Generation of a packaging cell line for prolonged large-scale production of high-titer HIV-1-based lentiviral vector.
BACKGROUND
A stable packaging cell line facilitates large-scale lentivirus vector manufacture. However, it has been difficult to produce clinical-scale HIV-1-based lentiviral vectors using a packaging cell line, in part due to toxicity of packaging genes, and gene silencing that occurs during the long culture period necessary for sequential addition of packaging constructs.


METHODS
To avoid these problems, we developed a three-level cascade gene regulation system designed to remove tetracycline transactivator (tTA) from cytomegalovirus immediate early promoter (CMV)-controlled expression to reduce cytotoxicity from constitutive expression of tTA and leaky expression of packaging genes. We also performed a one-step integration of the three packaging plasmids to shorten the culture time for clonal selection.


RESULTS
Although leaky expression of p24 and vector production still occurred despite the three-level regulation system, little cytotoxicity was observed and producer cells could be expanded for large-scale production. Producer cells yielded remarkably stable vector production over a period greater than 11 days with the highest titer 3.5 x 10(7) transducing units (TU)/ml and p24 300 ng/ml, yielding 2.2 x 10(11) TU and 1.8 milligram (mg) p24 from one cell factory. No replication-competent lentivirus (RCL) was detected. Long-term analysis demonstrated that, although the cells are genetically stable, partial gene silencing occurs after 2-3 months in culture; however, the one-step construct integration allowed prolonged vector production before significant gene silencing. Concentrated vector resulted in 90% transduction in CD4+ lymphocytes at 20 TU per cell. CD34+ progenitor cells were transduced at 41-46% efficiency, and long-term initiating culture (LTC-IC) was transduced at 45-51%.


CONCLUSIONS
These results demonstrate for the first time HIV-1-based lentiviral vector production on the large scale using a packaging cell line.
Ni
Yajin
Y
VIRxSYS Corporation, Gaithersburg, Maryland 20877 [correction] USA.
Sun
Susan
S
Oparaocha
Ibe
I
Humeau
Laurent
L
Davis
Brian
B
Cohen
Reuben
R
Binder
Gwendolyn
G
Chang
Yung-Nien
YN
Slepushkin
Vladimir
V
Dropulic
Boro
B
D001483:Base Sequence; D002460:Cell Line; D002999:Clone Cells; D003001:Cloning, Molecular; D003062:Codon; D004797:Enzyme-Linked Immunosorbent Assay; D015685:Fusion Proteins, gag-pol / Q000737:chemistry / Q000235:genetics; D005786:Gene Expression Regulation / Q000187:drug effects; D015694:Gene Products, rev / Q000737:chemistry / Q000235:genetics; D015696:Gene Products, tat / Q000737:chemistry / Q000235:genetics; D005818:Genetic Engineering; D005822:Genetic Vectors / Q000096:biosynthesis / Q000235:genetics*; D015497:HIV-1 / Q000235:genetics*; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D016086:Lentivirus / Q000235:genetics*; D008562:Membrane Glycoproteins / Q000378:metabolism; D008957:Models, Genetic; D008969:Molecular Sequence Data; D010957:Plasmids; D013752:Tetracycline / Q000494:pharmacology; D014161:Transduction, Genetic; D014162:Transfection; D014759:Viral Envelope Proteins / Q000378:metabolism; D014771:Virion / Q000378:metabolism; D014779:Virus Replication; D054321:rev Gene Products, Human Immunodeficiency Virus; D054322:tat Gene Products, Human Immunodeficiency Virus
D003062:Codon; D015685:Fusion Proteins, gag-pol; C028391:G protein, vesicular stomatitis virus; D015694:Gene Products, rev; D015696:Gene Products, tat; D008562:Membrane Glycoproteins; D014759:Viral Envelope Proteins; D054321:rev Gene Products, Human Immunodeficiency Virus; D054322:tat Gene Products, Human Immunodeficiency Virus; D013752:Tetracycline