title: Rapid and efficient nonviral gene delivery of CD154 to primary chronic lymphocytic leukemia cells.
abstract: Interactions between CD40 and CD40 ligand (CD154) are essential in the regulation of both humoral and cellular immune responses. Forced expression of human CD154 in B chronic lymphocytic leukemia (B-CLL) cells can upregulate costimulatory and adhesion molecules and restore antigen-presenting capacity. Unfortunately, B-CLL cells are resistant to direct gene manipulation with most currently available gene transfer systems. In this report, we describe the use of a nonviral, clinical-grade, electroporation-based gene delivery system and a standard plasmid carrying CD154 cDNA, which achieved efficient (64+/-15%) and rapid (within 3 h) transfection of primary B-CLL cells. Consistent results were obtained from multiple human donors. Transfection of CD154 was functional in that it led to upregulated expression of CD80, CD86, ICAM-I and MHC class II (HLA-DR) on the B-CLL cells and induction of allogeneic immune responses in MLR assays. Furthermore, sustained transgene expression was demonstrated in long-term cryopreserved transfected cells. This simple and rapid gene delivery technology has been validated under the current Good Manufacturing Practice conditions, and multiple doses of CD154-expressing cells were prepared for CLL patients from one DNA transfection. Vaccination strategies using autologous tumor cells manipulated ex vivo for patients with B-CLL and perhaps with other hematopoietic malignancies could be practically implemented using this rapid and efficient nonviral gene delivery system.
authors.affiliation: MaxCyte, Inc., Gaithersburg, Maryland, USA.
authors.forename: L H
authors.initials: LH
authors.lastname: Li
authors.forename: E
authors.initials: E
authors.lastname: Biagi
authors.forename: C
authors.initials: C
authors.lastname: Allen
authors.forename: R
authors.initials: R
authors.lastname: Shivakumar
authors.forename: J M
authors.initials: JM
authors.lastname: Weiss
authors.forename: S
authors.initials: S
authors.lastname: Feller
authors.forename: E
authors.initials: E
authors.lastname: Yvon
authors.forename: J C
authors.initials: JC
authors.lastname: Fratantoni
authors.forename: L N
authors.initials: LN
authors.lastname: Liu
mesh_terms: D023201:CD40 Ligand / Q000235:genetics / Q000378:metabolism* / Q000627:therapeutic use*; D018274:Electroporation / Q000379:methods; D005434:Flow Cytometry; D015972:Gene Expression Regulation, Neoplastic / Q000276:immunology*; D015316:Genetic Therapy / Q000379:methods*; D006801:Humans; D016233:Immunotherapy, Active / Q000379:methods*; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000276:immunology / Q000628:therapy*; D010957:Plasmids / Q000235:genetics; D014162:Transfection / Q000379:methods*; D019076:Transgenes / Q000235:genetics
chemical_list: D023201:CD40 Ligand