title: 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.
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.
authors.affiliation: Unit of Clinical and Tumor Immunology, Department of Medical Oncology, Erasmus MC-Daniel den Hoed Cancer Center, AE Rotterdam, The Netherlands. c.lamers@erasmusmc.nl
authors.forename: Cor H J
authors.initials: CH
authors.lastname: Lamers
authors.forename: Ralph A
authors.initials: RA
authors.lastname: Willemsen
authors.forename: Pascal M M L
authors.initials: PM
authors.lastname: van Elzakker
authors.forename: Jan Willem
authors.initials: JW
authors.lastname: Gratama
authors.forename: Reno
authors.initials: R
authors.lastname: Debets
mesh_terms: D000818:Animals; D001681:Biological Assay; D005822:Genetic Vectors / Q000235:genetics / Q000502:physiology*; D006801:Humans; D017989:Leukemia Virus, Gibbon Ape / Q000235:genetics / Q000502:physiology*; D012680:Sensitivity and Specificity; D013601:T-Lymphocytes / Q000821:virology*; D014161:Transduction, Genetic; D014779:Virus Replication