| Methods and compositions for enhancing the efficacy and specificity of single and double blunt-ended sirna -> Monitor Keywords |
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Methods and compositions for enhancing the efficacy and specificity of single and double blunt-ended sirnaRelated Patent Categories: Chemistry: Molecular Biology And Microbiology, Process Of Mutation, Cell Fusion, Or Genetic Modification, Introduction Of A Polynucleotide Molecule Into Or Rearrangement Of Nucleic Acid Within An Animal CellMethods and compositions for enhancing the efficacy and specificity of single and double blunt-ended sirna description/claimsThe Patent Description & Claims data below is from USPTO Patent Application 20060134787, Methods and compositions for enhancing the efficacy and specificity of single and double blunt-ended sirna. Brief Patent Description - Full Patent Description - Patent Application Claims
RELATED APPLICATIONS [0001] This patent application claims the benefit of U.S. Provisional Patent Application Ser. No. 60/532,116, entitled "Methods and Compositions for Enhancing the Efficacy and Specificity of Single and Double Blunt-Ended siRNA", filed Dec. 22, 2003. The entire contents of the above-referenced provisional patent application are incorporated herein by this reference. RELATED INFORMATION [0002] The contents of any patents, patent applications, and references cited throughout this specification are hereby incorporated by reference in their entireties. BACKGROUND OF THE INVENTION [0003] Small interfering RNAs (siRNAs) are produced by the cleavage of double-stranded RNA (dsRNA) precursors by Dicer, a member of the RNase III family of dsRNA-specific endonucleases. Typically, siRNAs result when transposons, viruses, or endogenous genes express long dsRNA or when dsRNA is introduced experimentally into plant or animal cells to trigger gene silencing, a process known as RNA interference (RNAi). [0004] siRNAs were first identified as the specificity determinants of the RNA interference (RNAi) pathway, where they act as guides to direct endonucleolytic cleavage of their target RNAs. Prototypical siRNA duplexes are 21 nucleotide, double-stranded RNAs that contain 19 base pairs, with two-nucleotide, 3' overhanging ends. Active siRNAs contain 5' phosphates and 3' hydroxyls. [0005] siRNAs are typically found in the RNA-induced silencing complex (RISC) that mediates both cleavage and translational control. siRNA duplexes can assemble into RISC in the absence of target mRNA, both in vivo and in vitro. Each RISC contains only one of the two strands of the siRNA duplex. Since siRNA duplexes have no foreknowledge of which siRNA strand will guide target cleavage, both strands must assemble with the appropriate proteins to form a RISC. [0006] It has been observed that both siRNA strands are competent to direct RNAi (Tuschl et al., Genes Dev 13, 3191-3197 (1999); Hammond et al., Nature 404, 293-296 (2000); Zamore et al., Cell 101, 25-33 (2000); Elbashir et al., Genes Dev 15, 188-200 (2001); Elbashir et al., EMBO J 20, 6877-6888 (2001); Nykanen et al., Cell 107, 309-321 (2001). That is, the antisense strand of an siRNA can direct cleavage of a corresponding sense RNA target, whereas the sense siRNA strand directs cleavage of an antisense target. In this way, siRNA duplexes appear to be functionally symmetric. [0007] The ability to control which strand of an siRNA duplex enters into the RISC complex to direct cleavage of a corresponding RNA target would provide a significant advance for both research and therapeutic applications of RNAi technology. SUMMARY OF THE INVENTION [0008] The invention solves the foregoing problems of siRNA gene targeting by determining the structural and functional characteristics of single and blunt-ended siRNAs and in particular, their strand specificity for a gene target. Accordingly, an entirely new constellation of single and double blunt-ended siRNA agents, e.g., siRNA duplexes, can be designed to efficiently and specifically modulate a sense and/or antisense gene target. [0009] In addition, the invention provides a method for introducing alterations in either the 5', 3', or both the 5' and 3' of a single or double blunt-ended siRNA such that either the sense, the antisense, or both the sense and antisense strand will enter the RNAi pathway (e.g., RISC) and target a cognate gene target(s) for cleavage and destruction. Typically, the alteration takes the form of a mismatched base pair that allows for a portion of the siRNA duplex, e.g., the 5' end of the antisense strand, to separate or fray. [0010] Accordingly, the invention has several advantages which include, but are not limited to, the following: [0011] providing methods for designing single and double blunt-ended siRNA agents, e.g., siRNA duplexes, have a characteristic strand specificity; [0012] providing single and double blunt-ended siRNA agents, e.g., siRNA duplexes or small hairpin RNAs (shRNAs) with at least one blunt end, suitable for gene modulation