Detection of replication competent virus formation during production and use of lenti- and gammaretroviral vectors

Research reports | 31.01.2023 | CGM 2023-01

COGEM commissioned research on the tests and methods that can be used to demonstrate replication-competent retro- and lentiviral vectors (RCV). The research, conducted by Perseus B.V., includes a literature review of currently available tests for the detection of RCV, including an overview of their characteristics, advantages and disadvantages, and validation data. In addition, through a survey, stakeholders were asked about their experience with the daily practice of RCV testing.

The research report provides an overview of currently available RCV tests, their suitability and limitations, available data on test validation and positive and negative controls used. A distinction is made in the report between so-called “structural tests,” where sequences or proteins of the RCV are demonstrated (immuno-assays or molecular assays), and “functional tests,” where the biological activity of any RCV present can be visualized (cellular assays).

The report highlights the importance of an amplification step prior to performing an RCV test. Such a step is useful to increase the amount of any RCVs present and reduce the number of replication-deficient vector particles. This increases the sensitivity of an assay and allows sampling at multiple time points to study an increase or decrease in a parameter over time.

From both the literature review and survey results, data on validation were limited and difficult to compare. Moreover, it became clear that RCV testing was more prominent in organizations where vectors are used for clinical applications than in a more basic research environment. In the former setting, RCV testing of vector material (and of transduced drugs) is mandatory in many cases for patient safety reasons. In research and development laboratories, on the other hand, vectors are usually applied under contained use conditions. Nevertheless, RCV testing in R&D laboratories can be of added value to help decide on lowering the required containment for handling transduced host cells or animals.

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