Epstein-Barr Virus Vectors Provide Prolonged Robust Factor IX Expression in Mice Christopher R. Sclimenti, Andrew S. Neviaser, ‡ Edward J. Baba, § Leonard Meuse, † Mark A. Kay, † and Michele P. Calos* Departments of Genetics and Pediatrics, Stanford University School of Medicine, Stanford, California 94305 We demonstrate that vectors incorporating components from Epstein-Barr virus (EBV) for retention and from human genomic DNA for replication greatly enhance the level and duration of marker gene expression in dividing cultured cells. The same types of vectors were tested in vivo by high-pressure tail vein injection of naked DNA in mice, resulting in liver delivery and expression. The therapeutic gene was a human factor IX (hFIX) minigene comprising genomically derived 5′,3′, and intronic sequences that provided relatively good gene expression in vivo. We demonstrated that addition of the EBV EBNA1 gene and its family of repeats binding sites provided a 10- to 100- fold increase in prolonged hFIX expression in mouse liver. A single 25-μg dose of vector DNA generated normal (>5 μg/mL) levels of hFIX throughout the 8 month duration of the experiment. Vector DNA with or without the EBV sequences was retained in liver cells, and vector replication was not a factor in these nondividing liver cells. Instead, it appears that enhancement of stable hFIX expression by the EBV components was responsible for the increased level and duration of therapeutic gene expression. The EBV sequences also significantly enhanced stable expression of a vector carrying the full genomic hFIX gene delivered to mouse liver. These results underline the crucial importance of appropriate gene expression signals on gene therapy vectors and the utility of EBV sequences in particular for increasing stable gene expression. Introduction A significant issue for any gene therapy vector is whether it can provide adequate gene expression for the prolonged periods required in most applications. Intro- duced DNA needs to be retained in target cells and must continue to be well-expressed over time. The safety and simplicity of nonintegrating plasmid vectors are appeal- ing, but these vectors present challenges for maintenance of stable gene expression (1). If the target cells are dividing, then conventional nonintegrated plasmid DNA will be lost during mitosis (2). In nondividing cells, even though plasmid DNA may be retained, it has become clear that persistence of introduced plasmid DNA does not necessarily equate with continued robust gene ex- pression. Instead, special sequences seem to be required in order to prevent silencing of vector gene expression. Various genomic sequences, including some genomic promoters and/or intronic sequences, e.g., refs 3-6, have shown positive effects in this regard. Certain viral sequences may also protect against silencing, as explored in this study. After delivery of plasmid DNA to mouse liver, a typical cDNA construct with a viral promoter, such as the human gene encoding R 1 -antitrypsin (hAAT) driven by the CMV promoter, gives rise to only a transient level of strong gene expression. Gene expression then declines rapidly over 2 weeks, even though plasmid DNA remains in the liver cells (7). This progressive silencing of expression of plasmid DNA is the usual result and is not confined to liver. Silencing is probably due to cellular mechanisms acting through chromatin features. In mouse lung similar results were seen with CMV, RSV, and SV40 viral promoters driving a luciferase reporter gene. Expression fell to low levels during the first 2 weeks, even while continued presence of plasmid DNA was documented (6). By using genomic promoters for the cellular polyubiq- uitin C or elongation factor 1R genes in place of the viral promoters, increased persistence of lung gene expression was obtained (6). Likewise, increased persistence of human factor IX (hFIX) was seen when cellular locus control region, promoter, and 3′ regions derived from various genes were used, in combination with a portion of the first intron from the native hFIX gene (3, 4). In a further example, greatly enhanced and prolonged expres- sion of hAAT was observed when the entire genomic gene, as opposed to a cDNA with a viral promoter, was present on the vector (5). These results all support the hypothesis that some genomic sequences are effective in endowing introduced genes with the ability to continue to be expressed in vivo. In addition to certain genomic sequences, observations with Epstein-Barr virus (EBV) vectors have implicated viral sequences such as the EBNA1 gene and its family of repeats binding sites in long-term expression of plas- mid DNA. In cases where the host cells are dividing, the special replication and nuclear retention properties of EBV vectors are beneficial and give a far better outcome * To whom correspondence should be addressed. Ph: 650-723- 5558. Fax: 650-723-5645. E-mail: calos@stanford.edu. † Department of Pediatrics. ‡ Present address: Case Western Reserve School of Medicine, 10900 Euclid Avenue, Cleveland, OH 44106-4920. § Present address: Syracuse University College of Law, Syra- cuse, NY 13244-1030. 144 Biotechnol. Prog. 2003, 19, 144-151 10.1021/bp0200907 CCC: $25.00 © 2003 American Chemical Society and American Institute of Chemical Engineers Published on Web 11/28/2002