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Modified polynucleotides for use in rna interferenceRelated Patent Categories: Drug, Bio-affecting And Body Treating Compositions, Designated Organic Active Ingredient Containing (doai), O-glycoside, , Nitrogen Containing Hetero Ring, Polynucleotide (e.g., Rna, Dna, Etc.)Modified polynucleotides for use in rna interference description/claimsThe Patent Description & Claims data below is from USPTO Patent Application 20070173476, Modified polynucleotides for use in rna interference. Brief Patent Description - Full Patent Description - Patent Application Claims
CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This patent application is a continuation of U.S. patent application Ser. No. 10/551,350, which is a nationalization of PCT application PCT/US2004/010343, filed Apr. 1, 2004, which claims benefit of U.S. Provisional Patent Application Ser. Nos. 60/543,640 and 60/543,661, each of which were filed Feb. 10, 2004, and Patent Application Ser. Nos. 60/542,646 and 60/542,668, each of which were filed Feb. 6, 2004, and claims benefit of both U.S. patent application Ser. No. 10/613,077, filed Jul. 1, 2003, and U.S. patent application Ser. No. 10/406,908, filed Apr. 2, 2003, wherein each of the foregoing applications is incorporated herein by reference. BACKGROUND OF THE INVENTION [0002] 1. The Field of the Invention [0003] The present invention relates to the field of modified polynucleotides. [0004] 2. The Relevant Technology [0005] RNA-induced gene silencing in mammalian cells is presently believed to implicate a minimum of three different levels of control: (i) transcription inactivation (siRNA-guided DNA and histone methylation); (ii) small interfering RNA (siRNA)-induced mRNA degradation; and (iii) mRNA-induced transcriptional attenuation. The RNA interference (RNAi) generated by siRNA can be long lasting and effective over multiple cell divisions. Consequently, the ability to assess gene function via siRNA mediated methods, as well as to develop therapies for over-expressed genes, represents an exciting and valuable tool that will accelerate gene function analysis, drug target validation, and genome-wide investigations. Moreover, RNAi has broad potential as a therapeutic tool. [0006] Relatively recent discoveries in the field of RNA metabolism have revealed that the uptake of duplex RNA (dsRNA) can induce RNAi. [0007] In these circumstances, a Type III RNase called Dicer processes the long ds RNA into siRNA that subsequently partner with the RNA Interfering Silencing Complex (RISC) to mediate the degradation of target transcripts in a sequence specific manner. This phenomenon has been observed in a diverse group of organisms. Unfortunately, in mammalian cells, the use of long dsRNA to induce RNAi has been met with only limited success. In large part, this ineffectiveness is due to induction of the interferon response, which results in a general, as opposed to targeted, inhibition of protein synthesis. [0008] Recently, it has been shown that when synthetic siRNAs are introduced into mammalian cells in culture, sequence-specific inhibition of target mRNA can be realized without inducing an interferon response. These short duplexes, can act catalytically at sub-molar concentrations to cleave greater than 95% of the target mRNA in a cell. A description of the mechanisms for siRNA activity, as well as some of its applications is provided in Provost et al., Ribonuclease Activity and RNA Binding of Recombinant Human Dicer, E.M.B.O.J., 2002 Nov., 1, 21(21): 5864-5874; Tabara et al., The dsRNA Binding Protein RDE-4 Interacts with RDE-1, DCR-1 and a DexH-box Helicase to Direct RNAi in C. elegans, Cell. 2002, Jun. 28, 109(7):861-71; Ketting et al., Dicer Functions in RNA Interference and in Synthesis of Small RNA Involved in Developmental Timing in C. elegans; and Martinez et al., Single-Stranded Antisense siRNAs Guide Target RNA Cleavage in RNAi, Cell 2002, Sep. 6, 110(5):563. [0009] There are four main issues that must be addressed when working with siRNA: (i) functionality; (ii) specificity; (iii) delivery methods; and (iv) stability. Functionality refers to the ability of a particular siRNA to silence the desired target. Methods for improving functionality are, for example, the subject of U.S. patent application Ser. No. 10/714,333. Specificity refers to the ability of a particular siRNA to silence a desired target and only the desired target. Thus, specificity refers to minimizing off-target effects. Delivery methods are the means by which a user introduces a particular siRNA into a cell and may, for example, include using vectors or modifications of the siRNA itself. Stabilization refers to the ability of an siRNA to resist degradation by enzymes and other harmful substances that exist in intra-cellular and extra-cellular environments. For example, when naked siRNA molecules are introduced into blood, serum, or serum-containing media, they are not stable and are almost immediately degraded, which reduces or eliminates their effectiveness. [0010] The present invention addresses the second and fourth issues; specificity and stability, by providing