title: "RCL-Pooling Assay": A Simplified Method for the Detection of Replication-Competent Lentiviruses in Vector Batches Using Sequential Pooling.
abstract: Nonreplicative recombinant HIV-1-derived lentiviral vectors (LV) are increasingly used in gene therapy of various genetic diseases, infectious diseases, and cancer. Before they are used in humans, preparations of LV must undergo extensive quality control testing. In particular, testing of LV must demonstrate the absence of replication-competent lentiviruses (RCL) with suitable methods, on representative fractions of vector batches. Current methods based on cell culture are challenging because high titers of vector batches translate into high volumes of cell culture to be tested in RCL assays. As vector batch size and titers are continuously increasing because of the improvement of production and purification methods, it became necessary for us to modify the current RCL assay based on the detection of p24 in cultures of indicator cells. Here, we propose a practical optimization of this method using a pairwise pooling strategy enabling easier testing of higher vector inoculum volumes. These modifications significantly decrease material handling and operator time, leading to a cost-effective method, while maintaining optimal sensibility of the RCL testing. This optimized "RCL-pooling assay" ameliorates the feasibility of the quality control of large-scale batches of clinical-grade LV while maintaining the same sensitivity.
authors.affiliation: 1 Genethon, Evry, France .|2 INSERM, UMR_S951, Evry, France .|3 University of Evry , UMR_S951, Evry, France .
authors.forename: Guillaume
authors.initials: G
authors.lastname: Corre
authors.affiliation: 4 Genosafe, Evry, France .
authors.forename: Michel
authors.initials: M
authors.lastname: Dessainte
authors.affiliation: 4 Genosafe, Evry, France .
authors.forename: Jean-Brice
authors.initials: JB
authors.lastname: Marteau
authors.affiliation: 1 Genethon, Evry, France .
authors.forename: Bruno
authors.initials: B
authors.lastname: Dalle
authors.affiliation: 1 Genethon, Evry, France .|2 INSERM, UMR_S951, Evry, France .|3 University of Evry , UMR_S951, Evry, France .
authors.forename: David
authors.initials: D
authors.lastname: Fenard
authors.affiliation: 1 Genethon, Evry, France .|2 INSERM, UMR_S951, Evry, France .|3 University of Evry , UMR_S951, Evry, France .
authors.forename: Anne
authors.initials: A
authors.lastname: Galy
mesh_terms: D001681:Biological Assay / Q000191:economics / Q000379:methods* / Q000592:standards; D002460:Cell Line; D003362:Cost-Benefit Analysis; D005822:Genetic Vectors / Q000737:chemistry / Q000276:immunology; D016655:HIV Core Protein p24 / Q000032:analysis* / Q000235:genetics / Q000276:immunology; D015497:HIV-1 / Q000235:genetics* / Q000276:immunology; D006801:Humans; D016086:Lentivirus / Q000235:genetics* / Q000276:immunology; D012680:Sensitivity and Specificity; D013601:T-Lymphocytes / Q000276:immunology / Q000821:virology*; D014161:Transduction, Genetic; D038301:Virus Inactivation; D014779:Virus Replication
chemical_list: D016655:HIV Core Protein p24