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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. ...


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.

FIG. 2 shows an RNAi assay in 3T3 cells expressing endogenous EGFP. The experimental procedure is described in Example 2. Measurements were made 4 days after treatment. The dose response curves for 21-mer duplex with 2-base 3′-overhang (SEQ ID No. 6/7), 25-mer duplex with 2-base 5′-overhang (SEQ ID Nos. 16/17), and blunt 27-mer duplex (SEQ ID Nos. 30/31) are shown.

FIG. 3 shows RNAi assays of various 27-mer RNA duplex formats as outlined in Example 2. Duplex 27+0UU (SEQ ID Nos. 30/31) was most potent.

FIG. 4 shows RNAi assays on HEK 293 cells that were either mock transfected (negative control), transfected with 200 ng EGFP reporter plasmid alone (positive control), or reporter plasmid+RNA duplexes at varying concentrations as described in Example 3.

FIG. 5 shows superior knockout of the HNRPH1 gene by a 27-mer of the invention as compared to a 21-mer directed to the same target. Western blots obtained from HEK 293 cells after transfection with EGFP-specific siRNA (SEQ ID No. 6/7; C) (negative control) and an HNRPH1 specific 21-mer siRNA duplex (SEQ ID Nos. 51/52; 21+2) at varying concentrations, or with an HNRPH1 specific 27-mer siRNA duplex (SEQ ID Nos. 53/54; 27+0) at varying concentrations, as described in Example 4.

FIG. 6 shows the reaction of Dicer with various length RNA duplexes as described in Example 5. Dicer was able to digest 25-29-mers primarily into about a 21 basepair duplex but did not digest the 21 nucleotide long test duplex.

FIG. 7 shows the relative expression of EGFP after RNAi assays using a 27-mer dsRNA versus shorter 21-mer siRNAs contained within the 27-mer sequence as described in more detail in Example 6. As shown a blunt ended 27-mer that covers a poor site for a 21 nucleotide RNAi can effectively target that site.

FIG. 8 shows the results of RNAi assays after treatment by various effector dsRNA molecules and pools of molecules as set forth in Example 6.

FIG. 9 shows the time course study of the duration of the RNAi effect with various effector molecules as described in Example 7. The study shows the duration of the RNAi effect is at least about twice as long with the 27-mer dsRNA of the invention as with 21-mers. The “27+0 UU” sequences are set forth in SEQ ID NOs:28 and 29. The “Mut-16” sequences are set forth in SEQ ID NOs:70 and 71. The “Mut-16,17” sequences are set forth in SEQ ID NOs:72 and 73. The “Mut-15,16,17” sequences are set forth in SEQ ID NOs:74 and 75.

FIG. 10 shows the images of cells in a time course study of the duration of the RNAi effect with various effector molecules as described in Example 7. The study shows the duration of the RNAi effect is at least about twice as long with the 27-mer dsRNA of the invention as with 21-mers.

FIG. 11 shows that neither interferon alpha (FIG. 11A) or interferon beta (FIG. 11B) are induced by the 27-mer dsRNA of the invention as described in more detail in Example 8.

FIG. 12 shows the results of a PKR activation assay in which long dsRNA resulted in strong PKR activation (positive control) while all of the short synthetic RNAs showed no evidence for PKR activation.

DETAILED DESCRIPTION

OF THE INVENTION

The invention is directed to compositions that contain double stranded RNA (“dsRNA”), and methods for preparing them, that are capable of reducing the expression of target genes in eukaryotic cells. One of the strands of the dsRNA contains a region of nucleotide sequence that has a length that ranges from about 19 to about 23 nucleotides that can direct the destruction of the RNA transcribed from the target gene.

For purposes of the invention a suitable dsRNA contains one oligonucleotide sequence, a first sequence, that is at least 25 nucleotides in length and no longer than about 30 nucleotides. More preferably this sequence of RNA is between about 26 and 29 nucleotides in length. Still more preferably this sequence is about 27 or 28 nucleotides in length, 27 nucleotides is most preferred. The second sequence of the dsRNA can be any sequence that anneals to the first sequence under biological conditions, such as within the cytoplasm of a eukaryotic cell. Generally, the second oligonucleotide sequence will have at least 19 complementary base pairs with the first oligonucleotide sequence, more typically the second oligonucleotides sequence will have about 21 or more complementary base pairs, and more preferably about 25 or more complementary base pairs with the first oligonucleotide sequence. In a preferred embodiment the second sequence is the same length as the first sequence.

