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. 2011 Mar;19(3):557-66.
doi: 10.1038/mt.2010.278. Epub 2010 Dec 21.

Replication-competent lentivirus analysis of clinical grade vector products

Kenneth Cornetta  1 , Jing Yao, Aparna Jasti, Sue Koop, Makhaila Douglas, David Hsu, Larry A Couture, Troy Hawkins, Lisa Duffy
Affiliations

Replication-competent lentivirus analysis of clinical grade vector products

Kenneth Cornetta et al. Mol Ther. 2011 Mar.

Abstract

Lentiviral vectors are now in clinical trials for a variety of inherited and acquired disorders. A challenge for moving any viral vector into the clinic is the ability to screen the vector product for the presence of replication-competent virus. Assay development for replication-competent lentivirus (RCL) is particularly challenging because recombination of vector packaging plasmids and cellular DNA leading to RCL has not been reported with the current viral vector systems. Therefore, the genomic structure of a RCL remains theoretical. In this report, we describe a highly sensitive RCL assay suitable for screening vector product and have screened large-scale vector supernatant, cells used in vector production, and cells transduced with clinical grade vector. We discuss the limitations and challenges of the current assay, and suggest modifications that may improve the suitability of this assay for screening US Food and Drug Administration (US FDA)-licensed products.

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Figures

Figure 1
Figure 1
Schematic representation of the replication-competent lentivirus (RCL) assay. Vector product is used to transduce C8166 cells. Small amount of virus which may be present in vector preparations will propagate in the C8166 culture over the 3 weeks of the amplification phase typically yielding virus concentration in excess of X ng/ml of p24. Cell-free media is harvested at the end of the amplification phase and used to transduce naive C8166 cells, which are propagated in the indicator phase. At the end of the indicator phase, the media is evaluated for virus using the p24 enzyme-linked immunosorbent assay (ELISA) assay. Cells are evaluated for evidence of virus using PCR amplification with primers within the viral packaging sequence and the gag gene region (psi-gag PCR).
Figure 2
Figure 2
Effect of concentrated lentiviral vector on the growth and infectivity of C8166 cells. (a) The effect on concentrated vector was assessed in the nonadherent C8166 cell line and the adherent MRC-5 cell line. Cells were exposed to concentrated CSCGW vector for 4 hours and cell number counted after 48 hours. Concentrated vector (~5,000 ng/ml) was used undiluted or diluted 1:10 and 1:100. Camptothecin was used as a positive toxin control. The data represents the average of three cultures ± SD. (b) The effect of concentrated vector on the growth of C8166 cells was measured at three concentration, 1,000, 5,000, and 10,000 ng/ml of p24 using the CSCGW vector. Duplicate cultures were exposed to vector preparations on day 0 and the total number of cells was determined in each culture after 5 or 6 days of growth (experiment 1 or 2, respectively). Data represents the average of two cultures ± SD for two independent experiments. (c) The inhibitory effect of concentrated vector on C8166 cell infectivity was measured by introducing increasing amounts of a null vector (CSO) into a lentiviral vector preparation of the green fluorescent protein (GFP)-expressing CSCGW vector. The x-axis represents the amount of CSO vector as measured by p24 ELISA. The y-axis represents the GFP-expressing cells as a percentage of control (no CSO vector). The percentage of control cells expressing GFP ranged between 13.5 and 20.1%. The data represents the average of three independent experiment ± SD.
Figure 3
Figure 3
Controls for the replication-competent lentivirus (RCL) assay: inoculation and acceptance criteria. (a) The initial version of the RCL assay utilized three negative controls setup at the start of the amplification and three at the start of the indicator phase. All cultures are assayed for RCL at the end of the indicator phase. Two sets of positive controls were utilized, both inoculating cultures with R8.71 attenuated HIV-1 virus at the TCID50. Three cultures are set up at the start of the amplification phase and five cultures inoculated at the start of the indicator phase. Five cultures were chosen for the indicator phase positive control since virus expansion will only be occurring for 7 days and the level of virus after this time is expected to be low. However, both the amplification phase positive controls and the indicator phase positive controls must have at least one culture positive for RCL for the assay to be valid. The assay is considered positive is the p24, psi-gag PCR, or both are positive. The established acceptance required all negative cultures to be negative for RCL. (b) The RCL assay was modified to remove the indicator phase negative and positive controls. The inoculum at the start of the amplification phase was increased to 5 IU per positive culture. An inhibitor control was added to the assay in which the test articles (at a concentration ≤1,000 ng/ml of p24) are spiked with 50 IU. At least one of the amplification phase positive controls and two of the inhibitor controls must be positive for the assay to be valid. IP, intraperitoneal.
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