Journal
HUMAN GENE THERAPY
Volume 12, Issue 1, Pages 61-70Publisher
MARY ANN LIEBERT, INC
DOI: 10.1089/104303401450979
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Funding
- NCRR NIH HHS [U42 RR11148] Funding Source: Medline
- NHLBI NIH HHS [P01 HL53586] Funding Source: Medline
- NIDDK NIH HHS [P50 DK49218] Funding Source: Medline
- NATIONAL CENTER FOR RESEARCH RESOURCES [U42RR011148] Funding Source: NIH RePORTER
- NATIONAL HEART, LUNG, AND BLOOD INSTITUTE [P01HL053586] Funding Source: NIH RePORTER
- NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES [P50DK049218] Funding Source: NIH RePORTER
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The potential pathogenicity of replication-competent retroviruses (RCR) requires vigilant testing to exclude inadvertent contamination of clinical gene therapy vector products with RCR. Pseudotyped vectors using the gibbon ape leukemia virus (GALV) envelope have entered into clinical trials but specific recommendations regarding methods for screening of vector product and analysis of clinical samples have not been set forth. Unfortunately, current screening assays used for detecting amphotropic RCR are not suitable for GALV-pseudotyped RCR. We modified the extended S+/L- assay for RCR detection by using human 293 cells for virus amplification. Of five cell fines tested, 293 cells were selected because they combined a high transduction efficiency and an ability to generate RCR at high titer. After optimizing the amplification assay, a dilution of GALV virus could consistently be detected at a dilution of 10(-6). In coculture experiments, one GALV-infected cell could be consistently detected in 10(6) uninfected cells. A PCR-based assay was developed that was capable of detecting 100 copies of a GALV envelope containing plasmid diluted in 1 mug of DNA obtained from uninfected cells. PCR was also able to detect one GALV-infected cell in 10(6) uninfected cells. These assays will be suitable for testing of vector preparations and for monitoring of clinical samples from patients treated in clinical gene therapy protocols. The assays developed are similar in methodology and sensitivity to those currently used for certification of amphotropic retroviral vectors.
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