Nonviral and viral gene transfer into different subsets of human dendritic cells yield comparable efficiency of transfection.
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.
Lundqvist
Andreas
A
Immune and Gene Therapy, Cancer Center Karolinska, R8:01, Karolinska Hospital, 17177 Stockholm, Sweden.
Noffz
Gabriele
G
Pavlenko
Maxim
M
Saebøe-Larssen
Stein
S
Fong
Timothy
T
Maitland
Norman
N
Pisa
Pavel
P
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
D004274:DNA, Recombinant; D008081:Liposomes; D008164:Luminescent Proteins; D012333:RNA, Messenger; D049452:Green Fluorescent Proteins