RNA interference (RNAi) technology is a promising approach for efficient silencing of a particular gene for cancer gene therapy. However, the main obstacle for the development of RNAi-based therapeutic approaches is the delivery of the RNAi effector molecules to target cells. One promising strategy to surmount this challenge is the application of nonpathogenic bacteria as a delivery vector to target cells. In this chapter, the design of invasive Escherichia coli is described. The strain carries a plasmid encoding short hairpin RNAs (shRNAs), a protein (invasin) necessary for endocytotic absorption of the bacteria by target cells, and listeriolysin O required for the lysis of endocytotic vesicles within the target cells.
The ATP-binding cassette (ABC)-transporter P-glycoprotein (Pgp, also known as ABCB1) is the best characterized factor involved in multidrug resistance (MDR) of cancer cells. Pgp, which is encoded by the MDR1 gene, acts as a membrane-embedded drug extrusion pump for multiple structurally unrelated cytotoxic drugs. Inhibition of the pump activity of Pgp by low-molecular weight pharmacologically active compounds as a method to reverse MDR in cancer patients has been studied extensively, but so far clinical trials have generally been disappointing. Thus, experimental strategies for overcoming MDR are under investigation. These approaches include the application of the RNA interference (RNAi) technology. RNAi is a physiological mechanism triggered by small double-stranded RNA molecules resulting in a sequence-specific gene-silencing. Besides its potential for development of novel therapeutics, RNAi also offers the possibility for specific inhibition of cellular targets in functional investigations. For specific inhibition of Pgp by triggering the RNAi pathway, transient gene-silencing by application of small interfering RNA (siRNA), and stable inhibition by transfection of MDR cancer cells with short hairpin RNA (shRNA) encoding expression cassettes encoded on plasmid DNA are described. Efficacy of RNAi on MDR1 mRNA expression level is determined by quantitative real-time RT-PCR and Northern blot. The consequences of RNAi on protein expression level are measured by Western blot and immunohistochemistry. The effects on the drug extrusion activity are measured by a drug accumulation assay based on flow cytometry, and reversal of the drug-resistant phenotype by assessment of drug-specific IC(50)-values by a cell proliferation assay based on colorimetry.
RNA interference (RNAi) represents a high effective mechanism for specific inhibition of mRNA expression. Besides its potential as a powerful laboratory tool, the RNAi pathway appears to be promising for therapeutic utilization. For development of RNA interference (RNAi)-based therapies, delivery of RNAi-mediating agents to target cells is one of the major obstacles. A novel strategy to overcome this hurdle is transkingdom RNAi (tkRNAi). This technology uses non-pathogenic bacteria, e.g. Escherichia coli, to produce and deliver therapeutic short hairpin RNA (shRNA) into target cells to induce RNAi. A first-generation tkRNAi-mediating vector, TRIP, contains the bacteriophage T7 promoter for expression regulation of a therapeutic shRNA of interest. Furthermore, TRIP has the Inv locus from Yersinia pseudotuberculosis that encodes invasin, which permits natural noninvasive bacteria to enter β1-integrin-positive mammalian cells and the HlyA gene from Listeria monocytogenes , which produces listeriolysin O. This enzyme allows the therapeutic shRNA to escape from entry vesicles within the cytoplasm of the target cell. TRIP constructs are introduced into a competent non-pathogenic Escherichia coli strain, which encodes T7 RNA polymerase necessary for the T7 promoter-driven synthesis of shRNAs. A well-characterized cancer-associated target molecule for different RNAi strategies is ABCB1 (MDR1/P-glycoprotein, MDR1/P-gp). This ABC-transporter acts as a drug extrusion pump and mediates the "classical" ABCB1-mediated multidrug resistance (MDR) phenotype of human cancer cells which is characterized by a specific cross resistance pattern. Different ABCB1-expressing MDR cancer cells were treated with anti-ABCB1 shRNA expression vector bearing E. coli. This procedure resulted in activation of the RNAi pathways within the cancer cells and a considerable down regulation of the ABCB1 encoding mRNA as well as the corresponding drug extrusion pump. Accordingly, drug accumulation was enhanced in the pristine drug-resistant cancer cells and the MDR phenotype was reversed. By means of this model the data provide the proof-of-concept that tkRNAi is suitable for modulation of cancer-associated factors, e.g. ABCB1, in human cancer cells. Protocol 1) Bacterial Delivery of shRNAs 2) Representative ResultsIf the protocol is performed correctly, the results should be comparable to the ones shown below. 1. Before starting with the bacterial culture, one has to prepare LB-medium and LB-agar. 2. For the LB-medium weigh out yeast extract (0.5 % w/v), bacto tryptone (1.0 % w/v), and NaCl (0.6 % w/v) and dilute these components in aqua bidest. The prepared solutions have to be sterilized in an autoclave and are then ready to use. 3. For LB-agar plates bacto agar (1.5 % w/v) has to be added to the LB-medium previous to sterilization. 4. Heat up LB-agar in the microwave until the LB-agar is completely dissolved. Let the solution cool down until the bottle can be touched easily without getting burned. Add kanamycin (100 mg/ml) for selection of ...
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