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Bacteria mediated gene silencing

a technology of gene silencing and bacteria, applied in the field of bacteria mediated gene silencing, to achieve the effect of reducing or inhibiting the expression of one or more hpv oncogenes, and reducing or inhibiting the expression of one or more genes of interes

Inactive Publication Date: 2008-12-18
CEQUENT PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0017]The expressed siRNAs can direct the multienzyme complex RNA-induced silencing complex of the cell to interact with the mRNA of one or more genes of interest. Preferably, the siRNAs interact with the mRNA of one or more HPV oncogenes. Preferably, the complex can degrade the mRNA. Preferably, the expression of one or more genes of interest is decreased or inhibited. The expression is decreased or inhibited as compared to the expression of the gene prior to administration or treatment with an invasive bacterium or BTP containing one or more siRNA or a DNA encoding for one or more siRNAs. Preferably, the expression of one or more HPV oncogenes is decreased or inhibited.

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.

Method used

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Examples

Experimental program
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Effect test

example 1

Knockdown of β-Catenin and k-Ras

[0243]Previous studies have demonstrated the powerful nature of the siRNA knockdown technology disclosed herein. For example, in vitro and in vivo knockdown of beta catenin and k-ras utilizing bacterial delivery is described in PCT Publication No. WO 06 / 066048, which is incorporated herein by reference in its entirety.

example 2

TRIP with Multiple shRNA Expression Cassettes

[0244]The TRIP described herein, and described in further detail in PCT Publication No. WO 06 / 066048, can be modified to produce a plasmid which allows targeting of multiple genes simultaneously or multiple sequences within one gene simultaneously. For example, TRIP with multiple hairpin expression cassettes to produce shRNA can target different sequences in a given gene, or target multiple genes through a simultaneous bacterial treatment.

[0245]The TRIP plasmid can incorporate multiple (up to ten) cloning sites to express different shRNA constructs (FIG. 1). The purpose of such a plasmid will be to allow silencing of various genes through a single therapeutic bacterium which will be empowered by the Multiple-expression cassette-TRIP (mec-TRIP) to synthesize short hairpin RNA against a variety of targets simultaneously.

[0246]These different hairpins can either be expressed competitively at high levels through the use of an identical high l...

example 3

Operator Repressor Titration System

[0248]The TRIP system (bacteria and plasmid) have been modified to include the ORT (Operator Repressor Titration) system from Cobra Biomanufacturing (Keele, UK). This adaptation helps to maintain the plasmid in suitable strains in the absence of selective antibiotics. The bacterial carrier strain has been modified accordingly to allow for the ORT system to function (deletion of the DAP gene and replacement with an ORT-controlled DAP gene expression system). The plasmid has been modified to remove the antibiotic selection sequences to support the ORT system. Further changes have been introduced to the bacterial genome, including for example, (a) deletion of the aroA gene (in some CEQ strains) to make the bacteria more susceptible to nutrient shortage, particularly in the intracellular compartment where they will die due to lack of nutrients; (b) insertion of T7RNA polymerase gene into the chromosome and or (c) integration of a shRNA expression casse...

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Abstract

Methods are described for the delivery of one or more small interfering RNAs (siRNAs) to a eukaryotic cell using a bacterium or BTP. Methods are also described for using this bacterium to regulate gene expression in eukaryotic cells using RNA interference, and methods for treating viral diseases and disorders. The bacterium or BTP 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

RELATED APPLICATIONS[0001]This application claims priority to, and the benefit of, U.S. Provisional Patent Application No. 60 / 934,751, filed Jun. 15, 2007 and U.S. Provisional Patent Application No. 61 / 010,028, filed Jan. 4, 2008. The contents of these applications are incorporated by reference in their entireties.BACKGROUND[0002]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)).[0003]Possible applications of RNAi for therapeutic purposes are exten...

Claims

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Application Information

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IPC IPC(8): A61K35/74C12N1/20C12N15/63C12N15/87C12N15/11C12N15/113
CPCC12N15/111C12N15/1131C12N15/1135C12N2310/111C12N15/113C12N2320/32C12N15/1136C12N2320/30C12N2310/14A61P37/02A61P37/08C12N15/63
Inventor FRUEHAUF, JOHANNESVAZE, MORESHWAR BHANUDASLAROUX, JR., FLOYD STEPHENSAUER, NOEL JOY
Owner CEQUENT PHARMA
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