Compositions for Bacterial Mediated Gene Silencing and Methods of Using the Same

Abstract

Methods are described for the delivery of one or more small interfering RNAs (siRNAs) to a eukaryotic cell using a bacterium. Methods are also described for using this bacterium to regulate gene expression in eukaryotic cells using RNA interference, and methods for treating cancer off cell proliferative disorders. The bacterium includes one or more siRNAs or one or more DNA molecules encoding one or more siRNAs. Vectors are also described for use with the bacteria of the invention for causing RNA interference in eukaryotic cells.

Description

BACKGROUND[0001]Gene silencing through RNAi (RNA-interference) by use of short interfering RNA (siRNA) has emerged as a powerful tool for molecular biology and holds the potential to be used for therapeutic gene silencing. Short hairpin RNA (shRNA) transcribed from small DNA plasmids within the target cell has also been shown to mediate stable gene silencing and achieve gene knockdown at levels comparable to those obtained by transfection with chemically synthesized siRNA (T. R. Brummelkamp, R. Bernards, R. Agami, Science 296, 550 (2002), P. J. Paddison, A. A. Caudiy, G. J. Hannon, PNAS 99, 1443 (2002)).[0002]Possible applications of RNAi for therapeutic purposes are extensive and include silencing and knockdown of disease genes such as oncogenes or viral genes. One major obstacle for the therapeutic use of RNAi is the delivery of siRNA to the target cell (Zamore P D, Aronin N. Nature Medicine 9, (3):266-8 (2003)). In fact, delivery has been described as the major hurdle now for RNA...

AI Technical Summary

Benefits of technology

[0006]The invention generally pertains to methods of delivering one or more siRNAs to a eukaryotic cell by introducing a b...

Problems solved by technology

One major obstacle for the therapeutic use of RNAi is the delivery of siRNA to the target cell (Zamore P D, Aronin N.

(1) Direct hydrodynamic intravenous injection of siRNA or shRNA-encoding plasmids: using this method, several authors have described application of RNAi against various conditions, e.g. hepatitis B (A. P. McCaffrey et al., Nat. Biotechnol. 2003 June; 21(6):639-44), fulminant hepatitis (E. Song, S. K. Lee, J. Wang, N. Ince, J. Min, J. Chen, P. Shankar, J. Lieberman. Nature Medicine 9, 347 (2003)), tumor xenograft (Spaenkuch B, et al. JNCI, 96(1): 862-72 (2004)), hepatic transgene expression (D. L. Lewis, J. E. Hagstrom, A. G. Loomis, J. A. Wolff, H. Hereijer, Nature Genetics, 32, 107 (2002), D. R. Sorensen D R, M. Leirdal, M. Sioud, JMB, 327, 761 (2003)). This method uses a high pressure and high volume injection (2.5 ml) into the mouse tail vein. The mechanism of siRNA / DNA uptake into the cells is not clear but probably mechanical damage to the vascular endothelial layer is involved. A clear disadvantage of this method is that this is not a method which could be developed into human application as it involves a massive volume charge and completely unknown mechanism of action.

(2) Direct injection, into the target tissue (brain) of an siRNA encoding adenoviral vector (H. Xia, Q. Mao, H. L. Paulson, B. L. Davidson, Nat Biotechnol, 20, 1006 (2002)). This method showed silencing of transgene (GFP) expression in the brain tissues reached by the adenoviral vector. However, the area of silencing could not be predicted reliably. This method might be developed further and might become applicable for local, e.g. intratumoral injection. Viral vectors have been used widely for gene therapy purposes, but one lesson learned from gene therapy experiments is that viral spreading can be unpredictable at times and lead to unwanted side effects (Marshall E. Science 286(5448): 2244-5 (1999)). A new method is needed for the safe and predictable administration of interfering RNAs to mammals.

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