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  Interfering rna duplex having blunt-ends and 3'-modificationsUSPTO Application #: 20070203084Title: Interfering rna duplex having blunt-ends and 3'-modifications Abstract: The present invention relates to double-stranded RNA compounds with at least one blunt end comprising at least one 3′-end of Formula (I): wherein X is O or S R1 and R2 are independently OH, NH2, SH, alkyl, aryl, alkyl-aryl, aryl-alkyl, where alkyl, aryl, alkyl-aryl, aryl-alkyl can be substituted by additional heteroatoms and functional groups, preferably a heteroatom selected from the group of N, O, or S or a functional group selected from the group OH, NH2, SH, carboxylic acid or ester; or R1 and R2 may be of formula Y-Z where Y is O, N, S and Z is H, alkyl, aryl, alkyl-aryl, aryl-alkyl, where alkyl, aryl, alkyl-aryl, aryl-alkyl can be substituted by additional heteroatoms, preferably a heteroatom selected from the group of N, O, or S; and wherein said double-stranded RNA mediates RNA interference. Preferred 3′end modifications are: phosphate, phosphorothioate, abasic ribonucleoside, hydroxyphopyl phosphodiester. (end of abstract)
Agent: Novartis Corporate Intellectual Property - East Hanover, NJ, US Inventors: Jan Weiler, Jonathan Hall, Jean-Charles Bologna, Francois Jean-Charles Natt, Robert Haner USPTO Applicaton #: 20070203084 - Class: 514044000 (USPTO) Related 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.) The Patent Description & Claims data below is from USPTO Patent Application 20070203084. Brief Patent Description - Full Patent Description - Patent Application Claims
FIELD OF THE INVENTION [0001] The invention relates to selective inhibition of target genes using double-stranded RNA and provides compounds useful for this purpose. BACKGROUND OF THE INVENTION [0002] Short RNA duplexes have been shown to be the effective guides that mediate RNA interference in many in vitro and in vivo models (Hamilton et al. 1999, Zamore et al., 2000, Caplen et al., 2001, Elbashir et al., 2001, Yang et al., 2000). [0003] Most commonly, synthetic siRNA duplexes are designed such as a stretch of 19 contiguous ribonucleotide base-pairs is flanked with 2-3 unpaired nucleotides at the 3'-end of each strand ("overhangs"). This 21-nt siRNA species has been found to be generated during DICER-mediated cleavage of long ds-RNA in mammalian and non-mammalian systems (Bernstein et al., 2001, Ketting et al., 2001). This particular 21-mer siRNA format has been firstly selected from a drosophila melanogaster model and then highlighted regarding its efficiency (Elbashir et al. 2001). Consequently, the major part of today's studies applying synthetic siRNAs as gene inhibitors is relying on this "wildtype" 21-mer siRNA derivative. For the overhangs usually 2'-deoxynucleotides are used, notably for cost reasons but also with regard to a potential protection against intracellular nuclease activity. [0004] The present invention now provides a new and inventive format for double-stranded RNA ("dsRNA") mediating RNAi. The blunt-ended siRNAs in accordance with the present invention overcome disadvantages of the synthetic siRNAs with 3'-overhangs which are currently used in the art. SUMMARY OF THE INVENTION [0005] The present invention provides double-stranded RNA with at least one blunt end comprising at least one 3'-end of formula: wherein [0006] X is O or S [0007] R.sub.1 and R.sub.2 are independently OH, NH.sub.2, SH, alkyl, aryl, alkyl-aryl, aryl-alkyl, where alkyl, aryl, alkyl-aryl, aryl-alkyl can be substituted by additional heteroatoms and functional groups, preferably a heteroatom selected from the group of N, O, or S or a functional group selected from the group OH, NH.sub.2, SH, carboxylic acid or ester; [0008] Also, R.sub.1 and R.sub.2 may be of formula Y-Z where Y is O, N, S and Z is H, alkyl, aryl, alkyl-aryl, aryl-alkyl, where alkyl, aryl, alkyl-aryl, aryl-alkyl can be substituted by additional heteroatoms, preferably a heteroatom selected from the group of N, O, or S; and wherein said double-stranded RNA mediates RNA