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RNA interference (RNAi) is a recently described biologic phenomenon that allows suppression of targeted genes in a highly specific and efficient manner and is being developed as a therapeutic modality with significant medical potential. RNAi can be initiated by direct delivery of short interfering RNAs (siRNAs) into the target cell or by short hairpin RNAs (shRNAs), which are either transcribed from DNA-based plasmids or viruses (i.e., lentivirus or adenovirus) or, in the case of tkRNAi, generated and delivered by live bacteria to the cytoplasm of eukaryotic target cells. Regardless of the delivery method, shRNAs are then processed into siRNAs in the cytoplasm by Dicer RNase III, a protein required for RNAi function. The generated siRNAs work in concert with a naturally occurring cellular protein complex called RNA-induced silencing complex (RISC). The RISC complex binds to the siRNA duplex, unwinds it and generates a stable complex between itself and the single-stranded antisense component of the siRNA. The single-stranded form of the siRNA, termed the guide strand, will pair with its complementary sequences within the targeted mRNA and facilitate cleavage of the mRNA by RISC, thus making it susceptible to complete degradation by cellular RNases. In the case of CEQ508, that complementary sequence is contained within the mRNA of ?-catenin (CTNNB1), and this is expected to result in the reduction of ?-catenin expression levels in infected cells.
CEQ508 is the first clinical drug candidate in a novel class of live-attenuated bacterial therapeutic agents employing a concept called transkingdom RNA interference (tkRNAi) that has been developed by Cequent Pharmaceuticals, Inc. (Cambridge, Mass.) The tkRNAi system consists of live non-pathogenic E. coli bacteria (strain CEQ221) genetically modified to possess two important novel properties: Production of high levels of intracellular shRNA under the control of a plasmid-based E. coli promoter system, and an ability to enter host cells and release the expressed shRNA through the use of two unrelated proteins, invasin and listeriolysin, that have been engineered into CEQ508 E. coli strain.
Invasin is encoded by the inv gene derived from Yersinia pseudotuberculosis. It is expressed on the bacterial surface and results in the uptake of invasin-expressing bacteria into non-phagocytic cells, (i.e., epithelial cells), through an interaction with 1-integrins expressed on the surface of these cells, where the bacteria subsequently enter in an endosomal vesicle and lyse due to an engineered nutrient auxotrophy. CEQ508 also produces listeriolysin O (LLO), which is encoded by the hly gene derived from Listeria monocytogenes. LLO is a pore forming protein that selectively ruptures the endosomal membrane membrane that, in conjunction with bacterial lysis, results in the release of shRNA into the host cell cytoplasm where it interacts with the RNAi machinery (the RISC complex) to induce degradation of the ?-catenin mRNA. Suppression of ?-catenin can arrest or slow the growth of colon cancer cells and has the potential to prevent polyp formation in the context of APC mutation.
Editor’s Note: On April 1, 2010, Cequent was acquired by MDRNA Inc. (Bothell, Wash.) for $46 million.
This article was published in Drug Discovery & Development magazine: Vol. 13, No. 3, April 2010, p. 31.
Filed Under: Drug Discovery
