Process development of lentiviral vectors for large scale production using a HEK293 producer cell line

Download
  1. Get@NRC: Process development of lentiviral vectors for large scale production using a HEK293 producer cell line (Opens in a new window)
DOIResolve DOI: http://doi.org/10.1016/S1525-0016(16)33516-X
AuthorSearch for: ; Search for: ; Search for: ; Search for: ; Search for:
TypeArticle
Journal titleMolecular Therapy
ISSN1525-0016
1525-0024
Volume24
IssueSupplement 1
PagesS279S280
AbstractLentiviral vectors (LVs) are promising vectors for gene therapy. Most often, they are used to deliver a functional copy of a gene to sustainably replace a defective or missing gene. However, processes for LVs must be improved to increase the yield, facilitate the scale up and satisfy Health regulatory agencies. For these reasons, we have developed and optimized a LVs production process in serum-free medium using an inducible HEK293 producer cell line which possesses the capacity to grow in suspension culture. By adding two inducing molecules, (cumate and doxycycline) this cell line produces LVs pseudotyped with the protein G of the vesicular stomatitis virus without the need of any transfection. Our tested LV carried an expression cassette for GFP to facilitate LV quantification. To optimize the process, a design of experiment (DoE) was prepared which included the study of different culture media, high cell density production using six cell boosts commercially available and the addition of sodium butyrate, caffeine and valproic acid. We found that two cell boosts were outperforming the other cell boosts tested. At the present time, two commercial media (Hycell TransFx-H and SFM4TransFx-293 media) were our best candidates to maximize viral titer by achieving high cell density culture. In parallel, a LV carrying the cDNA for a shorter version of dystrophin (mini-dystrophin) was constructed. The truncated version of the dystrophin was produced by transient transfection in 293A cells and its presence was confirmed by western blot. We are planning to evaluate if the optimal conditions for the production of LV-GFP will be also applicable to LV-mini-dystrophin, a LV encoding a much longer transgene than GFP (0.7 kb vs 5.8 kb). This LV could be first evaluated for cell therapy in animal models and later, in patients suffering from Duchenne muscular dystrophy, where the dystrophin gene is defective and the protein is absent.
Publication date
PublisherNature Publishing Group
LanguageEnglish
AffiliationHuman Health Therapeutics; National Research Council Canada
Peer reviewedYes
NPARC number23001309
Export citationExport as RIS
Report a correctionReport a correction
Record identifier64571471-5ffb-4516-bcf9-8685698f524a
Record created2017-01-16
Record modified2017-01-16
Bookmark and share
  • Share this page with Facebook (Opens in a new window)
  • Share this page with Twitter (Opens in a new window)
  • Share this page with Google+ (Opens in a new window)
  • Share this page with Delicious (Opens in a new window)