In certain embodiments the double-stranded RNA structure the first and second oligonucleotide sequences exist on separate oligonucleotide strands which can be and typically are chemically synthesized. In preferred embodiments both strands are between 26 and 30 nucleotides in length. In one preferred embodiment both strands are 27 nucleotides in length, are completely complementary and have blunt ends. The dsRNA can be from a single RNA oligonucleotide that undergoes intramolecular annealing or, more typically, the first and second sequences exist on separate RNA oligonucleotides.

Suitable dsRNA compositions that contain two separate oligonucleotides can be chemically linked outside their annealing region by chemical linking groups. Many suitable chemical linking groups are known in the art and can be used. Suitable groups will not block Dicer activity on the dsRNA and will not interfere with the directed destruction of the RNA transcribed from the target gene.

The first and second oligonucleotide sequences are not required to be completely complimentary. In fact, it is preferred that the 3′-terminus of the sense strand contains one or more mismatches. It is more preferred that two mismatches be incorporated at the 3′ terminus. In a most preferred embodiment the dsRNA of the invention is a double stranded RNA molecule containing two RNA oligonucleotides each of which is 27 nucleotides in length and, when annealed to each other, have blunt ends and a two nucleotide mismatch on the 3′-terminus of the sense strand (the 5′-terminus of the antisense strand).

One feature of the dsRNA compositions of the invention is that they can serve as a substrate for Dicer. Typically, the dsRNA compositions of this invention will not have been treated with Dicer, other RNAses, or extracts that contain them. Such treatments could digest the dsRNA to lengths of less than 25 nucleotides that are no longer Dicer substrates. Several methods are known and can be used for determining whether a dsRNA composition serves as a substrate for Dicer. For example, Dicer activity can be measured in vitro using the Recombinant Dicer Enzyme Kit (GTS, San Diego, Calif.) according to the manufacturer\'s instructions. Dicer activity can be measured in vivo by treating cells with dsRNA and maintaining them for 24 h before harvesting them and isolating their RNA. RNa can be isolated using standard methods, such as with the RNeasy™ Kit (Qiagen) according to the manufacturer\'s instructions. The isolated RNA can be separated on a 10% PAGE gel which is used to prepare a standard RNA blot that can be probed with a suitable labeled deoxyoligonucleotide, such as an oligonucleotide labeled with the Starfire™ Oligo Labeling System (Integrated DNA Technologies, Inc., Coralville, Iowa).

It has been found empirically that these longer dsRNA species of from 25 to about 30 nucleotides give unexpectedly improved results in terms of increased potency and increased duration of action over shorter prior art RNAi compositions. The dsRNA compositions of the invention are at least as active as any isolated 23 nucleotide dsRNA sequence contained within them and in preferred embodiments more active. Without wishing to be bound by the underlying theory of the invention, it is thought that the longer dsRNA species serve as a substrate for the enzyme Dicer in the cytoplasm of a cell. In addition to cleaving the dsRNA of the invention into shorter segments, Dicer is thought to facilitate the incorporation of a single-stranded cleavage product derived from the cleaved dsRNA into the RISC complex that is responsible for the destruction of the cytoplasmic RNA derived from the target gene. Studies have shown that the cleavability of a dsRNA species by Dicer corresponds with increased potency and duration of action of the dsRNA species.

Suitable dsRNA compositions of this invention do not induce apoptosis in the cells in which they are used. Apoptosis or “programmed cell death,” includes any non-necrotic, cell-regulated form of cell death, as defined by criteria well established in the art. Cells undergoing apoptosis show characteristic morphological and biochemical features. Once the process is triggered, or the cells are committed to undergoing apoptosis, morphological and physiological changes include cell shrinkage, chromatin condensation, nuclear and cytoplasmic condensation, membrane blebbing, partitioning of cytoplasm and nucleus into membrane bound vesicles which contain ribosomes (apoptotic bodies), and DNA degradation into a characteristic oligonucleosomal ladder composed of multiples of 200 base pairs, leading eventually to cell death. In vivo, these apoptotic bodies are rapidly recognized and phagocytized by either macrophages or adjacent epithelial cells. In vitro, the apoptotic bodies as well as the remaining cell fragments ultimately swell and finally lyse. This terminal phase of in vitro cell death has been termed “secondary necrosis.”

