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

Mol Ther. 2003 Aug;8(2):332-41. doi: 10.1016/s1525-0016(03)00167-9.


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 x 10(8) 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 x 10(10) 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.

MeSH terms

  • Animals
  • Cell Line
  • Enzyme-Linked Immunosorbent Assay
  • Genetic Vectors / biosynthesis
  • Genetic Vectors / genetics*
  • Genetic Vectors / physiology*
  • HIV / genetics*
  • HIV / growth & development*
  • Humans
  • Polymerase Chain Reaction
  • Sensitivity and Specificity
  • Time Factors
  • Virus Replication*