Transkingdom RNA interference (tkRNAi) uses engineered E. coli to express shRNA and knock down target genes after invasion into cancer cells in vitro and in vivo (Nature Biotech, 2006, 697). Here we show the improvement of this tkRNAi technology through the development of a multi-targeting hairpin expression construct which allows simultaneous expression of up to three different shRNA. Methods: E. Coli shRNA expression promoter and terminator were cloned into an expression vector which contains invasin expression cassette and LLO (HLY) expression cassette; kanamycin resistance gene was used as a selection marker. A small DNA oligo was further designed and cloned into the expression cassette to serve as template for expression of hairpin RNA which target three genes; beta-catenin, K-ras and cyclin D1. Another DNA oligo template for hairpin RNA targeting GFP, luciferase and LacZ served as negative control. For the bacterial invasion assay, SW480 cells were plated in 6-well plates and treated with invasive tkRNAi bacteria at different doses from Multiplicity of infection (MOI) 10 to 150 for 2hr, then washed and replenished with normal medium with antibiotics for 2 days. Total RNA was extracted for qPCR readout at mRNA level and protein was applied to Western Blot analysis of beta-catenin, K-ras and Cyclin D1. Bacterial extracts were analyzed for shRNA expression using Northern Blot. Results; shRNA at the expected length was stably expressed in tkRNAi E. coli. In situ hybridization of hairpin RNA showed the bacteria began to release hairpin RNA into the cytosol at 6hr after invasion. Bacteria invasion up to 200 MOI showed no obvious cytotoxicity. At 100 MOI, the bacteria of target hairpins effectively knocked down beta-catenin 70%, cyclin D1 80% and k-ras 40% at mRNA level although the bacteria expressing control hairpin showed some 20% non-specific knockdown. However, at protein level, we found treatment of cancer cells with tkRNAi bacteria expressing the multiple hairpin constructs dose-dependently reduced Beta-catenin, K-ras and cyclin D1 while control bacteria had no effect at all. We further observed that SW480 cells underwent cell death proportional to the level of target knockdown in the cells. These findings open the possibility of using tkRNAi for simultaneous targeting of multiple genes, which will be useful for indications in cancer therapy.

Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr LB-38.

100th AACR Annual Meeting-- Apr 18-22, 2009; Denver, CO