Adoptive T-cell transfer is recognized as an innovative treatment strategy for various malignant diseases. 1, 2 To improve the efficacy and sometimes the safety of this approach, T cells can be genetically manipulated to modify their antigen specificity, to enhance their in vivo survival and trafficking to specific tissues, or to be eliminated in the event of undesired toxic effects. 3 γ-retroviral vectors are frequently used to obtain robust and stable genetic modification of human T lymphocytes because these vectors can efficiently integrate within the genome and ensure that the inserted transgene is passed to the progeny of infected cells.

Although integration is the desired effect of retroviral-mediated gene transfer, it carries the risk of insertional mutagenesis, which can result in dysregulated gene expression and subsequent malignant transformation. 4 The potential for insertional mutagenesis and malignant transformation may be increased in cases where replication-competent retroviruses (RCRs) are present in the vector products, as continued viral replication could result in multiple integrations within the hostcell genome. When a high-titer vectorproducer packaging cell line (VPC) with a known level of RCR contamination of 103–104 virions/ml was used to transduce stem cells in 10 nonhuman primates, three animals developed virus-induced T-cell lymphoma. 5 This observation led to extensive public discussions and the adoption of the current US Food and Drug Administration (FDA) guidance for RCR testing. Almost all γ-retroviral vectors manufactured for clinical use are produced using stable VPCs derived from murine or human cell lines. The risk of RCR generation is minimized during the production of the retroviral vector supernatant by segregating vectors encoding the exogenous gene of interest from sequences encoding the viral proteins gag, pol, and env. These sequences are provided in trans by VPCs that stably express gag, pol, and env genes. Early VPCs that contained gag, pol, and env genes expressed from a single plasmid had a high incidence of RCR generation due to recombination between the vector and viral