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Exp Hematol. 2003 Dec;31(12):1223-9.

Enhancement of gene transfer with recombinant adeno-associated virus (rAAV) vectors into primary B-cell chronic lymphocytic leukemia cells by CpG-oligodeoxynucleotides.

Author information

  • 1KKG Gene Therapy, GSF-National Research Center for Environment and Health, Munich, Germany.



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.


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.


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).


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.

[PubMed - indexed for MEDLINE]
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