Optimization of the Generation and Propagation of Gutless Adenoviral Vectors
- 10 February 2003
- journal article
- research article
- Published by Mary Ann Liebert Inc in Human Gene Therapy
- Vol. 14 (3), 243-254
- https://doi.org/10.1089/10430340360535797
Abstract
Adenoviral vectors devoid of all viral coding regions are referred to by many names, including gutless vectors. Gutless vectors display reduced toxicity and immunogenicity, increased duration of transgene expression, and increased coding capacity compared to early generation vectors, which contain the majority of the viral backbone genes. However, the production of gutless vectors at a scale and purity suitable for clinical use has limited the utility of this technology. In this work we describe the optimization of the production of gutless vectors. We constructed an improved helper virus and generated an alternative gutless vector producer cell line, PERC6-Cre. We demonstrated increased gutless vector yields, minimal helper virus contamination, and no replication-competent adenovirus contamination using the optimized system. Furthermore, the PERC6-Cre cells were adapted to serum-free suspension culture and high-titer gutless vector preparations were produced using bioreactor technology, suggesting the feasibility of gutless vector scale-up for clinical use. Finally, we observed that helper virus lacking a packaging signal could be packaged at a low frequency, revealing an inherent limitation to the differential packaging strategy for gutless vector propagation.Keywords
This publication has 23 references indexed in Scilit:
- Gutted adenoviral vector growth using E1/E2b/E3‐deleted helper virusesThe Journal of Gene Medicine, 2002
- Cre Levels Limit Packaging Signal Excision Efficiency in the Cre/ loxP Helper-Dependent Adenoviral Vector SystemJournal of Virology, 2002
- Efficient Rescue of Gutted Adenovirus Genomes Allows Rapid Production of Concentrated Stocks Without Negative SelectionHuman Gene Therapy, 2002
- A novel system for the production of fully deleted adenovirus vectors that does not require helper adenovirusGene Therapy, 2001
- System for Efficient Helper-Dependent Minimal Adenovirus Construction and RescueHuman Gene Therapy, 2001
- Characterization of Replication-Competent Adenovirus Isolates from Large-Scale Production of a Recombinant Adenoviral VectorHuman Gene Therapy, 1999
- High Doses of a Helper-Dependent Adenoviral Vector Yield Supraphysiological Levels ofα1-Antitrypsin with Negligible ToxicityHuman Gene Therapy, 1998
- Genomic DNA transfer with a high-capacity adenovirus vector results in improved in vivo gene expression and decreased toxicityNature Genetics, 1998
- Emergence of Early Region 1-Containing Replication-Competent Adenovirus in Stocks of Replication-Defective Adenovirus Recombinants (ΔE1 + ΔE3) During Multiple Passages in 293 CellsHuman Gene Therapy, 1994
- The adenovirus type 5 E1A enhancer contains two functionally distinct domains: One is specific for E1A and the other modulates all early units in cisCell, 1986