title: Use of the piggyBac transposon to create stable packaging cell lines for the production of clinical-grade self-inactivating γ-retroviral vectors.
abstract: Efforts to improve the biosafety of γ-retroviral-mediated gene therapy have resulted in a shift toward the use of self-inactivating (SIN) γ-retroviral vectors. However, scale-up and manufacturing of such vectors requires significant optimization of transient transfection-based processes or development of novel platforms for the generation of stable producer cell clones. To that end, we describe the use of the piggybac transposon to generate stable producer cell clones for the production of SIN γ-retroviral vectors. The piggybac transposon is a universal tool allowing for the stable integration of SIN γ-retroviral constructs into murine (PG13) and human 293-based Phoenix (GALV and RD114, respectively) packaging cell lines without reverse transcription. Following transposition, a high-titer clone is selected for manufacture of a master cell bank and subsequent γ-retroviral vector supernatant production. Packaging cell clones created using the piggybac transposon have comparable titers to non-SIN vectors generated via conventional methods. We describe herein the use of the piggybac transposon for the production of stable packaging cell clones for the manufacture of clinical-grade SIN γ-retroviral vectors for ex vivo gene therapy clinical trials.
authors: 1 Surgery Branch, National Cancer Institute , Bethesda, MD 20892-1201.
authors: Steven A
authors: SA
authors: Feldman
authors: Hui
authors: H
authors: Xu
authors: Mary A
authors: MA
authors: Black
authors: Tristen S
authors: TS
authors: Park
authors: Paul F
authors: PF
authors: Robbins
authors: James N
authors: JN
authors: Kochenderfer
authors: Richard A
authors: RA
authors: Morgan
authors: Steven A
authors: SA
authors: Rosenberg
mesh_terms: D000818:Animals; D002460:Cell Line; D016207:Cytokines / Q000378:metabolism; D004251:DNA Transposable Elements / Q000235:genetics; D005434:Flow Cytometry; D005822:Genetic Vectors / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000166:cytology / Q000378:metabolism; D051379:Mice; D012190:Retroviridae / Q000235:genetics*; D014162:Transfection / Q000379:methods*
chemical_list: D016207:Cytokines; D004251:DNA Transposable Elements; D049452:Green Fluorescent Proteins