modifications to siRNA that can either increase or decrease specificity and/or increase stability. BRIEF SUMMARY OF THE INVENTION [0011] The present invention is directed to compositions and methods for performing RNA interference. In general the siRNA chemical modifications described herein affect two critical properties of the molecules to which they are associated: stability and specificity. Those that affect stability are particularly advantageous for use in applications that require exposure to blood, serum, serum-containing media, and other biological material that contain nucleases or other factors that tend to degrade nucleic acids. Modifications that reduce the level of off-target effects induced by a siRNA directed against a specific target are particularly valuable in research and therapeutic settings. Multiple distinct combinations of modifications that substantially improve RNAi applications are disclosed and are applicable in the design of optimum silencing reagents, transfection controls, and exaequo reagents. [0012] According to a first embodiment, the present invention provides an siRNA having a sense strand comprising a polynucleotide comprised of at least one orthoester modified nucleotide, and an antisense strand comprising a polynucleotide comprised of at least one 2' modified nucleotide unit. [0013] According to a second embodiment, the present invention provides an siRNA having: a sense strand comprising a polynucleotide comprised of at least one orthoester modified nucleotide; an antisense strand comprising a polynucleotide comprised of at least one 2' modified nucleotide; and a conjugate. [0014] According to a third embodiment, the present invention provides an siRNA having: a sense strand comprising at least one orthoester modified nucleotide; an antisense strand; and a conjugate. [0015] According to a fourth embodiment, the present invention provides an siRNA having: a sense strand; an antisense strand; and a conjugate, wherein the sense strand and/or the antisense strand have at least one 2' modified nucleotide. [0016] According to a fifth embodiment, the present invention provides an siRNA having a sense strand comprising at least one orthoester modified nucleotide, an antisense strand comprising at least one 2' modified nucleotide selected from the group consisting of a 2' halogen modified nucleotide, a 2' amine modified nucleotide, a 2'-O-alkyl modified nucleotide, and a 2' alkyl modified nucleotide, and a conjugate selected from the group consisting of amino acids, peptides, polypeptides, proteins, sugars, carbohydrates, lipids, polymers, nucleotides, polynucleotides, and combinations thereof, wherein the polyribonucleotide comprises between 18 and 30 nucleotide base pairs. [0017] According to a sixth embodiment, the present invention provides a composition comprising one of the structures below: [0018] In the structure, each of B1 and B2 is a nitrogenous base, carbocycle, or heterocycle; X is selected from the group consisting of O, S, C, and N; W is selected from the group consisting of an OH, a phosphate, a phosphate ester, a phosphodiester, a phosphotriester, a modified internucleotide linkage, a conjugate, a nucleotide, and a polynucleotide; R1 is an orthoester; R2 is selected from the group consisting of a 2'-O-alkyl group, an alkyl group, an amine and a halogen; and Y is a nucleotide or polynucleotide. The dashed lines between B1 and B2 indicate interaction by hydrogen bonding between nitrogenous bases. [0019] According to a seventh embodiment, the present invention provides a method of performing RNA interference. This method is comprised of exposing an siRNA to a target nucleic acid. The siRNA is comprised of a sense strand and an antisense strand, and at least one of said sense strand and said antisense strand comprises at least one orthoester modified nucleotide. [0020] According to an eighth embodiment, the present invention provides another method of performing RNA interference. This method is comprised of exposing an siRNA to a target nucleic acid, wherein the siRNA is comprised of a sense strand, an antisense strand, and a conjugate. According to this embodiment, either the sense strand or the antisense strand comprises a 2' modified nucleotide. [0021] The compositions of the first through eighth embodiments of the present invention can render siRNAs resistant to nuclease degradation, while maintaining biological functionality. By, for example, using siRNAs with at least one orthoester modified nucleotide, such as on the sense strand, and at least one other modification, such as at an appropriate position on the antisense strand, one can enhance stability while retaining functionality in RNA interference applications. Additionally, using siRNAs with one or more 2' modifications, and/or modified internucleotide linkages, in conjunction with conjugates, in RNA interference applications, can also provide enhanced stability while retaining functionality, even in the absence of an orthoester modification on either strand. Continue reading about Modified polynucleotides for use in rna interference... 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