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.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 285-294 |
| Number of pages | 10 |
| Journal | European Journal of Haematology |
| Volume | 73 |
| Issue number | 4 |
| DOIs | |
| State | Published - Oct 2004 |
| Externally published | Yes |
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Keywords
- AAV
- CD34 hematopoietic progenitor cells
- E2A
- E4
- Lentivirus
- Packaging cell
- Rep/Cap
ASJC Scopus subject areas
- Hematology
Cite this
Development of packaging cell lines for generation of adeno-associated virus vectors by lentiviral gene transfer of trans-complementary components. / Nakamura, Satoki; Nakamura, Reiko; Shibata, Kiyoshi; Kobayashi, Miki; Sahara, Naohi; Shigeno, Kazuyuki; Shinjo, Kaori; Naito, Kensuke; Ohnishi, Kazunori; Kasahara, Noriyuki; Iwaki, Yuichi.
In: European Journal of Haematology, Vol. 73, No. 4, 10.2004, p. 285-294.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Development of packaging cell lines for generation of adeno-associated virus vectors by lentiviral gene transfer of trans-complementary components
AU - Nakamura, Satoki
AU - Nakamura, Reiko
AU - Shibata, Kiyoshi
AU - Kobayashi, Miki
AU - Sahara, Naohi
AU - Shigeno, Kazuyuki
AU - Shinjo, Kaori
AU - Naito, Kensuke
AU - Ohnishi, Kazunori
AU - Kasahara, Noriyuki
AU - Iwaki, Yuichi
PY - 2004/10
Y1 - 2004/10
N2 - 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.
AB - 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.
KW - AAV
KW - CD34 hematopoietic progenitor cells
KW - E2A
KW - E4
KW - Lentivirus
KW - Packaging cell
KW - Rep/Cap
UR - http://www.scopus.com/inward/record.url?scp=4644241220&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=4644241220&partnerID=8YFLogxK
U2 - 10.1111/j.1600-0609.2004.00290.x
DO - 10.1111/j.1600-0609.2004.00290.x
M3 - Article
VL - 73
SP - 285
EP - 294
JO - European Journal of Haematology
JF - European Journal of Haematology
SN - 0902-4441
IS - 4
ER -