Document Detail

Large-scale manufacture and characterization of a lentiviral vector produced for clinical ex vivo gene therapy application.
MedLine Citation:
PMID:  21043787     Owner:  NLM     Status:  MEDLINE    
From the perspective of a pilot clinical gene therapy trial for Wiskott-Aldrich syndrome (WAS), we implemented a process to produce a lentiviral vector under good manufacturing practices (GMP). The process is based on the transient transfection of 293T cells in Cell Factory stacks, scaled up to harvest 50 liters of viral stock per batch, followed by purification of the vesicular stomatitis virus glycoprotein-pseudotyped particles through several membrane-based and chromatographic steps. The process leads to a 200-fold volume concentration and an approximately 3-log reduction in protein and DNA contaminants. An average yield of 13% of infectious particles was obtained in six full-scale preparations. The final product contained low levels of contaminants such as simian virus 40 large T antigen or E1A sequences originating from producer cells. Titers as high as 2 × 10(9) infectious particles per milliliter were obtained, generating up to 6 × 10(11) infectious particles per batch. The purified WAS vector was biologically active, efficiently expressing the genetic insert in WAS protein-deficient B cell lines and transducing CD34(+) cells. The vector introduced 0.3-1 vector copy per cell on average in CD34(+) cells when used at the concentration of 10(8) infectious particles per milliliter, which is comparable to preclinical preparations. There was no evidence of cellular toxicity. These results show the implementation of large-scale GMP production, purification, and control of advanced HIV-1-derived lentiviral technology. Results obtained with the WAS vector provide the initial manufacturing and quality control benchmarking that should be helpful to further development and clinical applications.
Otto-Wilhelm Merten; Sabine Charrier; Nicolas Laroudie; Sylvain Fauchille; Céline Dugué; Christine Jenny; Muriel Audit; Maria-Antonietta Zanta-Boussif; Hélène Chautard; Marina Radrizzani; Giuliana Vallanti; Luigi Naldini; Patricia Noguiez-Hellin; Anne Galy
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Publication Detail:
Type:  Journal Article     Date:  2011-03-07
Journal Detail:
Title:  Human gene therapy     Volume:  22     ISSN:  1557-7422     ISO Abbreviation:  Hum. Gene Ther.     Publication Date:  2011 Mar 
Date Detail:
Created Date:  2011-03-15     Completed Date:  2011-06-21     Revised Date:  2011-11-24    
Medline Journal Info:
Nlm Unique ID:  9008950     Medline TA:  Hum Gene Ther     Country:  United States    
Other Details:
Languages:  eng     Pagination:  343-56     Citation Subset:  IM    
Généthon, F-91002 Evry, France.
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MeSH Terms
Cell Culture Techniques
Cell Line
Drug Contamination / legislation & jurisprudence,  prevention & control
Gene Expression Regulation
Gene Order
Gene Therapy*
Genetic Vectors / biosynthesis*,  genetics*,  physiology
HEK293 Cells
Hematopoietic Stem Cells / metabolism
Industrial Microbiology / methods*
Lentivirus / genetics*,  physiology
Plasmids / genetics
Proviruses / genetics
Quality Control
Transduction, Genetic
Transgenes / genetics
Wiskott-Aldrich Syndrome / therapy
Grant Support

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine

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