title: Non-replicating Epstein-Barr virus-based plasmids extend gene expression and can improve gene therapy in vivo.
abstract: To date, no gene transfer vector has produced prolonged gene expression following a single intravenous injection and then efficiently re-expressed the delivered gene following repeated systemic injection into immunocompetent hosts. To overcome these limitations, a gene therapy regimen using non-replicating Epstein-Barr virus (EBV)-based expression plasmids was developed. One plasmid contains the FR (EBV family of repeats) sequence and the expressed gene. The other encodes Epstein-Barr nuclear antigen 1 (EBNA-1), but lacks FR. Although unable to replicate in mice, intravenous co-injection of EBV-based plasmids in cationic liposome-DNA complexes (CLDCs) substantially prolonged luciferase gene expression. The use of a two-vector system limited host exposure to the EBNA-1 gene product. Furthermore, this EBV-based vector system could be intravenously re-injected multiple times into immunocompetent mice without loss of transfection efficiency. Use of this vector system significantly improved the therapeutic efficacy of the biologically important human granulocyte colony-stimulating factor gene. Delivery of the human granulocyte colony-stimulating factor gene in EBV-based plasmids increased circulating white blood counts for at least 2 months following a single CLDC-based intravenous co-injection. Conversely, white blood counts were never elevated following injection of CLDCs lacking EBV-derived elements. Thus, this EBV-based plasmid vector system both markedly prolongs gene expression at therapeutic levels and efficiently and repeatedly re-transfects immunocompetent hosts. These properties of EBV-based plasmid vectors appear to be due, at least in part, to the documented abilities of the EBNA-1 protein both to retain FR-containing DNA intracellularly and within the nucleus and to block anti-EBNA-1 cytotoxic T cell responses.
authors.affiliation: California Pacific Medical Research Institute, San Francisco, California 94115, USA.
authors.forename: G
authors.initials: G
authors.lastname: Tu
authors.forename: A L
authors.initials: AL
authors.lastname: Kirchmaier
authors.forename: D
authors.initials: D
authors.lastname: Liggitt
authors.forename: Y
authors.initials: Y
authors.lastname: Liu
authors.forename: S
authors.initials: S
authors.lastname: Liu
authors.forename: W H
authors.initials: WH
authors.lastname: Yu
authors.forename: T D
authors.initials: TD
authors.lastname: Heath
authors.forename: A
authors.initials: A
authors.lastname: Thor
authors.forename: R J
authors.initials: RJ
authors.lastname: Debs
mesh_terms: D000818:Animals; D004279:DNA, Viral / Q000235:genetics*; D004337:Drug Carriers; D019309:Epstein-Barr Virus Nuclear Antigens / Q000235:genetics; D005260:Female; D015870:Gene Expression; D017930:Genes, Reporter; D015316:Genetic Therapy / Q000379:methods*; D016179:Granulocyte Colony-Stimulating Factor / Q000008:administration & dosage* / Q000096:biosynthesis / Q000235:genetics; D004854:Herpesvirus 4, Human / Q000235:genetics* / Q000254:growth & development; D006801:Humans; D007275:Injections, Intravenous; D008081:Liposomes; D008156:Luciferases / Q000096:biosynthesis / Q000235:genetics; D051379:Mice; D008815:Mice, Inbred Strains; D010957:Plasmids / Q000235:genetics*; D012091:Repetitive Sequences, Nucleic Acid; D014779:Virus Replication
chemical_list: D004279:DNA, Viral; D004337:Drug Carriers; D019309:Epstein-Barr Virus Nuclear Antigens; D008081:Liposomes; D016179:Granulocyte Colony-Stimulating Factor; D008156:Luciferases