title: High level of transgene expression in primary chronic lymphocytic leukemia cells using helper-virus-free recombinant Epstein-Barr virus vectors.
abstract: OBJECTIVE
  Epstein-Barr virus (EBV)-based vectors have favorable features for gene transfer, including a high transduction efficiency especially for B cells, large packaging capacity up to 150 kb pairs, and ability to infect postmitotic cells. Recombinant EBV was explored for transduction of primary human B-cell chronic lymphocytic leukemia (CLL) cells.
  
  
  METHODS
  EBV vectors deleted for all oncogenic sequences and encoding terminal repeats (TR) essential for encapsidation, the lytic origin of replication (oriLyt) for DNA amplification, and the enhanced green fluorescent protein (EGFP) were packaged using an optimized, helper-virus-free method. Infectious EBV virions encoding EGFP (EBV/EGFP) with an infectious titer up to 2 x 10(6) per milliliter were generated. Primary leukemic cells from 14 patients with CLL were successfully transduced with EBV/EGFP at a very low multiplicity of infection (< 1).
  
  
  RESULTS
  Transgene expression was detected in up to 85% of cells 48 hours after infection. Transduction was specifically mediated by EBV vectors because gene transfer was inhibited by an antibody (72A1) directed against the viral envelope glycoprotein gp350/220. Furthermore, transduction of CLL cells with packaged EBV vectors coding for EGFP but deleted for TR sequences (TR-) did not result in EGFP expression compared to TR+ vector constructs (p = 0.009).
  
  
  CONCLUSIONS
  Helper-virus-free EBV-based gene transfer vectors hold promise for development of genetic therapies for CLL patients.
authors.affiliation: Medical Clinic III, University Hospital Grosshadern, Ludwig-Maximilians-University, Marchioninistrasse 15, D-81377 Munich, Germany. Clemens.Wendtner@med3.med.uni-muenchen.de
authors.forename: Clemens-Martin
authors.initials: CM
authors.lastname: Wendtner
authors.forename: Christian
authors.initials: C
authors.lastname: Kurzeder
authors.forename: Hans D
authors.initials: HD
authors.lastname: Theiss
authors.forename: David M
authors.initials: DM
authors.lastname: Kofler
authors.forename: Jens
authors.initials: J
authors.lastname: Baumert
authors.forename: Henri-Jacques
authors.initials: HJ
authors.lastname: Delecluse
authors.forename: Annette
authors.initials: A
authors.lastname: Janz
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Hammerschmidt
authors.forename: Michael
authors.initials: M
authors.lastname: Hallek
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D001402:B-Lymphocytes / Q000378:metabolism / Q000473:pathology; D005260:Female; D015870:Gene Expression; D017930:Genes, Reporter; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D006378:Helper Viruses; D004854:Herpesvirus 4, Human / Q000235:genetics*; D006801:Humans; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000473:pathology* / Q000628:therapy; D008164:Luminescent Proteins / Q000096:biosynthesis / Q000235:genetics; D008297:Male; D008875:Middle Aged; D014161:Transduction, Genetic / Q000379:methods; D019076:Transgenes / Q000235:genetics*
chemical_list: D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins