'RCL-Pooling Assay': A Simplified Method for the Detection of Replication-Competent Lentiviruses in Vector Batches Using Sequential Pooling

Autor: Jean-Brice Marteau, Guillaume Corre, Anne Galy, Bruno Dalle, David Fenard, Michel Dessainte
Přispěvatelé: Approches génétiques intégrées et nouvelles thérapies pour les maladies rares (INTEGRARE), École pratique des hautes études (EPHE)-Université d'Évry-Val-d'Essonne (UEVE)-GENETHON 3-Institut National de la Santé et de la Recherche Médicale (INSERM), Laboratoire d'Onco-Hématologie (LOH), Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Departments of Hematology, Université Paris Diderot - Paris 7 (UPD7), Genethon - Inserm UMR_S951, Immunologie moléculaire et biothérapies innovantes, École pratique des hautes études (EPHE)-Université d'Évry-Val-d'Essonne (UEVE)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Genethon-École pratique des hautes études (EPHE)-Université d'Évry-Val-d'Essonne (UEVE)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Genethon-Généthon, Association française contre les myopathies (AFM-Téléthon)-Association française contre les myopathies (AFM-Téléthon), Généthon, Association française contre les myopathies (AFM-Téléthon), École pratique des hautes études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Université d'Évry-Val-d'Essonne (UEVE)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Généthon, École Pratique des Hautes Études (EPHE), GENOSAFE S.A, GENOSAFE, Yposkesi
Jazyk: angličtina
Rok vydání: 2016
Předmět:
0301 basic medicine
Virus inactivation
[SDV.BIO]Life Sciences [q-bio]/Biotechnology
Cost-Benefit Analysis
T-Lymphocytes
Genetic Vectors
Pooling
HIV Core Protein p24
Computational biology
[SDV.BC]Life Sciences [q-bio]/Cellular Biology
Biology
Virus Replication
Sensitivity and Specificity
Cell Line
03 medical and health sciences
Transduction (genetics)
Transduction
Genetic
Genetics
Humans
[SDV.BBM]Life Sciences [q-bio]/Biochemistry
Molecular Biology
Purification methods
Molecular Biology
ComputingMilieux_MISCELLANEOUS
[SDV.GEN]Life Sciences [q-bio]/Genetics
Lentivirus
Virology
[SDV.BIBS]Life Sciences [q-bio]/Quantitative Methods [q-bio.QM]
3. Good health
030104 developmental biology
HIV-1
Virus Inactivation
Molecular Medicine
[SDV.IMM]Life Sciences [q-bio]/Immunology
Biological Assay
Material handling
Zdroj: Human Gene Therapy
Human Gene Therapy, Mary Ann Liebert, 2016, 27 (2), pp.202-210. ⟨10.1089/hum.2015.166⟩
Human Gene Therapy, 2016, 27 (2), pp.202-210. ⟨10.1089/hum.2015.166⟩
ISSN: 1043-0342
DOI: 10.1089/hum.2015.166⟩
Popis: 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.
Databáze: OpenAIRE
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