title: Enhancement of gene transfer with recombinant adeno-associated virus (rAAV) vectors into primary B-cell chronic lymphocytic leukemia cells by CpG-oligodeoxynucleotides.
abstract: OBJECTIVE
  Transduction of primary B-cell chronic lymphocytic leukemia (B-CLL) cells with recombinant adeno-associated virus (rAAV) vectors is dependent on preactivation of leukemic cells by CD40L. CpG-oligodeoxynucleotides (CpG-ODNs) are able to activate cytokine production and proliferation of B-CLL cells. Therefore CpG-ODNs were tested for their potential to enhance transgene expression in CLL cells.
  
  
  METHODS
  Using an optimized adenovirus-free packaging system, rAAV vectors coding for the enhanced green fluorescent protein (AAV/EGFP) were packaged and highly purified resulting in infectious titers up to 5 x 10(9)/mL. Cells obtained from patients with B-CLL were infected with AAV/EGFP at a multiplicity of infection of 100 while being stimulated with CpG-ODNs and/or CD40L-expressing HeLa/SF cells. Transgene expression was assessed after 48 hours by flow cytometry.
  
  
  RESULTS
  Stimulation of B-CLL cells by CpG-ODNs resulted in up-regulation of costimulatory molecules and G(1)/S-phase transition at similar levels compared to activation by HeLa/SF cells, but use of CpG-ODNs alone did not result in any efficient AAV/EGFP transduction. Combined stimulation of B-CLL cells with HeLa/SF cells and CpG-ODNs during AAV/EGFP transduction significantly enhanced transgene expression compared to feeder stimulation alone (p=0.004). In addition, the copy number per single cell was significantly increased by addition of CpG-ODNs as detected by quantitative real-time PCR (p=0.04). Use of self-complementary AAV vectors that are not dependent on target cell DNA synthesis did not result in increased transgene expression compared to single-stranded AAV vectors (p=0.30).
  
  
  CONCLUSIONS
  Stimulation by CD40L is crucial for efficient gene transfer into B-CLL cells by rAAV vectors, whereas transduction efficiency can be significantly enhanced by CpG-ODNs.
authors.affiliation: KKG Gene Therapy, GSF-National Research Center for Environment and Health, Munich, Germany.
authors.forename: Hans D
authors.initials: HD
authors.lastname: Theiss
authors.forename: David M
authors.initials: DM
authors.lastname: Kofler
authors.forename: Hildegard
authors.initials: H
authors.lastname: Büning
authors.forename: Anna-Lena
authors.initials: AL
authors.lastname: Aldenhoff
authors.forename: Bernhard
authors.initials: B
authors.lastname: Kaess
authors.forename: Thomas
authors.initials: T
authors.lastname: Decker
authors.forename: Jens
authors.initials: J
authors.lastname: Baumert
authors.forename: Michael
authors.initials: M
authors.lastname: Hallek
authors.forename: Clemens-Martin
authors.initials: CM
authors.lastname: Wendtner
mesh_terms: D023201:CD40 Ligand / Q000494:pharmacology; D004274:DNA, Recombinant; D000229:Dependovirus / Q000235:genetics; D016193:G1 Phase / Q000187:drug effects; D015870:Gene Expression / Q000187:drug effects; D005822:Genetic Vectors / Q000235:genetics*; D049452:Green Fluorescent Proteins; D006801:Humans; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000473:pathology*; D008164:Luminescent Proteins / Q000235:genetics; D009838:Oligodeoxyribonucleotides / Q000494:pharmacology*; D014161:Transduction, Genetic / Q000379:methods* / Q000592:standards; D019076:Transgenes / Q000235:genetics; D014407:Tumor Cells, Cultured; D015854:Up-Regulation / Q000187:drug effects
chemical_list: C408982:CPG-oligonucleotide; D004274:DNA, Recombinant; D008164:Luminescent Proteins; D009838:Oligodeoxyribonucleotides; D023201:CD40 Ligand; D049452:Green Fluorescent Proteins