Bacterial delivery of functional messenger RNA to mammalian cells.
The limited access to the nuclear compartment may constitute one of the major barriers after bacteria-mediated expression plasmid DNA delivery to eukaryotic cells. Alternatively, a self-destructing Listeria monocytogenes strain was used to release translation-competent mRNA directly into the cytosol of epithelial cells, macrophages and human dendritic cells. Enhanced green fluorescent protein (EGFP)-encoding mRNA, adapted for translation in mammalian cells by linking an IRES element to the 5'-end of the egfp coding sequence, was produced by T7 RNA polymerase in the carrier bacteria upon entry into the cytosol where the mRNA is efficiently released from the lysed bacteria and immediately translated in eukaryotic host cells. Besides the much earlier expression of EGFP being detectable already 4 h after infection, the number of EGFP expressing mammalian cells obtained with this novel RNA delivery technique is comparable to or - especially in phagocytic cells - even higher than that obtained with the expression plasmid DNA delivery strategy. Accordingly, bacteria-mediated delivery of ovalbumin-encoding mRNA to macrophages resulted in efficient antigen processing and presentation in vitro indicating that this approach may also be adapted for the in vivo delivery of antigen-encoding mRNA leading to a more efficient immune response when applied to vaccine development.
Schoen
Christoph
C
Lehrstuhl für Mikrobiologie, Biozentrum der Universität Würzburg, Am Hubland, Germany.
Kolb-Mäurer
Annette
A
Geginat
Gernot
G
Löffler
Daniela
D
Bergmann
Birgit
B
Stritzker
Jochen
J
Szalay
Aladar A
AA
Pilgrim
Sabine
S
Goebel
Werner
W
D000818:Animals; D002460:Cell Line; D003600:Cytosol / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000235:genetics* / Q000378:metabolism; D003713:Dendritic Cells / Q000378:metabolism; D004847:Epithelial Cells / Q000378:metabolism; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D006801:Humans; D008089:Listeria monocytogenes / Q000235:genetics*; D008264:Macrophages / Q000378:metabolism; D051379:Mice; D010957:Plasmids; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics*; D014764:Viral Proteins / Q000235:genetics* / Q000378:metabolism
D012333:RNA, Messenger; D014764:Viral Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases