ENABLING THE USE OF LONG dsRNA FOR GENE TARGETING IN MAMMALIAN AND OTHER SELECTED ANIMAL CELLS

Abstract

The present invention provides a method of enabling the use of long dsRNA for gene silencing in mammalian cells through bacteria, preferably non-pathogenic or therapeutic strains of bacteria. DNA that encodes long double-strand RNAs are transformed into bacteria and processed in the bacterial cells into a mixture of smaller RNA duplexes and then released into the cytoplasm of the target cells, resulting in modulation of gene expression in the target cells. The methods overcome the incompatibility between long strong dsRNA and mammalian cells by eliminating, or mitigating, the non-specific innate immune response. The eukaryotic cells can be mammalian cells or avian cells. The gene of interest can be a mammalian, avian, bacterial, eukaryotic, or viral gene.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This application claims priority to and the benefit of U.S. provisional patent application Ser. No. 60 / 947,311 filed Jun. 29, 2007, the content of which application is incorporated herein by reference in its entirety.BACKGROUND OF THE INVENTION[0002]RNA interference (RNAi) is a catalytic mechanism of gene-specific silencing in eukaryotic organisms with profound implications for biology and medicine. However, the inducers of such a powerful mechanism of gene-targeting are very different in mammalian cells and in non-mammalian cells.[0003]Long double-strand RNAs (dsRNAs) triggered potent sequence-specific gene silencing in C. elegans and Drosophila Melanogaster. In contrast, long dsRNAs induce sequence-nonspecific response in mammalian cells due to activation of interferon related pathways. RNAi mechanism was considered non-functional in mammalian cells until siRNA duplexes were discovered.[0004]The use of short interfering RNA (siRNA) to s...

AI Technical Summary

Benefits of technology

"The present invention provides a solution to the problem of incompatibility between long dsRNA and mammalian cells by using bacteria as an intermediary. Bacteria with cell-invasive properties are transformed with vectors that contain long dsRNA or lhRNA that is complementary to a target eukaryotic gene or viral gene. The bacteria produce and process the long dsRNA, which is then delivered to the target cell. The long dsRNA is shielded from recognition by immune sensors on the cell surface, preventing strong innate immune responses. The invention also provides a powerful tool for target identification and validation, and a method for gene-targeting in vivo. The RNAi effects from the invention are more potent and specific than currently-available RNAi technologies."

Problems solved by technology

For some genes, identifying an efficacious siRNA motif has been unachievable.

After an effective siRNA is designed and developed, a mutation can take place in the targeted motif of the gene that can render the siRNA completely ineffective.

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