Prolonged in vivo gene silencing by electroporation-mediated plasmid delivery of small interfering RNA.
For the successful application of RNA interference in vivo, it is desired to achieve (local) delivery of small interfering RNAs (siRNAs) and long-term gene silencing. Nonviral electrodelivery is suitable to obtain local and prolonged expression of transgenes. By intramuscular electrodelivery of a plasmid in which two opposing human polymerase III promoters (H1 and U6) drive the expression of siRNA constructs that form functional double-stranded siRNAs, in combination with in vivo bioluminescence imaging, we were able to knock down exogenous delivered luciferase for at least 100 days in murine calf muscles. This effect was sequence specific, because scrambled siRNA had no effect. Moreover, we were able to demonstrate in vivo reduction of endogenous TLR4 expression for at least 1 week, using a similar vector expressing an siRNA for TLR4 in the muscle. In this study, we demonstrate that in vivo suppression of both endogenous (for at least 1 week) and introduced genes (>100 days) is feasible via plasmid-driven siRNA expression after electroporation-mediated intramuscular gene transfer. With this approach the short-term effect of oligonucleotides and the drawbacks of viral gene delivery, like immunological responses, could be circumvented. Therefore, this application of RNA interference is a useful tool with which to investigate gene function and might be promising as a therapeutic tool for locally acting diseases such as restenosis or tumors.
Eefting
Daniël
D
Gaubius Laboratory, TNO Quality of Life, 2301 CE Leiden, The Netherlands.
Grimbergen
Jos M
JM
de Vries
Margreet R
MR
van Weel
Vincent
V
Kaijzel
Eric L
EL
Que
Ivo
I
Moon
Randall T
RT
Löwik
Clemens W
CW
van Bockel
J Hajo
JH
Quax
Paul H A
PH
D000818:Animals; D002417:Cattle; D002461:Cell Line, Transformed; D002472:Cell Transformation, Viral; D018274:Electroporation; D005240:Feasibility Studies; D020868:Gene Silencing; D017930:Genes, Reporter; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics; D008070:Lipopolysaccharides / Q000494:pharmacology; D008156:Luciferases / Q000378:metabolism; D008163:Luminescent Measurements; D008297:Male; D051379:Mice; D008815:Mice, Inbred Strains; D018482:Muscle, Skeletal / Q000166:cytology / Q000378:metabolism; D041681:NIH 3T3 Cells; D010957:Plasmids / Q000235:genetics*; D034622:RNA Interference; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics* / Q000378:metabolism; D013997:Time Factors; D051197:Toll-Like Receptor 4 / Q000378:metabolism
D008070:Lipopolysaccharides; D012333:RNA, Messenger; D034741:RNA, Small Interfering; C493487:Tlr4 protein, mouse; D051197:Toll-Like Receptor 4; D008156:Luciferases; D007041:Hypoxanthine Phosphoribosyltransferase