Genetic design of an optimized packaging cell line for gene vectors transducing human B cells.
Viral gene vectors often rely on packaging cell lines, which provide the necessary factors in trans for the formation of virus-like particles. Previously, we reported on a first-generation packaging cell line for gene vectors, which are based on the B-lymphotropic Epstein-Barr virus (EBV), a human gamma-herpesvirus. This 293HEK-derived packaging cell line harbors a helper virus genome with a genetic modification that prevents the release of helper virions, but efficiently packages vector plasmids into virus-like particles with transducing capacity for human B cells. Here, we extended this basic approach towards a non-transforming, virus-free packaging cell line, which harbors an EBV helper virus genome with seven genetic alterations. In addition, we constructed a novel gene vector plasmid, which is devoid of a prokaryotic antibiotic resistance gene, and thus more suitable for in vivo applications in human gene therapy. We demonstrate in this paper that EBV-based gene vectors can be efficiently generated with this much-improved packaging cell line to provide helper virus-free gene vector stocks with transducing capacity for established human B-cell lines and primary B cells.
Hettich
E
E
Department of Gene Vectors, GSF-National Research Center for Environment and Health, München, Germany.
Janz
A
A
Zeidler
R
R
Pich
D
D
Hellebrand
E
E
Weissflog
B
B
Moosmann
A
A
Hammerschmidt
W
W
D001402:B-Lymphocytes / Q000821:virology*; D002460:Cell Line; D004279:DNA, Viral / Q000032:analysis; D005434:Flow Cytometry; D015870:Gene Expression; D005818:Genetic Engineering; D005822:Genetic Vectors / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000235:genetics; D006378:Helper Viruses; D004854:Herpesvirus 4, Human; D006801:Humans; D016133:Polymerase Chain Reaction; D011995:Recombination, Genetic; D014161:Transduction, Genetic / Q000379:methods*; D014162:Transfection / Q000379:methods; D019065:Virus Assembly
D004279:DNA, Viral; D049452:Green Fluorescent Proteins