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.
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.
KKG Gene Therapy, GSF-National Research Center for Environment and Health, Munich, Germany.
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Kofler
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Büning
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Mayr
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Bund
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Baumert
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Hallek
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Wendtner
D023201:CD40 Ligand / Q000235:genetics; D045744:Cell Line, Tumor; D000229:Dependovirus / Q000235:genetics*; D005434:Flow Cytometry; D015870:Gene Expression; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000008:administration & dosage* / Q000235:genetics / Q000378:metabolism; D019812:Heparan Sulfate Proteoglycans / Q000235:genetics / Q000378:metabolism; D006493:Heparin / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D016130:Immunophenotyping / Q000379:methods; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000378:metabolism / Q000628:therapy*; D011505:Protein-Tyrosine Kinases / Q000235:genetics*; D011947:Receptors, Antigen, B-Cell / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000008:administration & dosage / Q000235:genetics / Q000378:metabolism; D014161:Transduction, Genetic / Q000379:methods*; D051746:ZAP-70 Protein-Tyrosine Kinase
D019812:Heparan Sulfate Proteoglycans; D011947:Receptors, Antigen, B-Cell; D011993:Recombinant Fusion Proteins; D023201:CD40 Ligand; D006493:Heparin; D011505:Protein-Tyrosine Kinases; D051746:ZAP-70 Protein-Tyrosine Kinase; C497261:ZAP70 protein, human