title: Efficient gene transfer of CD40 ligand into primary B-CLL cells using recombinant adeno-associated virus (rAAV) vectors.
abstract: B cells of chronic lymphocytic leukemia (B-CLL) are resistant to transduction with most currently available vector systems. Using an optimized adenovirus-free packaging system, recombinant adeno-associated virus (rAAV) vectors coding for the enhanced green fluorescent protein (AAV/EGFP) and CD40 ligand (AAV/CD40L) were packaged and highly purified resulting in genomic titers up to 3 x 10(11)/mL. Cells obtained from 24 patients with B-CLL were infected with AAV/EGFP or AAV/CD40L at a multiplicity of infection (MOI) of 100 resulting in transgene expression in up to 97% of cells as detected by flow cytometry 48 hours after infection. Viral transduction could be specifically blocked by heparin. Transduction with AAV/CD40L resulted in up-regulation of the costimulatory molecule CD80 not only on infected CLL cells but also on noninfected bystander leukemia B cells, whereas this effect induced specific proliferation of HLA-matched allogeneic T cells. Vaccination strategies for patients with B-CLL using leukemia cells infected ex vivo by rAAV vectors now seems possible in the near future.
authors.affiliation: Medical Clinic III, University Hospital Grosshadern and Gene Center, Ludwig-Maximilians-University, Munich, Germany.
authors.forename: Clemens-Martin
authors.initials: CM
authors.lastname: Wendtner
authors.forename: David M
authors.initials: DM
authors.lastname: Kofler
authors.forename: Hans D
authors.initials: HD
authors.lastname: Theiss
authors.forename: Christian
authors.initials: C
authors.lastname: Kurzeder
authors.forename: Raymund
authors.initials: R
authors.lastname: Buhmann
authors.forename: Carmen
authors.initials: C
authors.lastname: Schweighofer
authors.forename: Luca
authors.initials: L
authors.lastname: Perabo
authors.forename: Susanne
authors.initials: S
authors.lastname: Danhauser-Riedl
authors.forename: Jens
authors.initials: J
authors.lastname: Baumert
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Hiddemann
authors.forename: Michael
authors.initials: M
authors.lastname: Hallek
authors.forename: Hildegard
authors.initials: H
authors.lastname: Büning
mesh_terms: D000328:Adult; D018122:B7-1 Antigen / Q000276:immunology; D023201:CD40 Ligand / Q000235:genetics*; D019496:Cancer Vaccines; D003602:Cytotoxicity, Immunologic; D000229:Dependovirus; D005260:Female; D015316:Genetic Therapy; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D007167:Immunotherapy; D066298:In Vitro Techniques; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000235:genetics* / Q000276:immunology; D008164:Luminescent Proteins; D008297:Male; D008875:Middle Aged; D014161:Transduction, Genetic
chemical_list: D018122:B7-1 Antigen; D019496:Cancer Vaccines; D008164:Luminescent Proteins; D023201:CD40 Ligand; D049452:Green Fluorescent Proteins