Jet-injection of short hairpin RNA-encoding vectors into tumor cells

Methods Mol Biol. 2010:629:123-39. doi: 10.1007/978-1-60761-657-3_9.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • ATP Binding Cassette Transporter, Subfamily B, Member 1 / genetics
  • ATP Binding Cassette Transporter, Subfamily B, Member 1 / metabolism
  • Animals
  • Base Sequence
  • Blotting, Northern
  • Down-Regulation / genetics
  • Gene Expression Regulation, Neoplastic
  • Genetic Vectors / genetics*
  • Humans
  • Injections, Jet / methods*
  • Mice
  • Molecular Sequence Data
  • Neoplasms / metabolism*
  • Neoplasms / pathology
  • Plasmids / genetics
  • RNA Interference
  • RNA, Messenger / genetics
  • RNA, Messenger / metabolism
  • RNA, Small Interfering / genetics*
  • RNA, Small Interfering / metabolism
  • Reverse Transcriptase Polymerase Chain Reaction
  • Xenograft Model Antitumor Assays

Substances

  • ATP Binding Cassette Transporter, Subfamily B, Member 1
  • RNA, Messenger
  • RNA, Small Interfering