Development of a sensitive assay for detection of replication-competent recombinant lentivirus in large-scale HIV-based vector preparations

Lentiviral vectors have demonstrated great potential as gene therapy vectors mediating efficient ex vivo and in vivo gene delivery and long-term transgene expression in both dividing and nondividing cells. However, for clinical studies it must be demonstrated that lentiviral vector preparations are...

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Bibliographic Details
Published in:Molecular therapy 2003-08, Vol.8 (2), p.332-341
Main Authors: Escarpe, Paul, Zayek, Nathalie, Chin, Peggy, Borellini, Flavia, Zufferey, Romain, Veres, Gabor, Kiermer, Veronique
Format: Article
Language:English
Subjects:
Animals
biological assay
Cell Line
Clinical trials
Design
Enzyme-Linked Immunosorbent Assay
Gene therapy
Genetic Vectors - biosynthesis
Genetic Vectors - genetics
Genetic Vectors - physiology
HIV - genetics
HIV - growth & development
Humans
Infections
lentiviral vectors
Leukemia
Polymerase Chain Reaction
safety
Sensitivity and Specificity
Stem cells
Time Factors
Vectors (Biology)
Virus Replication
Viruses
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Summary:Lentiviral vectors have demonstrated great potential as gene therapy vectors mediating efficient ex vivo and in vivo gene delivery and long-term transgene expression in both dividing and nondividing cells. However, for clinical studies it must be demonstrated that lentiviral vector preparations are safe and not contaminated by replication-competent recombinants related to the parental pathogenic virus. Here we describe a sensitive assay for the detection of replication-competent lentiviruses (RCL) in large-scale preparations of HIV-based lentiviral vectors. This RCL assay for lentiviral vectors is based on the principles used for retroviral vectors, using a highly permissive cell line, C8166-45, for RCL amplification and an appropriate positive control virus to establish the assay sensitivity. The assay is capable of detecting 1 RCL infectious unit in a background of 2.5 × 108 transducing units of vector in a single test culture. Statistically representative samples from large-scale lentiviral vector productions were assayed using multiple test cultures for each lot. Overall, a total of 1.4 × 1010 transducing units of vector from 10 independent 14-liter production lots were screened and no RCL was detected. We propose to implement this assay as a release testing for clinical-grade lentiviral vector preparations intended for gene therapy clinical trials.
ISSN:1525-0016
1525-0024
DOI:10.1016/S1525-0016(03)00167-9