Efficient FLPe recombinase enables scalable production of helper-dependentadenoviral vectors with negligible helper-virus contamination

Citation
P. Umana et al., Efficient FLPe recombinase enables scalable production of helper-dependentadenoviral vectors with negligible helper-virus contamination, NAT BIOTECH, 19(6), 2001, pp. 582-585
Citations number
20
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Biotecnology & Applied Microbiology",Microbiology
Journal title
NATURE BIOTECHNOLOGY
ISSN journal
1087-0156 → ACNP
Volume
19
Issue
6
Year of publication
2001
Pages
582 - 585
Database
ISI
SICI code
1087-0156(200106)19:6<582:EFRESP>2.0.ZU;2-6
Abstract
Helper-dependent (HD), high-capacity adenoviruses are one of the most effic ient and safe gene therapy vectors, capable of mediating long-term expressi on(1-12). Currently, the most widely used system for HD vector production a voids significant contamination with helper virus by using producer cells s tably expressing a nuclear-targeted Cre recombinase and an engineered first -generation helper virus with parallel loxP sites flanking its packaging si gnal(1,3-12). The system requires a final, density-based separation of HD a nd residual helper viruses by ultracentrifugation to reduce contaminating h elper virus to low levels. This separation step hinders large-scale product ion of clinical-grade HD virus(13), By using a very efficient recombinase, in vitro-evolved FLPe (ref. 14), to excise the helper virus packaging signa l 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 pla smid backbone(1,7,8,10-12), the FLPe-based system reproducibly yielded HD v irus with the same low levels of helper virus contamination before any dens ity-based separation by ultracentrifugation. This should allow large-scale production of HD vectors using column chromatography-based virus purificati on(13).