title: Human primary T lymphocytes have a low capacity to amplify MLV-based amphotropic RCR and the virions produced are largely noninfectious.
abstract: The presence of replication-competent retrovirus (RCR) in retroviral-based gene therapy products poses a potential safety risk for patients. Therefore, RCR testing of clinical gene therapy products and monitoring of patients enrolled in gene therapy trials is required to assure viral safety. The requirement to test ex vivo-transduced cells originates from the presumed amplification of adventitious RCR during the transduction procedure. However, data on the capacity of different cell types to do so are lacking. In this study, we sought to analyze the amplification potential of primary human T lymphocytes after infection with amphotropic MLV-based RCR. The total number of viral particles produced after 1 or 2 weeks was measured by a quantitative 4070A env-specific RT-PCR assay. The fraction of infectious replication-competent viral particles was analyzed in the PG-4 S+L- assay. From this study, we conclude that the total number of viral particles RCR produced by T lymphocytes is 2-4 logs lower than the number produced by NIH-3T3 cells. Surprisingly, less than 1% of the viral particles produced by primary T lymphocytes appeared to be infectious, while nearly all virions produced by NIH-3T3 were. We conclude that primary human T lymphocytes are low producers of MLV-based amphotropic RCR.
authors.affiliation: Jordan Laboratory for Hemato-Oncology, Department of Hematology, University Medical Center Utrecht, Utrecht, The Netherlands.
authors.forename: S B
authors.initials: SB
authors.lastname: Ebeling
authors.forename: E R
authors.initials: ER
authors.lastname: Simonetti
authors.forename: H P E
authors.initials: HP
authors.lastname: Borst
authors.forename: A
authors.initials: A
authors.lastname: Blok
authors.forename: A M
authors.initials: AM
authors.lastname: Schelen
authors.forename: E
authors.initials: E
authors.lastname: Braakman
authors.forename: J
authors.initials: J
authors.lastname: Ederveen
authors.forename: A
authors.initials: A
authors.lastname: Hagenbeek
mesh_terms: D015870:Gene Expression; D015316:Genetic Therapy / Q000009:adverse effects*; D005822:Genetic Vectors; D006801:Humans; D008979:Moloney murine leukemia virus / Q000502:physiology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013601:T-Lymphocytes / Q000821:virology*; D014161:Transduction, Genetic / Q000379:methods; D014779:Virus Replication