title: Efficient FLPe recombinase enables scalable production of helper-dependent adenoviral vectors with negligible helper-virus contamination.
abstract: Helper-dependent (HD), high-capacity adenoviruses are one of the most efficient and safe gene therapy vectors, capable of mediating long-term expression. 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 signal. 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 virus. 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 backbone, 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 purification.
authors.affiliation: Molecular Medicine and Gene Therapy Unit, Room 1.302 Stopford Building, School of Medicine, University of Manchester, Oxford Road, Manchester M13 9PT, United Kingdom. pablo.umana@glycart.com
authors.forename: P
authors.initials: P
authors.lastname: Umaña
authors.forename: C A
authors.initials: CA
authors.lastname: Gerdes
authors.forename: D
authors.initials: D
authors.lastname: Stone
authors.forename: J R
authors.initials: JR
authors.lastname: Davis
authors.forename: D
authors.initials: D
authors.lastname: Ward
authors.forename: M G
authors.initials: MG
authors.lastname: Castro
authors.forename: P R
authors.initials: PR
authors.lastname: Lowenstein
mesh_terms: D000256:Adenoviridae / Q000235:genetics*; D000818:Animals; D001709:Biotechnology / Q000379:methods; D001921:Brain / Q000378:metabolism; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D004254:DNA Nucleotidyltransferases / Q000378:metabolism*; D005822:Genetic Vectors; D006378:Helper Viruses / Q000378:metabolism*; D006801:Humans; D008156:Luciferases / Q000378:metabolism; D008957:Models, Genetic; D010957:Plasmids / Q000378:metabolism; D051381:Rats; D014162:Transfection; D001616:beta-Galactosidase / Q000378:metabolism
chemical_list: D008156:Luciferases; D004254:DNA Nucleotidyltransferases; C045119:FLP recombinase; D001616:beta-Galactosidase