LB-198

Background

RNA Interference (RNAi) is a promising tool for both discovery and therapy. The first clinical trials are already being conducted with RNAi-based therapies. Targeting oncogenes required for cancer maintenance may become a successful tool for future cancer therapy. Delivery of RNAi remains a great challenge, which needs to be solved before the broad potential of this novel technology can be harnessed for clinical use.
 >Hypothesis
 >We hypothesized that bacteria could express hairpin RNA and deposit them in target cells, triggering RNA interference.
 >Methods
 >We designed a plasmid, called TRIP (Transkingdom RNA Interference Plamsid) which confers three novel properties to laboratory strains of E.Coli:

  • Hairpin expression cassette (with unique restriction sites allowing for the rapid switch between different siRNA targets)

  • Invasin (inv) expression cassette. This bacterial gene is derived from Yersinia enterocolitica and triggers the uptake of the bacteria into epithelial cells

  • Listeriolysin O (hly) expression cassette. This bacterial gene is derived from Listeria monocytogenes. It functions to destroy the endosome in which the bacteria are contained following invasion, to allow the bacterial content to reach the host cell cytoplasm

Results

We show that this method, called “transkingdom RNA Interference” (tkRNAi) is able to trigger specific and potent gene silencing in a variety of cultured cell lines. tkRNAi was used to knock down cancer specific oncogenes (beta-catenin and k-ras) and significantly inhibited colon cancer cell proliferation in vitro.
 
 In a xenograft model of colon cancer, silencing of beta-catenin with tkRNAi lead to a significant decrease in the fraction of proliferating cells (PCNA stain). Beta-catenin expression was found suppressed in tumors of treated animals on both mRNA and protein levels, and tumor growth was decreased by more than 50%.
 
 Conclusion
 
 We conclude that tkRNAi is a useful method for the targeted delivery of therapeutic RNAi in vitro and in vivo and merits further research efforts.

98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA