GTMB 7 - Gene Therapy & Molecular Biology

Gene Therapy and Molecular Biology Vol 7, page 211Gene Ther Mol Biol Vol 7, 211-219, 2003The role of EBV and genomic sequences in geneexpression from extrachromosomal gene therapyvectors in mouse liverResearch ArticleStephanie M. Stoll 1 , Leonard Meuse 2§ , Mark A. Kay 1,2 , and Michele P. Calos* 1Departments of 1 Genetics and 2 Pediatrics, Stanford University School of Medicine, Stanford, CA 94305-5120__________________________________________________________________________________*Correspondence: Michele P. Calos, phone 650-723-5558, fax 650-725-1534, e-mail calos@stanford.edu§ Present address: University of Washington, Department of Neurology, Box 357720, Seattle, WA 98195-7720.Key words: Epstein-Barr virus (EBV); extrachromosomal gene therapy, SERPINA1 sequence, α1-antitrypsin (AAT)Received: 18 September 2003; Accepted: 29 October 2003; electronically published: November 2003SummaryA plasmid vector containing Epstein-Barr virus (EBV) sequences and the full genomic SERPINA1 locus encodingthe gene for α 1 -antitrypsin is capable of providing long-term, high-level expression when transfected into mouseliver. It was unclear which viral and genomic sequences were required for efficient expression of this transgene invivo. We tested here the requirement for EBV sequences for retention and expression of plasmid DNA in normaland replicating liver in vivo. The results showed that EBV sequences provided increased retention and expression ofan extrachromosomal vector containing the full SERPINA1 transgene, in addition to the expression provided by thefull gene alone. We also minimized the SERPINA1 sequence and determined which portions were necessary forpersistent, high expression levels. Finally, we demonstrated that the SERPINA1 sequence can act to enhanceexpression of a heterologous gene cloned within it. Expression from a factor IX minigene was increased ~50-foldwhen it was expressed from within the SERPINA1 sequence, compared to a vector containing the factor IXminigene alone. The results presented here demonstrate that a significant amount of genomic sequence may berequired for persistent, high levels of expression in vivo and that the persistence of plasmid DNA in dividing tissuesand expression levels are enhanced by inclusion of EBV sequences on the vector.I. IntroductionThe ability to achieve persistent, regulated, highlevels of transgene expression in vivo is often necessaryfor the success of a gene therapy vector. Unfortunately,with most gene therapy vectors used to date, expression istemporary, often falling to non-therapeutic or undetectablelevels within a few weeks after treatment. For viralvectors, transience may be due to the immunogenicity ofthe vector, resulting in loss of transfected cells with aconcurrent reduction in transgene expression. In the caseof non-integrating vectors, viral or non-viral, transiencecan result from vector loss as the cells divide.For both integrating and non-integrating systems,decreased transgene expression may also be attributable toDNA silencing. For example, when mouse hepatocyteswere transfected in vivo with naked plasmid DNAencoding the AAT cDNA under control of thecytomegalovirus (CMV) promoter, day 1 expression levelsof 500 µg/ml were observed. These levels fell to 300 µg/ml in vivo that persisted atthese high levels for > 9 months. However, similarconstructs carrying the AAT cDNA driven by the RSVpromoter gave equivalent day 1 expression levels, but theexpression dropped >100-fold within two weeks. Again,Southern analysis showed that plasmid DNA wasmaintained extrachromosomally in these relativelyquiescent liver cells. In addition to the SERPINA1 locus,the successful genomic AAT vector also possessed211