.  A trans-lentiviral packaging cell line for high-titer conditional self-inactivating HIV-1 vectors. , 2006, Molecular therapy : the journal of the American Society of Gene Therapy.

[13]  Clelia Di Serio,et al.  Hematopoietic stem cell gene transfer in a tumor-prone mouse model uncovers low genotoxicity of lentiviral vector integration , 2006, Nature Biotechnology.

[14]  Anne E Carpenter,et al.  A Lentiviral RNAi Library for Human and Mouse Genes Applied to an Arrayed Viral High-Content Screen , 2006, Cell.

[15]  K. Cornetta,et al.  Product-enhanced reverse transcriptase assay for replication-competent retrovirus and lentivirus detection. , 2005, Human gene therapy.

[16]  Brian Davis,et al.  Generation of a packaging cell line for prolonged large‐scale production of high‐titer HIV‐1‐based lentiviral vector , 2005, The journal of gene medicine.

[17]  H. Lodish,et al.  Murine hematopoietic stem cells change their surface phenotype during ex vivo expansion. , 2005, Blood.

[18]  Christine Kinnon,et al.  Mutations in TNFRSF13B Encoding TACI Are Associated With Common Variable Immunodeficiency in Humans , 2006, Pediatrics.

[19]  K. Keyvanfar,et al.  Efficient gene transfer into rhesus repopulating hematopoietic stem cells using a simian immunodeficiency virus-based lentiviral vector system. , 2004, Blood.

[20]  M. Kay,et al.  Silencing of episomal transgene expression by plasmid bacterial DNA elements in vivo , 2004, Gene Therapy.

[21]  F. Cosset,et al.  Continuous high-titer HIV-1 vector production , 2003, Nature Biotechnology.

[22]  Jody A. Vandergriff,et al.  Comparison of various envelope proteins for their ability to pseudotype lentiviral vectors and transduce primitive hematopoietic cells from human blood. , 2002, Molecular therapy : the journal of the American Society of Gene Therapy.

[23]  A. Consiglio,et al.  A new-generation stable inducible packaging cell line for lentiviral vectors. , 2001, Human gene therapy.

[24]  H. Schaal,et al.  Generation of a flexible cell line with regulatable, high-level expression of HIV Gag/Pol particles capable of packaging HIV-derived vectors. , 2001, Molecular therapy : the journal of the American Society of Gene Therapy.

[25]  Y. Ron,et al.  An inducible packaging cell system for safe, efficient lentiviral vector production in the absence of HIV-1 accessory proteins. , 2001, Virology.

[26]  I. Verma,et al.  Generation of a stable cell line producing high-titer self-inactivating lentiviral vectors. , 2001, Molecular therapy : the journal of the American Society of Gene Therapy.

[27]  L. Naldini,et al.  Lentiviral vectors: excellent tools for experimental gene transfer and promising candidates for gene therapy , 2000, The journal of gene medicine.

[28]  D. Trono,et al.  A stable system for the high-titer production of multiply attenuated lentiviral vectors. , 2000, Molecular therapy : the journal of the American Society of Gene Therapy.

[29]  F. Deist,et al.  Gene therapy of human severe combined immunodeficiency (SCID)-X1 disease. , 2000, Science.

[30]  Tal Kafri,et al.  A Packaging Cell Line for Lentivirus Vectors , 1999, Journal of Virology.

[31]  I. Weissman,et al.  HIV, but not murine leukemia virus, vectors mediate high efficiency gene transfer into freshly isolated G0/G1 human hematopoietic stem cells. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[32]  Peter Gedeck,et al.  Reviews in Computational Chemistry, Volume 9 , 1997 .

[33]  R. Mulligan,et al.  Versatile Retrovirus Vector Systems for Regulated Gene Expression In Vitro and In Vivo , 1997, Molecular medicine.

[34]  G. Felsenfeld,et al.  Characterization of the chicken beta-globin insulator. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[35]  F. Gage,et al.  In Vivo Gene Delivery and Stable Transduction of Nondividing Cells by a Lentiviral Vector , 1996, Science.