The effect that a dsRNA has on a cell can depend upon the cell itself. In some circumstances a dsRNA could induce apoptosis or gene silencing in one cell type and not another. Thus, it is possible that a dsRNA could be suitable for use in one cell and not another. To be considered “suitable” a dsRNA composition need not be suitable under all possible circumstances in which it might be used, rather it need only be suitable under a particular set of circumstances.

Modifications can be included in the disclosed dsRNA so long as the dsRNA remains sufficiently chemically stable, does not induce apoptosis, does not substantially interrupt annealing of the first and second strands, and otherwise does not substantially interfere with the directed destruction of the RNA transcribed from the target gene. Modifications can be incorporated in the 3′-terminal region, the 5′-terminal region, in both the 3′-terminal and 5′-terminal region or in some instances throughout the sequence. With the restrictions noted above in mind any number and combination of modifications can be incorporated into the dsRNA. Where multiple modifications are present, they may be the same or different. Modifications to bases, sugar moieties, the phosphate backbone, and their combinations are contemplated.

For example, either the 3′ or 5′ terminal regions of the sequences in a dsRNA can be phosphorylated or biotinylated. Examples of modifications contemplated for the phosphate backbone include phosphonates, including methylphosphonate, phosphorothioate, and phosphotriester modifications such as alkylphosphotriesters, and the like. Examples of modifications contemplated for the sugar moiety include 2′-alkyl pyrimidine, such as 2′-O-methyl, 2′-fluoro, amino, and deoxy modifications and the like. Examples of modifications contemplated for the base groups include abasic sugars, 2-O-alkyl modified pyrimidines, 4-thiouracil, 5-bromouracil, 5-iodouracil, and 5-(3-aminoallyl)-uracil and the like. Many other modifications are known and can be used so long as the above criteria are satisfied

The double-stranded RNA sample can be suitably formulated and introduced into the environment of the cell by any means that allows for a sufficient portion of the sample to enter the cell to induce gene silencing, if it is to occur. Many formulations for dsRNA are known in the art and can be used so long as dsRNA gains entry to the target cells so that it can act. For example, dsRNA can be formulated in buffer solutions such as phosphate buffered saline solutions, liposomes, micellar structures, and capsids. Formulations of dsRNA with cationic lipids can be used to facilitate transfection of the dsRNA into cells. Suitable lipids include Oligofectamine, Lipofectamine (Life Technologies), NC388 (Ribozyme Pharmaceuticals, Inc., Boulder, Colo.), or FuGene 6 (Roche) all of which can be used according to the manufacturer\'s instructions.

It can be appreciated that the method of introducing dsRNA into the environment of the cell will depend on the type of cell and the make up of its environment. For example, when the cells are found within a liquid, one preferable formulation is with a lipid formulation such as in lipofectamine and the dsRNA can be added directly to the liquid environment of the cells. Lipid formulations can also be administered to animals such as by intravenous, intramuscular, or intraperitoneal injection, or orally or by inhalation or other methods as are known in the art. When the formulation is suitable for administration into animals such as mammals and more specifically humans, the formulation is also pharmaceutically acceptable. Pharmaceutically acceptable formulations for administering oligonucleotides are known and can be used. In some instances, it may be preferable to formulate dsRNA in a buffer or saline solution and directly inject the formulated dsRNA into cells, as in studies with oocytes. The direct injection of dsRNA duplexes

Suitable amounts of dsRNA must be introduced and these amounts can be empirically determined using standard methods. Typically, effective concentrations of individual dsRNA species in the environment of a cell will be about 50 nanomolar or less 10 nanomolar or less, more preferred are compositions in which concentrations of about 1 nanomolar or less can be used. Even more preferred are methods that utilize a concentration of about 200 picomolar or less and even a concentration of about 50 picomolar or less can be used in many circumstances.

The method can be carried out by addition of the dsRNA compositions to any extracellular matrix in which cells can live provided that the dsRNA composition is formulated so that a sufficient amount of the dsRNA can enter the cell to exert its effect. For example, the method is amenable for use with cells present in a liquid such as a liquid culture or cell growth media, in tissue explants, or in whole organisms, including animals, such as mammals and especially humans.



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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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