Methods and Compositions Relating to Gene Silencing

Abstract

A method for obtaining a mixture of heterogenous short double-stranded RNA molecules suitable for use in gene silencing (hsiRNA) by subjecting large double-stranded RNA to enzymatic cleavage under specified conditions. The resulting mixture consistently includes enhanced representation of fragments having a size of 21-22 nucleotides absent any fractionation step. The fragments contain sequences that collectively span the entire length of the large double-stranded RNA from which they are derived. Double-stranded RNA with sequences that individually represent segments of a target mRNA may be analyzed using the methods described herein to identify the most active subset of hsiRNA fragments or individual siRNA fragments for achieving gene silencing for any gene or transcribed sequences. A method is additionally provided for preparing and cloning DNA encoding selected siRNA, hsiRNA mixtures or hairpin sequences to provide a continuous supply of a gene silencing reagent derived from any long double-stranded RNA.

Description

CROSS REFERENCE[0001]This application is a Continuation of U.S. application Ser. No. 10 / 622,240 filed Jul. 18, 2003, which claims priority from U.S. Provisional Application Ser. No. 60 / 402,769 filed Aug. 12, 2002, U.S. Provisional Application Ser. No. 60 / 407,543 filed Aug. 30, 2002 and U.S. Provisional Application Ser. No. 60 / 467,541 filed May 2, 2003. These Applications are herein incorporated by reference.BACKGROUND OF THE INVENTION[0002]RNA interference (RNAi) employing short double-stranded RNA (siRNA) is a powerful tool for silencing gene expression in mammalian cells (see for example, U.S. Pat. No. 6,506,559, International Publication No. WO 01 / 29058, International Publication No. WO 01 / 68836, International Publication No. WO 01 / 75164, U.S. Publication No. 20020114784, U.S. Publication No. 20030125281, U.S. Publication No. 2002162126, U.S. Publication No. 20030108923, U.S. Publication No. 20020173478, Fire, et al. Nature 391:806-811 (1998); Yang, et al., Mol. Cell. Biol. 21:78...

AI Technical Summary

Benefits of technology

[0016]The DNA clones produced by the above methods may be used to reduce expression of one or more target genes in a eukaryotic cell. Reducing expression of a target gene in a cell or organism provides a means of analyzing a resulting phenotypic change either in the cell or in tissues containing the cell or in an organism as a whole. Understanding the role of gene expression in a phenotype can provide insights into mechanism of disease and methods of treating diseases and for diagnosis. It can also provide a means to enhance a desired characteristic in the organism. Altering gene expression by gene silencing using DNA clones or mixtures of hsiRNA described above can provide valuable tools for analyzing a biochemical pathway in which the gene product functions and can be used in conjunction with other reagents such as antibodies.

[0020]In an embodiment of the invention, a rapid discovery method is provided for identifying an hsiRNA mixture which is capable of increased gene silencing of a target gene and includes: (a) synthesizing a plurality of large dsRNAs each large dsRNA having a sequence complementary to a segment of a target gene; (b) digesting each of the large dsRNA with RNaseIII in the presence of a manganese ions to produce a corresponding hsiRNA mixture; (c) introducing each hsiRNA mixture into a eukaryotic cell to determine whether gene silencing occurs; and (d) determining which of the hsiRNA mixtures caused increased gene silencing. Gene silencing may be further enhanced by combining a pre-selected hsiRNA mixture with a selected second hsiRNA mixture or by combining individual siRNA fragments selected from the hsiRNA mixtures or subsets thereof on the basis of silencing activity. These fragments can then be combined to form a novel mixture of desired gene silencing activity.

Problems solved by technology

A standard method for generating siRNA relies on an inherently expensive chemical synthesis of a pre-determined short sequence.

Problems associated with using crude cell extracts containing a putative cleavage enzyme are for example, that it is unclear what proteins in the mixture of proteins are necessary and sufficient to generate the observed effect.

In addition, the extract is relatively inefficient at cleaving large double-stranded RNA with only a relatively small amount of the starting material being cleaved to the desired size in vitro even under extended incubation times. (Paddison et al., Proc. Natl. Acad. Sci.

Limitations of this approach include the cost of baculovirus expression systems, the incomplete digestion of double-stranded RNA starting material and the need for gel based or other purification step to eliminate precursor RNA prior to performing silencing experiments.

Problems associated with this approach include low recovery amounts of the double-stranded fragments in a specific size range larger than about 15 nt and the associated inconvenience of titration to avoid over or under-digestion.

A problem with this approach is the lack of certainty with respect to (a) an end product where the end product relates to yield of a dsRNA having a particular size larger than about 15 nucleotides and (b) the extent of representation of the large double-strand RNA sequence in the cleavage products.

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