in plant or animal cells; and [0013] methods for modulating gene expression in a subject in need thereof using the single or double blunt-ended siRNA compositions of the invention, e.g., in the form of a pharmaceutical composition suitable for administering to a patient. [0014] Accordingly, in one aspect, the invention provides methods for improving the efficiency (or specificity) of an RNAi reaction comprising modifying (e.g., increasing) the asymmetry of an RNAi agent (i.e., an RNA duplex having at least one blunt end) such that the ability of the sense or second strand to mediate RNAi (e.g., mediate cleavage of a target RNA) is lessened. [0015] In one embodiment, the asymmetry is increased in favor of the 5' end of the first strand, e.g., by lessening the bond strength (e.g., the strength of the interaction) between the 5' end of the first strand and 3' end of the second strand relative to the bond strength (e.g., the strength of the interaction) between the 5' end of the second strand and the 3' end of the first strand. [0016] In another embodiment, the asymmetry is increased in favor of the 5' end of the first strand by increasing bond strength (e.g., the strength of the interaction) between the 5' end of the second or sense strand and the 3' end of the first or antisense strand, relative to the bond strength (e.g., the strength of the interaction) between the 5' end of the first and the 3' end of the second strand. [0017] In another embodiment, the bond strength is increased, e.g., the hydrogen bonding is increased between nucleotides or analogs at the 5' end, e.g., within 5 nucleotides of the second or sense strand (numbered from the 5' end of the second strand) and complementary nucleotides of the first or antisense strand. It is understood that the asymmetry can be zero (i.e., no asymmetry), for example, when the bonds or base pairs between the 5' and 3' terminal bases are of the same nature, strength or structure. More routinely, however, there exists some asymmetry due to the different nature, strength or structure of at least one nucleotide (often one or more nucleotides) between terminal nucleotides or nucleotide analogs. [0018] Accordingly, in one aspect, the instant invention provides a method of enhancing the ability of a first strand of a single or double blunt-ended RNAi agent to act as a guide strand in mediating RNAi, involving lessening the base pair strength between the 5' end of the first strand and the 3' end of a second strand of the duplex as compared to the base pair strength between the 3' end of the first strand and the 5' end of the second strand. [0019] In a related aspect, the invention provides a method of enhancing the efficacy of a single or double blunt-ended siRNA duplex, the siRNA duplex comprising a sense and an antisense strand, involving lessening the base pair strength between the antisense strand 5' end (AS 5') and the sense strand 3' end (S 3') as compared to the base pair strength between the antisense strand 3' end (AS 3') and the sense strand 5' end (S '5), such that efficacy is enhanced. [0020] In another aspect of the invention, a method is provided for promoting entry of a desired strand of an single or double blunt-ended siRNA duplex into a RISC complex, comprising enhancing the asymmetry of the single or double blunt-ended siRNA duplex, such that entry of the desired strand is promoted. In one embodiment of this aspect of the invention, the asymmetry is enhanced by lessening the base pair strength between the 5' end of the desired strand and the 3' end of a complementary strand of the duplex as compared to the base pair strength between the 3' end of the desired strand and the 5' end of the complementary strand. [0021] In another aspect of the invention, a single or double blunt-ended siRNA duplex is provided comprising a sense strand and an antisense strand, wherein the base pair strength between the antisense strand 5' end (AS 5') and the sense strand 3' end (S 3') is less than the base pair strength between the antisense strand 3' end (AS 3') and the sense strand 5' end (S '5), such that the antisense strand preferentially guides cleavage of a target mRNA. [0022] In one embodiment of these aspects of the invention, the base-pair strength is less due to fewer G:C base pairs between the 5' end of the first or antisense strand and the 3' end of the second or sense strand than between the 3' end of the first or antisense strand and the 5' end of the second or sense strand. [0023] In another embodiment, the base pair strength is less due to at least one mismatched base pair between the 5' end of the first or antisense strand and the 3' end of the second or sense strand. Preferably, the mismatched or wobble base pair is selected from the group consisting of G:A, C:A, C:U, G:G, A:A, C:C, U:U, I:A, I:U, and I:C. Continue reading about Methods and compositions for enhancing the efficacy and specificity of single and double blunt-ended sirna... 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