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Methods and compositions for the specific inhibition of gene expression by double-stranded rna

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Title: Methods and compositions for the specific inhibition of gene expression by double-stranded rna.
Abstract: The invention provides compositions and methods for selectively reducing the expression of a gene product from a desired target gene, as well as treating diseases caused by expression of the gene. The method involves introducing into the environment of a cell an amount of a double-stranded RNA (dsRNA) such that a sufficient portion of the dsRNA can enter the cytoplasm of the cell to cause a reduction in the expression of the target gene. The dsRNA has a first oligonucleotide sequence that is between 26 and about 30 nucleotides in length and a second oligonucleotide sequence that anneals to the first sequence under biological conditions. In addition, a region of one of the sequences of the dsRNA having a sequence length of from about 19 to about 23 nucleotides is complementary to a nucleotide sequence of the RNA produced from the target gene. ...


Browse recent City Of Hope patents - Duarte, CA, US
Inventors: John J. ROSSI, Mark A. BEHLKE, Dongho KIM
USPTO Applicaton #: #20110065908 - Class: 536 245 (USPTO) - 03/17/11 - Class 536 
Organic Compounds -- Part Of The Class 532-570 Series > Azo Compounds Containing Formaldehyde Reaction Product As The Coupling Component >Carbohydrates Or Derivatives >Nitrogen Containing >Dna Or Rna Fragments Or Modified Forms Thereof (e.g., Genes, Etc.) >Nucleic Acid Expression Inhibitors

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The Patent Description & Claims data below is from USPTO Patent Application 20110065908, Methods and compositions for the specific inhibition of gene expression by double-stranded rna.

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CROSS-REFERENCE TO RELATED APPLICATION

The present application is a continuation of U.S. patent application Ser. No. 12/137,914 filed 12 Jun. 2008, which in turn is a division of U.S. patent application Ser. No. 11/079,476 filed 15 Mar. 2005, which in turn is related to and claims priority under 35 U.S.C. §119(e) to U.S. provisional patent application No. 60/553,487 filed 15 Mar. 2004. Each application is incorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

This invention was made in part with Government support under Grant Numbers AI29329 and HL074704 awarded by the National Institute of Health. The Government has certain rights in this invention.

SEQUENCE SUBMISSION

The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is entitled 1954—546_Sequence_Listing.txt, was created on 29 Nov. 2010 and is 17 kb in size. The information in the electronic format of the Sequence Listing is part of the present application and is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

This invention pertains to compositions and methods for gene-specific inhibition of gene expression by double-stranded ribonucleic acid (dsRNA) effector molecules. The compositions and methods are useful in modulating gene expression in a variety of applications, including therapeutic, diagnostic, target validation, and genomic discovery.

BACKGROUND OF THE INVENTION

Suppression gene expression by double-stranded RNA (dsRNA) has been demonstrated in a variety of systems including plants (post-transcriptional gene suppression) (Napoli et al., 1990, Plant Cell. 2:279-289), fungi (quelling) (Romano and Marcino, 1992, Mol. Microbiol. 6:3343-53), and nematodes (RNA interference) (Fire et al., 1998, Nature 391:806-811). Early attempts to similarly suppress gene expression using long dsRNAs in mammalians systems failed due to activation of interferon pathways that do not exist in lower organisms. Interferon responses are triggered by dsRNAs (Stark et al., 1998, Annu. Rev. Biochem., 67:227-264). In particular, the protein kinase PKR is activated by dsRNAs of greater than 30 bp long (Manche et al., 1992, Mol Cell Biol., 12:5238-48) and results in phosphorylation of translation initiation factor eIF2α which leads to arrest of protein synthesis and activation of 2′5′-oligoadenylate synthetase (2′-5′-OAS), which leads to RNA degradation (Minks et al., 1979, J. Biol. Chem. 254:10180-10183).

In Drosophila cells and cell extracts, dsRNAs of 150 bp length or greater were seen to induce RNA interference while shorter dsRNAs were ineffective (Tuschl et al., 1999, Genes & Dev., 13:3191-3197). Long double-stranded RNA, however, is not the active effecter molecule; long dsRNAs are degraded by an RNase III class enzyme called Dicer (Bernstein et al., 2001, Nature, 409:363-366) into very short 21-23 bp duplexes that have 2-base 3′-overhangs (Zamore et al., 2000, Cell, 101:25-33). These short RNA duplexes, called siRNAs, direct the RNAi response in vivo and transfection of short chemically synthesized siRNA duplexes of this design permits use of RNAi methods to suppress gene expression in mammalian cells without triggering unwanted interferon responses (Elbashir et al., 2001, Nature, 411:494-498). The antisense strand of the siRNA duplex serves as a sequence-specific guide that directs activity of an endoribonuclease function in the RNA induced silencing complex (RISC) to degrade target mRNA (Martinez et al., 2002, Cell, 110:563-574).

In studying the size limits for RNAi in Drosophila embryo extracts in vitro, a lower threshold of around 38 bp double-stranded RNA was established for activation of RNA interference using exogenously supplied double-stranded RNA and duplexes of 36, 30, and 29 bp length were without effect (Elbashir et al., 2001, Genes & Dev., 15:188-200). The short 30-base RNAs were not cleaved into active 21-23-base siRNAs and therefore were deemed inactive for use in RNAi (Elbashir et al., 2001, Genes & Dev., 15:188-200). Continuing to work in the Drosophila embryo extract system, the same group later carefully mapped the structural features needed for short chemically synthesized RNA duplexes to function as siRNAs in RNAi pathways. RNA duplexes of 21-bp length with 2-base 3′-overhangs were most effective, duplexes of 20, 22, and 23-bp length had slightly decreased potency but did result in RNAi mediated mRNA degradation, and 24 and 25-bp duplexes were inactive (Elbashir et al., 2001, EMBO J., 20:6877-6888).

