title: Generation of a stable packaging cell line producing high-titer PPT-deleted integration-deficient lentiviral vectors.
abstract: The risk of insertional mutagenesis inherent to all integrating exogenous expression cassettes was the impetus for the development of various integration-defective lentiviral vector (IDLV) systems. These systems were successfully employed in a plethora of preclinical applications, underscoring their clinical potential. However, current production of IDLVs by transient plasmid transfection is not optimal for large-scale production of clinical grade vectors. Here, we describe the development of the first tetracycline-inducible stable IDLV packaging cell line comprising the D64E integrase mutant and the VSV-G envelope protein. A conditional self-inactivating (cSIN) vector and a novel polypurine tract (PPT)-deleted vector were incorporated into the newly developed stable packaging cell line by transduction and stable transfection, respectively. High-titer (~10(7) infectious units (IU)/ml) cSIN vectors were routinely generated. Furthermore, screening of single-cell clones stably transfected with PPT-deleted vector DNA resulted in the identification of highly efficient producer cell lines generating IDLV titers higher than 10(8) IU/mL, which upon concentration increased to 10(10) IU/ml. IDLVs generated by stable producer lines efficiently transduce CNS tissues of rodents. Overall, the availability of high-titer IDLV lentivirus packaging cell line described here will significantly facilitate IDLV-based basic science research, as well as preclinical and clinical applications.
authors.affiliation: Gene Therapy Center, University of North Carolina at Chapel Hill , Chapel Hill, North Carolina, USA ; Department of Microbiology and Immunology, University of North Carolina at Chapel Hill , Chapel Hill, North Carolina, USA.
authors.forename: Peirong
authors.initials: P
authors.lastname: Hu
authors.affiliation: Department of Internal Medicine, Washington University School of Medicine , St. Louis, Missouri, USA.
authors.forename: Yedda
authors.initials: Y
authors.lastname: Li
authors.affiliation: Department of Internal Medicine, Washington University School of Medicine , St. Louis, Missouri, USA ; Genetics, Washington University School of Medicine , St. Louis, Missouri, USA.
authors.forename: Mark S
authors.initials: MS
authors.lastname: Sands
authors.affiliation: Gene Therapy Center, University of North Carolina at Chapel Hill , Chapel Hill, North Carolina, USA ; Department of Psychiatry, University of North Carolina at Chapel Hill , Chapel Hill, North Carolina, USA.
authors.forename: Thomas
authors.initials: T
authors.lastname: McCown
authors.affiliation: Gene Therapy Center, University of North Carolina at Chapel Hill , Chapel Hill, North Carolina, USA ; Department of Microbiology and Immunology, University of North Carolina at Chapel Hill , Chapel Hill, North Carolina, USA.
authors.forename: Tal
authors.initials: T
authors.lastname: Kafri