title: Development of an equine-tropic replication-competent lentivirus assay for equine infectious anemia virus-based lentiviral vectors.
abstract: The release of lentiviral vectors for clinical use requires the testing of vector material, production cells, and, if applicable, ex vivo-transduced cells for the presence of replication-competent lentivirus (RCL). Vectors derived from the nonprimate lentivirus equine infectious anemia virus (EIAV) have been directly administered to patients in several clinical trials, with no toxicity observed to date. Because EIAV does not replicate in human cells, and because putative RCLs derived from vector components within human vector production cells would most likely be human cell-tropic, we previously developed an RCL assay using amphotropic murine leukemia virus (MLV) as a surrogate positive control and human cells as RCL amplification/indicator cells. Here we report an additional RCL assay that tests for the presence of theoretical "equine-tropic" RCLs. This approach provides further assurance of safety by detecting putative RCLs with an equine cell-specific tropism that might not be efficiently amplified by the human cell-based RCL assay. We tested the ability of accessory gene-deficient EIAV mutant viruses to replicate in a highly permissive equine cell line to direct our choice of a suitable EIAV-derived positive control. In addition, we report for the first time the mathematical rationale for use of the Poisson distribution to calculate minimal infectious dose of positive control virus and for use in monitoring assay positive/spike control failures in accumulating data sets. No RCLs have been detected in Good Manufacturing Practice (GMP)-compliant RCL assays to date, further demonstrating that RCL formation is highly unlikely in contemporary minimal lentiviral vector systems.
authors.affiliation: Oxford BioMedica, Oxford OX4 4GA, United Kingdom. d.farley@oxfordbiomedica.co.uk
authors.forename: Daniel C
authors.initials: DC
authors.lastname: Farley
authors.forename: Richard
authors.initials: R
authors.lastname: Bannister
authors.forename: Marie A
authors.initials: MA
authors.lastname: Leroux-Carlucci
authors.forename: Nerys E
authors.initials: NE
authors.lastname: Evans
authors.forename: James E
authors.initials: JE
authors.lastname: Miskin
authors.forename: Kyriacos A
authors.initials: KA
authors.lastname: Mitrophanous
mesh_terms: D000818:Animals; D001681:Biological Assay; D002460:Cell Line; D023061:Gene Order; D005822:Genetic Vectors / Q000235:genetics*; D006736:Horses; D006801:Humans; D004860:Infectious Anemia Virus, Equine / Q000235:genetics* / Q000502:physiology; D009052:Leukemia Virus, Murine; D051379:Mice; D015203:Reproducibility of Results; D014161:Transduction, Genetic; D056189:Viral Tropism; D014779:Virus Replication