title: Development of packaging cell lines for generation of adeno-associated virus vectors by lentiviral gene transfer of trans-complementary components.
abstract: Adeno-associated virus (AAV) vector system has several useful advantages with regard to in vitro and in vivo gene transfer. However, their usages have been limited by cumbersome and labor-intensive vector production in the traditional method. To overcome limitations in AAV production, in this report, we explored the possibility of generating AAV packaging cell line, 293T R/C.VA.E2A.E4. cells, by using lentivirus-mediated transduction of Rep/Cap gene of AAV-2, VA RNA, E2A, and E4 genes of Ad5 into 293T cells. In packaging cell lines, it is important that supply of the AAV vector can be stably performed for long time. We showed that the 293T R/C.VA.E2A.E4. cells have stably maintained the transduced components after more than 10 passages and yielded high-titer AAV vectors, and the titer of AAV vectors did not decline even if culture of the packaging cells was continued for long time. The Rep/Cap and E4 gene products caused no remarkable cytotoxicity. The 293T R/C.VA.E2A.E4. cells might be able to tolerate the Rep/Cap and E4 gene products, or have less copy numbers of the Rep/Cap and E4 genes than the traditional method. Moreover, we showed that the AAV vectors derived from 293T R/C.VA.E2A.E4. cells infected the primary human CD34+ haematopoietic progenitor cells with high efficiency (50-70%). In the 293T R/C.VA.E2A.E4. cells, the AAV vectors can be generated by the transfection of one AAV vector plasmid, and large-scale AAV production can be easily achieved. It is important that cumbersome, variable, and costly transfection is avoided.
authors.affiliation: Department of Internal Medicine III, Hamamatsu University School of Medicine, Handayama, Japan. satonaka@hama-med.ac.jp
authors.forename: Satoki
authors.initials: S
authors.lastname: Nakamura
authors.forename: Reiko
authors.initials: R
authors.lastname: Nakamura
authors.forename: Kiyoshi
authors.initials: K
authors.lastname: Shibata
authors.forename: Miki
authors.initials: M
authors.lastname: Kobayashi
authors.forename: Naohi
authors.initials: N
authors.lastname: Sahara
authors.forename: Kazuyuki
authors.initials: K
authors.lastname: Shigeno
authors.forename: Kaori
authors.initials: K
authors.lastname: Shinjo
authors.forename: Kensuke
authors.initials: K
authors.lastname: Naito
authors.forename: Kazunori
authors.initials: K
authors.lastname: Ohnishi
authors.forename: Noriyuki
authors.initials: N
authors.lastname: Kasahara
authors.forename: Yuichi
authors.initials: Y
authors.lastname: Iwaki
mesh_terms: D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers; D000229:Dependovirus / Q000235:genetics*; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D006801:Humans; D007150:Immunohistochemistry
chemical_list: D017931:DNA Primers