Integrated strategy for the production of therapeutic retroviral vectors.
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Coroadinha, Ana Sofia
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AbstractThe broad application of retroviral vectors for gene delivery is still hampered by the difficulty to reproducibly establish high vector producer cell lines generating sufficient amounts of highly concentrated virus vector preparations of high quality. To enhance the process for producing clinically relevant retroviral vector preparations for therapeutic applications, we have integrated novel and state-of-the-art technologies in a process that allows rapid access to high-efficiency vector-producing cells and consistent production, purification, and storage of retroviral vectors. The process has been designed for various types of retroviral vectors for clinical application and to support a high-throughput process. New modular helper cell lines that permit rapid insertion of DNA encoding the therapeutic vector of interest at predetermined, optimal chromosomal loci were developed to facilitate stable and high vector production levels. Packaging cell lines, cultivation methods, and improved medium composition were coupled with vector purification and storage process strategies that yield maximal vector infectivity and stability. To facilitate GMP-grade vector production, standard of operation protocols were established. These processes were validated by production of retroviral vector lots that drive the expression of type VII collagen (Col7) for the treatment of a skin genetic disease, dystrophic epidermolysis bullosa. The potential efficacy of the Col7-expressing vectors was finally proven with newly developed systems, in particular in target primary keratinocyte cultures and three-dimensional skin tissues in organ culture.
CitationIntegrated strategy for the production of therapeutic retroviral vectors. 2011, 22 (3):370-9 Hum. Gene Ther.
AffiliationInstituto de Biologia Experimental e Tecnológica/Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, P-2781-901 Oeiras, Portugal.
JournalHuman gene therapy
The following license files are associated with this item:
- A new generation of retroviral producer cells: predictable and stable virus production by Flp-mediated site-specific integration of retroviral vectors.
- Authors: Schucht R, Coroadinha AS, Zanta-Boussif MA, Verhoeyen E, Carrondo MJ, Hauser H, Wirth D
- Issue date: 2006 Aug
- HEK293-based production platform for γ-retroviral (self-inactivating) vectors: application for safe and efficient transfer of COL7A1 cDNA.
- Authors: Hennig K, Raasch L, Kolbe C, Weidner S, Leisegang M, Uckert W, Titeux M, Hovnanian A, Kuehlcke K, Loew R
- Issue date: 2014 Dec
- Fibroblasts show more potential as target cells than keratinocytes in COL7A1 gene therapy of dystrophic epidermolysis bullosa.
- Authors: Goto M, Sawamura D, Ito K, Abe M, Nishie W, Sakai K, Shibaki A, Akiyama M, Shimizu H
- Issue date: 2006 Apr
- The use of recombinase mediated cassette exchange in retroviral vector producer cell lines: predictability and efficiency by transgene exchange.
- Authors: Coroadinha AS, Schucht R, Gama-Norton L, Wirth D, Hauser H, Carrondo MJ
- Issue date: 2006 Jul 13