Epstein-Barr virus/human vector provides high-level, long-term expression of alpha(1)-antitrypsin in mice

We have constructed plasmid DNA vectors that contain Epstein-Barr virus (EB V) sequences and the human gene (SERPINA1) encoding alpha (1)-antitrypsin ( AAT). We demonstrate that a plasmid carrying the full SERPINA1 on a 19-kb g enomic fragment and the EBV gene EBNA1 and its family of repeats binding si tes undergoes efficient extrachromosomal replication in dividing mammalian tissue culture cells. Therefore, use of a whole genomic therapeutic gene to provide both replication and gene expression may be an effective gene ther apy vector design, if the target cells are dividing. The efficacy of this s ame vector for expression of AAT in vivo in the nondividing cells of mouse liver was determined by hydrodynamic injection of naked plasmid DNA by mean s of the tail vein. A single injection of an EBV/genomic SERPINA1 vector pr ovided > 300 mug/ml of AAT, which approached normal plasma levels and persi sted for the >9-month duration of the experiment. These data exceed most pr eviously reported values, probably due to sequences in the genomic DNA that resist silencing of gene expression, possibly in combination with favorabl e effects on expression provided by the EBV sequences. These results demons trate that plasmid DNA with the correct cis-acting sequences can provide in vivo long-term expression of protein at high levels that are therapeutical ly relevant for gene therapy.