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| Role for highly regulated rep gene expression in adeno-associated virus vector production |
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| Author(s): Li JA, Samulski RJ, Xiao XA |
| Source: JOURNAL OF VIROLOGY Volume: 71 Issue: 7 Pages: 5236-5243 Published: JUL 1997 |
| Times Cited: 128 References: 57 |
| Abstract: Recent success achieving long-term in vivo gene transfer without a significant immune response by using adeno-associated virus (AAV) vectors (X. Xiao, J, Li, and R. J. Samulski, J, Virol. 70:8098-8108, 1995) has encouraged further development of this vector for human gene therapy. Currently, studies focus on the generation of high-titer vectors by using the two-plasmid helper-vector system in adenovirus (Ad)-infected cells. To examine the effects of the AAV replication (rep) genes on recombinant AAV (rAAV) vector production, we have constructed a series of AAV helper plasmids that contain strong heterologous promoters in place of the endogenous p5 promoter. Although high-level rep gene expression was achieved, rAAV DNA failed to replicate in the absence of Ad infection. Moreover, unregulated overexpression of Rep78/68 led to substantially lower rAAV yields in the presence of Ad (10(4-5) versus 10(7-8)). In contrast, under similar conditions, reduced Rep78/68 expression resulted in much higher rAAV yields (10(9)). Molecular characterization showed that overexpression of the rep gene decreased rAAV DNA replication and severely inhibited capsid (eap) gene expression. Interestingly, a reduced rep level enhanced cnp gene expression and supported normal rAAV DNA replication. These studies suggest a critical role for regulated rep gene expression in rAAV production and have facilitated the development of a new AAV helper plasmid that increases vector production eightfold over currently used constructs. |
| Document Type: Article |
| Language: English |
Addresses:
1. UNIV N CAROLINA, GENE THERAPY CTR, CHAPEL HILL, NC 27599 USA
2. UNIV N CAROLINA, DEPT PHARMACOL, CHAPEL HILL, NC 27599 USA
3. SOMATIX THERAPY CORP, ALAMEDA, CA 94501 USA |
| Publisher: AMER SOC MICROBIOLOGY, 1325 MASSACHUSETTS AVENUE, NW, WASHINGTON, DC 20005-4171 |
| Subject Category: Virology |
| IDS Number: XD829 |
| ISSN: 0022-538X |
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