Some of the conclusions of these earlier studies may be specific to the Drosophila system employed. Other investigators established that longer siRNAs can work in human cells. However, duplexes in the 21-23-bp range have been shown to be more active and have become the accepted design (Caplen et al., 2001, Proc. Natl. Acad. Sci. USA, 98:9742-9747). Essentially, chemically synthesized duplex RNAs that mimicked the natural products that result from Dicer degradation of long duplex RNAs were identified to be the preferred compound for use in RNAi. Approaching this problem from the opposite direction, investigators studying size limits for RNAi in C. elegans found that although a microinjected 26-bp RNA duplex could function to suppress gene expression, it required a 250-fold increase in concentration compared with an 81-bp duplex (Parrish et al., 2000, Mol. Cell, 6:1077-1087).

Despite the attention given to RNAi research recently, the field is still in the early stages of development. Not all siRNA molecules are capable of targeting the destruction of their complementary RNAs in a cell. As a result, complex sets of rules have been developed for designing RNAi molecules that will be effective. Those having skill in the art expect to test multiple siRNA molecules to find functional compositions. (Ji et al. 2003) Some artisans pool several siRNA preparations together to increase the chance of obtaining silencing in a single study. (Ji et al. 2003) Such pools typically contain 20 nM of a mixture of siRNA oligonucleotide duplexes or more (Ji et al. 2003), despite the fact that a siRNA molecule can work at concentrations of 1 nM or less (Holen et al. 2002). This technique can lead to artifacts caused by interactions of the siRNA sequences with other cellular RNAs (“off target effects”). (Scherer et al. 2003) Off target effects can occur when the RNAi oligonucleotides have homology to unintended targets or when the RISC complex incorporates the unintended strand from and RNAi duplex. (Scherer et al. 2003) Generally, these effects tend to be more pronounced when higher concentrations of RNAi duplexes are used. (Scherer et al. 2003)

In addition, the duration of the effect of an effective RNAi treatment is limited to about 4 days (Holen et al. 2002). Thus, researchers must carry out siRNA experiments within 2-3 days of transfection with an siRNA duplex or work with plasmid or viral expression vectors to obtain longer term silencing.

Additional physical studies are needed to more completely characterize the structural requirements of RNAi active oligonucleotide duplexes to identify more potent and longer lasting compositions and/or methods that simplify site-selection difficulties. These studies should also include a detailed analysis of the interferon response. Ideally, such studies will be useful in identifying new RNAi active compounds that are more potent, that simplify the site selection process, and decrease “off target effects.”

The invention provides RNAi compositions with increased potency, duration of action, and decreased “off target effects” that do not activate the interferon response and provides methods for their use. In addition, the compositions ease site selection criteria and provide a duration of action that is about twice as long as prior known compositions. These and other advantages of the invention, as well as additional inventive features, will be apparent from the description of the invention provided herein.

BRIEF

SUMMARY

OF THE INVENTION

The invention provides improved compositions and methods for selectively reducing the expression of a gene product from a desired target gene in a eukaryotic cell, as well as for treating diseases caused by the expression of the gene. The method involves introducing into the environment of a cell an amount of a double-stranded RNA (dsRNA) such that a sufficient portion of the dsRNA can enter the cytoplasm of the cell to cause a reduction in the expression of the target gene. The dsRNA has a first oligonucleotide sequence that is between 26 and about 30 nucleotides in length and a second oligonucleotide sequence that anneals to the first sequence under biological conditions, such as the conditions found in the cytoplasm of a cell. In addition, a region of one of the sequences of the dsRNA having a sequence length of from about 19 to about 23 nucleotides is complementary to a nucleotide sequence of the RNA produced from the target gene. A dsRNA composition of the invention is at least as active as any isolated 19, 20, 21, 22, or 23 basepair sequence that is contained within it. Pharmaceutical compositions containing the disclosed dsRNA compositions are also contemplated. The compositions and methods give a surprising increase in the potency and duration of action of the RNAi effect. Although the invention is not intended to be limited by the underlying theory on which it is believed to operate, it is thought that this increase in potency and duration of action are caused by the fact the dsRNA serves as a substrate for Dicer which appears to facilitate incorporation of one sequence from the dsRNA into the RISC complex that is directly responsible for destruction of the RNA from the target gene.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides a comparison of RNAi efficacy using several dsRNAs having variable length and formats including a two nucleotide 3′ overhang (+2), a two nucleotide 5′ overhang (−2), and blunt ends (+0). The sequences are disclosed in the Example 2. In each panel A-D 200 μg of reporter vector was co-transfected with the indicated concentration of dsRNA. Each bar represents the average of three duplicate experiments. In FIG. 1A, 50 nM of each dsRNA was used. In FIG. 1B, 1 nM of each dsRNA was used. In FIG. 1C, 200 pM of each dsRNA was used. In FIG. 1D, 50 pM of each dsRNA was used.



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stats Patent Info
Application #
US 20110065908 A1
Publish Date
03/17/2011
Document #
12955241
File Date
11/29/2010
USPTO Class
536 245
Other USPTO Classes
International Class
07H21/04
Drawings
14


Cytoplasm
Gene Product


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