title: RD2-MolPack-Chim3, a packaging cell line for stable production of lentiviral vectors for anti-HIV gene therapy.
abstract: Over the last two decades, several attempts to generate packaging cells for lentiviral vectors (LV) have been made. Despite different technologies, no packaging clone is currently employed in clinical trials. We developed a new strategy for LV stable production based on the HEK-293T progenitor cells; the sequential insertion of the viral genes by integrating vectors; the constitutive expression of the viral components; and the RD114-TR envelope pseudotyping. We generated the intermediate clone PK-7 expressing constitutively gag/pol and rev genes and, by adding tat and rd114-tr genes, the stable packaging cell line RD2-MolPack, which can produce LV carrying any transfer vector (TV). Finally, we obtained the RD2-MolPack-Chim3 producer clone by transducing RD2-MolPack cells with the TV expressing the anti-HIV transgene Chim3. Remarkably, RD114-TR pseudovirions have much higher potency when produced by stable compared with transient technology. Most importantly, comparable transduction efficiency in hematopoietic stem cells (HSC) is obtained with 2-logs less physical particles respect to VSV-G pseudovirions produced by transient transfection. Altogether, RD2-MolPack technology should be considered a valid option for large-scale production of LV to be used in gene therapy protocols employing HSC, resulting in the possibility of downsizing the manufacturing scale by about 10-fold in respect to transient technology.
authors.affiliation: MolMed S.p.A., Milano 20132, Italy.
authors.forename: Anna
authors.initials: A
authors.lastname: Stornaiuolo
authors.forename: Bianca Maria
authors.initials: BM
authors.lastname: Piovani
authors.forename: Sergio
authors.initials: S
authors.lastname: Bossi
authors.forename: Eleonora
authors.initials: E
authors.lastname: Zucchelli
authors.forename: Stefano
authors.initials: S
authors.lastname: Corna
authors.forename: Francesca
authors.initials: F
authors.lastname: Salvatori
authors.forename: Fulvio
authors.initials: F
authors.lastname: Mavilio
authors.forename: Claudio
authors.initials: C
authors.lastname: Bordignon
authors.forename: Gian Paolo
authors.initials: GP
authors.lastname: Rizzardi
authors.forename: Chiara
authors.initials: C
authors.lastname: Bovolenta
mesh_terms: D000818:Animals; D015685:Fusion Proteins, gag-pol / Q000235:genetics / Q000378:metabolism; D015694:Gene Products, rev / Q000235:genetics / Q000378:metabolism; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics* / Q000378:metabolism; D057809:HEK293 Cells; D015658:HIV Infections / Q000628:therapy; D006412:Hematopoietic Stem Cells / Q000378:metabolism / Q000821:virology; D006801:Humans; D016086:Lentivirus / Q000235:genetics* / Q000378:metabolism / Q000502:physiology; D061987:Sf9 Cells; D018411:Spodoptera; D014161:Transduction, Genetic / Q000379:methods*; D019076:Transgenes / Q000235:genetics; D019065:Virus Assembly
chemical_list: D015685:Fusion Proteins, gag-pol; D015694:Gene Products, rev