title: Nonviral and viral gene transfer into different subsets of human dendritic cells yield comparable efficiency of transfection.
abstract: Among the many promising cancer immunotherapeutic strategies, dendritic cells (DC) have become of particular interest. This study aims to optimize a clinical grade protocol for culture and transfection of human DC. Monocytes and CD34(+) hematopoietic stem cells (HSC) from same donor were differentiated under serum-free conditions and analyzed for their susceptibility to several recently described nonviral transfection methods as compared with established virally mediated gene transfer. Nonviral gene transfer methods studied were square-wave electroporation, lipofection, and particle-mediated transfer of plasmid DNA or in vitro transcribed mRNA. We conclude that DNA is not suitable for transduction of DC using nonviral methods. In contrast, mRNA and square-wave electroporation reproducibly yields 60% and 50% transfected monocyte- and CD34(+)-derived DC, respectively, measured at protein level, without affecting the cell viability. Thus, the transfection efficiency of this method is comparable with the 40-90% transgene expression obtained using retroviral (RV) or adenoviral (AdV) vectors in CD34(+)- and monocyte-derived DC, respectively. In monocyte-derived DC, however, the amount of protein expressed per-cell basis was higher after AdV (MOI = 1000) compared with mRNA electroporation-mediated transfer. This is the first study directly demonstrating side-by-side that mRNA electroporation into DC of different origin indeed results in a comparable number of transduced cells as when using virus-mediated gene transfer.
authors.affiliation: Immune and Gene Therapy, Cancer Center Karolinska, R8:01, Karolinska Hospital, 17177 Stockholm, Sweden.
authors.forename: Andreas
authors.initials: A
authors.lastname: Lundqvist
authors.forename: Gabriele
authors.initials: G
authors.lastname: Noffz
authors.forename: Maxim
authors.initials: M
authors.lastname: Pavlenko
authors.forename: Stein
authors.initials: S
authors.lastname: Saebøe-Larssen
authors.forename: Timothy
authors.initials: T
authors.lastname: Fong
authors.forename: Norman
authors.initials: N
authors.lastname: Maitland
authors.forename: Pavel
authors.initials: P
authors.lastname: Pisa
mesh_terms: D000256:Adenoviridae / Q000235:genetics*; D019470:Biolistics; D002454:Cell Differentiation / Q000187:drug effects; D002478:Cells, Cultured / Q000378:metabolism / Q000821:virology; D004274:DNA, Recombinant / Q000008:administration & dosage / Q000235:genetics; D003673:Defective Viruses / Q000235:genetics; D003713:Dendritic Cells / Q000145:classification / Q000378:metabolism* / Q000821:virology; D018274:Electroporation; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000187:drug effects; D006801:Humans; D008081:Liposomes; D008164:Luminescent Proteins / Q000235:genetics; D007959:Lymphocyte Culture Test, Mixed; D009000:Monocytes / Q000166:cytology / Q000187:drug effects; D010957:Plasmids / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D015203:Reproducibility of Results; D014161:Transduction, Genetic; D014162:Transfection
chemical_list: D004274:DNA, Recombinant; D008081:Liposomes; D008164:Luminescent Proteins; D012333:RNA, Messenger; D049452:Green Fluorescent Proteins