Abstract The Food and Drug Administration/Center for Biologics Evaluation and Research has defined that for retroviral gene therapy, the vector-producing cell, the vector preparation, and the ex vivo gene-transduced cells have to be tested for absence of replication-competent retrovirus (RCR) if the transduced cells are cultured for >4 days. We assessed the sensitivity of the "extended PG4(S+L-) assay" to detect gibbon ape leukemia virus (GALV) RCR, and applied this assay to measure GALV RCR spread in retrovirally transduced T cells. To this end, T cells were expanded for 12 days after transduction with a GALV-envelope pseudotyped retroviral vector expressing single chain variable fragment (anticarbonic anhydrase IX) in presence or absence of GALV RCR. Results showed that: (1) the "extended PG4(S+L-) assay" detects 1 focus-forming unit (ffu) GALV RCR and thus is applicable and sufficiently sensitive to screen human T-cell cultures for absence of infectious GALV RCR; (2) although GALV RCR infect human T cells, it very poorly replicate in T cells; (3) GALV RCR, when present at low levels immediately upon gene transduction (ie, 100 ffu/20x10 T cells in 100 mL), did not spread during a 12-day T-cell culture at clinical scale. Our observation that GALV RCR poorly spreads in primary human T-cell cultures questions the relevance of testing T-cell transductants for RCR on top of testing the vector-producing cells and the clinical vector batch for RCR and warrants evaluation of the current policy for safety testing of ex vivo retrovirally transduced T lymphocytes for GALV RCR.

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Keywords Animals, Biological Assay, Genetic Vectors/genetics/ physiology, Humans, Leukemia Virus, Gibbon Ape/genetics/ physiology, Sensitivity and Specificity, T-Lymphocytes/ virology, Transduction, Genetic, Virus Replication
Persistent URL dx.doi.org/10.1097/CJI.0b013e318199840a, hdl.handle.net/1765/21377
Lamers, C.H.J, Willemsen, R.A, van Elzakker, P.M.M.L, Gratama, J.W, & Debets, J.E.M.A. (2009). Gibbon ape leukemia virus poorly replicates in primary human T lymphocytes: implications for safety testing of primary human T lymphocytes transduced with GALV-pseudotyped vectors. Journal of Immunotherapy, 32(3), 272–279. doi:10.1097/CJI.0b013e318199840a