Infectious delivery of 120-kilobase genomic DNA by an epstein-barr virus amplicon vector.
It has been shown in a wide variety of contexts that persistent gene expression can best be obtained by using the genomic locus of a transgene. However, the size of most genomic loci precludes their use in current viral gene therapy vectors. Large transgene capacity and extrachromosomal persistence make Epstein-Barr virus (EBV) a promising vector to deliver genomic transgenes for gene therapy. We constructed an EBV amplicon vector that contains the EBV lytic origin of replication, the terminal repeats for viral packaging, and the EBV latent origin of replication for episomal persistence. This vector was able to deliver inserts of 60-123 kb to B-cell lines in culture in three steps. First, clonal packaging cells lines were generated that produce infectious amplicons at a titer of approximately 3-4x10(6) transducing units/ml after concentration. Second, we show infectious vector delivery to the Loukes B-cell line and three different EBV-immortalized lymphoblastoid cell lines. This infectious delivery system was 2000 times more efficient than transfection in B cells. Third, clonal cell lines from infection of Loukes contained persistent episomes of recircularized infectious vector. This first demonstration of infectious delivery of 120 kb of genomic DNA shows the potential of this high-capacity vector system.
White
Robert E
RE
Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford, OX3 7BN, UK.
Wade-Martins
Richard
R
James
Michael R
MR
D001402:B-Lymphocytes / Q000378:metabolism / Q000821:virology; D022202:Chromosomes, Artificial, Bacterial; D004274:DNA, Recombinant; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D016678:Genome; D049452:Green Fluorescent Proteins; D004854:Herpesvirus 4, Human / Q000235:genetics* / Q000472:pathogenicity; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics; D014162:Transfection; D019076:Transgenes; D014407:Tumor Cells, Cultured; D014771:Virion / Q000235:genetics / Q000472:pathogenicity
D004274:DNA, Recombinant; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins