Engagement of the B-cell antigen receptor (BCR) allows efficient transduction of ZAP-70-positive primary B-CLL cells by recombinant adeno-associated virus (rAAV) vectors

University of Cologne, Köln, North Rhine-Westphalia, Germany
Gene Therapy (Impact Factor: 3.1). 10/2004; 11(18):1416-24. DOI: 10.1038/
Source: PubMed


Engagement of the B-cell antigen receptor (BCR) by crosslinking of the surface immunoglobulin (sIg) homodimer was studied for recombinant adeno-associated virus (rAAV)-mediated gene transfer into B-cell chronic lymphocytic leukaemia (B-CLL) cells. Leukemic cells obtained from 20 patients were stimulated with anti-sIg-directed antibodies and transduced with rAAV vectors coding for enhanced green fluorescent protein (EGFP) (AAV/EGFP) or CD40L (AAV/CD40L). Transduction of B-CLL cells was enhanced after BCR engagement compared to unstimulated controls (P=0.0356). BCR crosslinking induced a significant, dose- and time-dependent upregulation of heparan sulfate proteoglycan (HSPG), the primary receptor for AAV, on B-CLL cells (mean: 38.2 versus 1.7%; P=0.0006). A correlation of HSPG expression after BCR crosslinking with transduction efficiency by AAV/EGFP (P=0.0153) and AAV/CD40L (P=0.0347) was observed. High expression of zeta-associated protein 70 (ZAP-70) in B-CLL cells correlated with a better transduction efficiency by AAV/EGFP (P<0.0001) and AAV/CD40L (P=0.002), respectively: 48 h after transduction of ZAP-70-positive samples, transgene expression was seen in a mean of 33.8% (s.e.m. 3.7%) and 28.9% (s.e.m. 6.7%) of cells, respectively, and could be specifically blocked by heparin, a soluble competitor of HSPG (P<0.0001). In summary, engagement of the BCR on ZAP-70 positive B-CLL cells allows efficient rAAV-mediated gene delivery.

Download full-text


Available from: Clemens-Martin Wendtner, Mar 10, 2014
  • Source
    • "Cells were cultured as described before [20]. BCR stimulation was performed as described by Kofler et al. [21] Anti-IgM-polyacrylamid immunobead (anti-IgM) reagent (Irvine Scientific, Santa Ana, CA, USA) was added to the PBMC cultures at a concentration of 100 µg/mL for 3 or 24 hours. Anti-IgA-immunobeads (anti-IgA, Irvine Scientific) served as a negative control. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Chronic lymphocytic leukemia (CLL) is a disease with variable clinical outcome. Several prognostic factors such as the immunoglobulin heavy chain variable genes (IGHV) mutation status are linked to the B-cell receptor (BCR) complex, supporting a role for triggering the BCR in vivo in the pathogenesis. The miRNA profile upon stimulation and correlation with IGHV mutation status is however unknown. To evaluate the transcriptional response of peripheral blood CLL cells upon BCR stimulation in vitro, miRNA and mRNA expression was measured using hybridization arrays and qPCR. We found both IGHV mutated and unmutated CLL cells to respond with increased expression of MYC and other genes associated with BCR activation, and a phenotype of cell cycle progression. Genome-wide expression studies showed hsa-miR-132-3p/hsa-miR-212 miRNA cluster induction associated with a set of downregulated genes, enriched for genes modulated by BCR activation and amplified by Myc. We conclude that BCR triggering of CLL cells induces a transcriptional response of genes associated with BCR activation, enhanced cell cycle entry and progression and suggest that part of the transcriptional profiles linked to IGHV mutation status observed in isolated peripheral blood are not cell intrinsic but rather secondary to in vivo BCR stimulation.
    Full-text · Article · Apr 2013 · PLoS ONE
  • Source

    Preview · Article · Mar 2005
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The efficient gene transfer of immunostimulatory cytokines into autologous tumor cells or the transfer of tumor-associated antigens into professional antigen-presenting cells is a prerequisite for many immunotherapeutic approaches. In particular with B cells, the efficiency of gene uptake is one of the limiting factors in cell-based vaccine strategies, since normal and malignant human B cells are commonly refractory to transducing gene vectors. Due to its natural tropism for human B cells, Epstein-Barr virus (EBV), a human herpes virus, might be an option, which we wanted to explore. EBV efficiently infects human B cells and establishes a latent infection, while the viral genome is maintained extrachromosomally. Although these characteristics are attractive, EBV is an oncogenic virus. Here, we present a novel EBV-derived vector, which lacks three EBV genes including two viral oncogenes and an essential lytic gene, and encodes granulocyte-macrophage colony-stimulating factor (GM-CSF) as a cytokine of therapeutic interest. We could show that EBV vectors efficiently transduce different B-cell lines, primary resting B cells, and tumor cells of B-cell lineage. Vector-derived GM-CSF was expressed in sufficient amounts to support the maturation of dendritic cells and their presentation of model antigens to cognate T-cell clones in autologous settings and an allogeneic, HLA-matched assay. We conclude that the EBV vector system might offer an option for ex vivo manipulation of B cells and gene therapy of B-cell lymphomas.
    Full-text · Article · Feb 2006 · Gene Therapy
Show more