Partial reconstitution of a replication-competent retrovirus in helper cells with partial overlaps between vector and helper cell genomes.
Contamination of retroviral vector preparations with replication-competent retroviruses is a major safety concern in human gene therapy. These can arise by recombination between retroviral vectors and packaging cell sequences. Recently, we constructed new packaging lines, derived from spleen necrosis virus (SNV) that do not contain overlapping regions of homology between these two components (DSH134G and DSH29B cells). These cell lines were tested for the presence of recombination products and replication-competent viruses in comparison to a similar packaging line (DSN) that contains a partial overlap between vector and viral protein coding regions. No recombination products were detected in DSH cells. However, we found that recombination had occurred in DSN cells, partially reconstituting a provirus-like structure that was capable of being spread, although inefficiently, through an infected cell culture. Our data indicate that even small regions of sequence homology eventually allow homologous recombination between vector and helper cell genomes.
Martinez
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Graduate Program in Microbiology and Molecular Genetics, Rutgers University, New Brunswick, NJ 08903, USA.
Dornburg
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R
D000818:Animals; D015139:Blotting, Southern; D004285:Dogs; D005822:Genetic Vectors / Q000235:genetics*; D016133:Polymerase Chain Reaction; D012194:RNA-Directed DNA Polymerase / Q000032:analysis; D011995:Recombination, Genetic; D012107:Research Design; D017986:Reticuloendotheliosis Viruses, Avian / Q000235:genetics* / Q000254:growth & development; D006377:T-Lymphocytes, Helper-Inducer / Q000502:physiology*; D014407:Tumor Cells, Cultured; D014779:Virus Replication
D012194:RNA-Directed DNA Polymerase