Generation of a stable packaging cell line producing high-titer PPT-deleted integration-deficient lentiviral vectors

• Published in: Molecular therapy. Methods & clinical development Vol. 2; p. 15025
• Main Authors: Hu, Peirong, Li, Yedda, Sands, Mark S, McCown, Thomas, Kafri, Tal
• Format: Journal Article
• Language: English
• Published: United States Elsevier Limited 01.01.2015; Nature Publishing Group; Elsevier
• Subjects: Lentivirus
• ISSN: 2329-0501; 2329-0501
• DOI: 10.1038/mtm.2015.25

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.