Efficient FLPe recombinase enables scalable production of helper-dependent adenoviral vectors with negligible helper-virus contamination
- 1 June 2001
- journal article
- technical report
- Published by Springer Nature in Nature Biotechnology
- Vol. 19 (6) , 582-585
- https://doi.org/10.1038/89349
Abstract
Helper-dependent (HD), high-capacity adenoviruses are one of the most efficient and safe gene therapy vectors, capable of mediating long-term expression1,2,3,4,5,6,7,8,9,10,11,12. Currently, the most widely used system for HD vector production avoids significant contamination with helper virus by using producer cells stably expressing a nuclear-targeted Cre recombinase and an engineered first-generation helper virus with parallel loxP sites flanking its packaging signal1,3,4,5,6,7,8,9,10,11,12. The system requires a final, density-based separation of HD and residual helper viruses by ultracentrifugation to reduce contaminating helper virus to low levels. This separation step hinders large-scale production of clinical-grade HD virus13. By using a very efficient recombinase, in vitro–evolved FLPe (ref. 14), to excise the helper virus packaging signal in the producer cells, we have developed a scalable HD vector production method. FLP has previously been shown to mediate maximum levels of excision close to 100% compared to 80% for Cre (ref. 15). Utilizing a common HD plasmid backbone1,7,8,10,11,12, the FLPe-based system reproducibly yielded HD virus with the same low levels of helper virus contamination before any density-based separation by ultracentrifugation. This should allow large-scale production of HD vectors using column chromatography–based virus purification13.Keywords
This publication has 21 references indexed in Scilit:
- Preexisting Antiadenoviral Immunity Is Not a Barrier to Efficient and Stable Transduction of the Brain, Mediated by Novel High-Capacity Adenovirus VectorsHuman Gene Therapy, 2001
- Strong Promoters Are the Key to Highly Efficient, Noninflammatory and Noncytotoxic Adenoviral-Mediated Transgene Delivery into the Brain in VivoMolecular Therapy, 2000
- An improved anion-exchange HPLC method for the detection and purification of adenoviral particlesGene Therapy, 2000
- Prolonged Expression and Effective Readministration of Erythropoietin Delivered with a Fully Deleted Adenoviral VectorHuman Gene Therapy, 2000
- Comparative kinetic analysis of FLP and cre recombinases: mathematical models for DNA binding and recombinationJournal of Molecular Biology, 1998
- Improved properties of FLP recombinase evolved by cycling mutagenesisNature Biotechnology, 1998
- Genomic DNA transfer with a high-capacity adenovirus vector results in improved in vivo gene expression and decreased toxicityNature Genetics, 1998
- A helper-dependent adenovirus vector system: Removal of helper virus by Cre-mediated excision of the viral packaging signalProceedings of the National Academy of Sciences, 1996
- A new adenoviral vector: Replacement of all viral coding sequences with 28 kb of DNA independently expressing both full-length dystrophin and beta-galactosidase.Proceedings of the National Academy of Sciences, 1996
- An efficient and flexible system for construction of adenovirus vectors with insertions or deletions in early regions 1 and 3.Proceedings of the National Academy of Sciences, 1994