Jet-injection of short hairpin RNA-encoding vectors into tumor cells.
The use of the RNA interference (RNAi) through the expression of small hairpin RNA (shRNA) is a promising approach for efficient gene silencing for therapeutic applications. In this chapter, we describe the in vivo reversal of the classical MDR1/P-glycoprotein (MDR1/P-gp)-mediated multidrug resistance (MDR) phenotype by shRNA. For local intratumoral delivery of naked shRNA-encoding vector constructs, the nonviral jet-injection was used. This jet-injector system uses compressed air to inject small volumes (5-10 muL) of naked nucleic acid solutions into tumor tissues. Furthermore, the design of the jet-injector allows multiple injections. Under our experimental design, the delivery of plasmid DNA encoding anti-MDR shRNA by jet-injection into human MDR1/P-gp overexpressing MaTu/ADR breast cancer xenografts resulted in a decrease of MDR1 mRNA expression level to more than 90%. Accordingly, the corresponding MDR1/P-gp protein is no longer detectable in the tumors after anti-MDR1 shRNA vector injection. Furthermore, combination of two intratumoral jet-injections of anti-MDR1 shRNA vectors with two intravenous administrations of doxorubicin is sufficient for a complete reversal of the MDR phenotype in association with tumor growth inhibition.
Walther
Wolfgang
W
Max-Delbrück-Center for Molecular Medicine and Experimental Clinical Research Center, Charité Berlin, Berlin, Germany.
Stein
Ulrike
U
Lage
Hermann
H
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000235:genetics / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D015536:Down-Regulation / Q000235:genetics; D015972:Gene Expression Regulation, Neoplastic; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D007277:Injections, Jet / Q000379:methods*; D051379:Mice; D008969:Molecular Sequence Data; D009369:Neoplasms / Q000378:metabolism* / Q000473:pathology; D010957:Plasmids / Q000235:genetics; D034622:RNA Interference; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D023041:Xenograft Model Antitumor Assays
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D012333:RNA, Messenger; D034741:RNA, Small Interfering