Bacterial Delivery of RNAi Effectors: Transkingdom RNAi

Summary

For development of RNA interference (RNAi)-based therapies, a novel strategy was developed, transkingdom RNAi (tkRNAi). This technology uses non-pathogenic bacteria to produce and deliver therapeutic short hairpin RNA (shRNA) into target cells. Here, tkRNAi was successfully applied for reversal of classical ABCB1-mediated multidrug resistance (MDR) of cancer cells.

Abstract

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

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....

Discussion

The cell number seeded for infection and the corresponding MOI used, critically depend on the cell lines under observation and their speed of growth. To find optimal cell numbers for seeding, pre-experiments to determine the speed of growth are strongly recommended. Besides this, different MOIs should be tested due to the limited extend to which the cells can stand the bacterial invasion without dying of stress. The optimal point of time where the down regulation of the gene under observation may vary. It is recommended .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Agar	Biochrom AG	214050
Amphotericin B	Biochrom AG	A2612
Dulbecco's Phosphate Buffered Saline-PBS (10 x)	Invitrogen GmbH	14080048
E. coli ceq221	Cequent Pharmaceuticals Inc.		No catalogue available, direct order
Gentamycin	Biochrom AG	A2710
Penicillin/Streptomycin	Invitrogen GmbH		Contains 5,000 units of penicillin (base) and 5,000 μg of streptomycin (base)/ml utilizing penicillin G (sodium salt) and streptomycin sulfate in 0.85% saline.
Sodium chloride	Merck KgaA	1064060500
Tryptone	Difco Laboratories	211705
Yeast Extract	Difco Laboratories	212750