TY - JOUR
T1 - Generation of a stable packaging cell line producing high-titer PPT-deleted integration-deficient lentiviral vectors
AU - Hu, Peirong
AU - Li, Yedda
AU - Sands, Mark S.
AU - McCown, Thomas
AU - Kafri, Tal
N1 - Publisher Copyright:
© 2015 American Society of Gene & Cell Therapy
PY - 2015/4/29
Y1 - 2015/4/29
N2 - 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 (∼107 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 108 IU/mL, which upon concentration increased to 1010 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.
AB - 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 (∼107 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 108 IU/mL, which upon concentration increased to 1010 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.
UR - http://www.scopus.com/inward/record.url?scp=84966588701&partnerID=8YFLogxK
U2 - 10.1038/mtm.2015.25
DO - 10.1038/mtm.2015.25
M3 - Article
C2 - 26229972
AN - SCOPUS:84966588701
SN - 2329-0501
VL - 2
SP - 15025
JO - Molecular Therapy - Methods and Clinical Development
JF - Molecular Therapy - Methods and Clinical Development
ER -