interference. DETAILED DESCRIPTION OF THE INVENTION [0009] The present invention is based on the surprising finding that synthetic double-stranded RNA (dsRNA) molecules with at least one blunt end comprising a certain type of chemical modification efficiently mediate RNA interference. The dsRNAs according to the present invention are particularly useful for high-throughput approaches using siRNAs due to their simplified synthetic procedure, such as for instance the use of a universal solid support. [0010] In one aspect, the present invention relates to double-stranded RNA with at least one blunt end comprising at least one 3'-end of the formula: wherein [0011] X is O or S [0012] R.sub.1 and R.sub.2 are independently OH, NH.sub.2, SH, alkyl, aryl, alkyl-aryl, aryl-alkyl, where alkyl, aryl, alkyl-aryl, aryl-alkyl can be substituted by additional heteroatoms and functional groups, preferably a heteroatom selected from the group of N, O, or S or a functional group selected from the group OH, NH.sub.2, SH, carboxylic acid or ester; [0013] Also, R.sub.1 and R.sub.2 may be of formula Y-Z where Y is O, N, S and Z is H, alkyl, aryl, alkyl-aryl, aryl-alkyl, where alkyl, aryl, alkyl-aryl, aryl-alkyl can be substituted by additional heteroatoms, preferably a heteroatom selected from the group of N, O, or S; [0014] and wherein said double-stranded RNA mediates RNA interference. [0015] R.sub.1 and R.sub.2 may also form a cyclic structure, e.g. a carbocyclic or heterocyclic ring, the ring structure preferably having from 3 to 7 members. [0016] In a preferred embodiment, Z is one or more abasic nucleoside, preferable ribonucleoside, moieties. The nucleoside moieties may be linked for instance by a phosphodiester or a phosphorothioate group. [0017] In another preferred embodiment, R.sub.1 is OH. In another preferred embodiment, R.sub.1 and R.sub.2 together comprise from 1 to 24 C-atoms more preferably from 1 to 12, or from 2 to 10 and most preferably from 1 to 8 or from 2 to 6. In another preferred embodiment, R.sub.1 and R.sub.2 are independently OH, lower alkyl, lower aryl, lower alkyl-aryl, lower aryl-alkyl, where lower alkyl, lower aryl, lower alkyl-aryl, lower aryl-alkyl can be substituted by additional heteroatoms and functional groups as defined above. In another preferred embodiment, R.sub.1 and R.sub.2 are not both OH. [0018] The term "lower" in connection with organic radicals or compounds means a compound or radical which may be branched or unbranched with up to and including 7 carbon atoms, preferably 1-4 carbon atoms. Lower alkyl represents, for example, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, n-pentyl and branched pentyl, n-hexyl and branched hexyl. [0019] In related aspect, the present invention relates to double-stranded RNA with at least one blunt end comprising at least one 3'-end of the formula: wherein R.sub.3 is OH, NH.sub.2, SH, alkyl, aryl, alkyl-aryl, aryl-alkyl, where alkyl, aryl, alkyl-aryl, aryl-alkyl can be substituted by additional heteroatoms, preferably a heteroatom selected from the group of N, O, or S, and R.sub.4 is independently alkyl, aryl, alkyl-aryl, aryl-alkyl, where alkyl, aryl, alkyl-aryl, aryl-alkyl can be substituted by additional heteroatoms, preferably a heteroatom selected from the group of N, O, or S or a functional group selected from the group OH, NH.sub.2, SH, carboxylic acid or ester, and wherein said double-stranded RNA mediates RNA interference. R.sub.3 and R.sub.4 may also form a cyclic structure, e.g. a carbocyclic or heterocyclic ring, the ring structure preferably having from 3 to 7 members. [0020] R.sub.3 and R.sub.4 may further comprise additional heteroatoms, preferably a heteroatom selected from the group of N, O, or S. [0021] In preferred embodiment, R.sub.3 is OH. In another preferred embodiment, R.sub.3 and R.sub.4, respectively, together comprise from 1 to 24 C-atoms more preferably from 1 to 12, or from 2 to 10 and most preferably from 1 to 8 or from 2 to 6. In another preferred embodiment, R.sub.3 is lower alkyl, lower aryl, lower alkyl-aryl, lower aryl-alkyl, where lower alkyl, lower aryl, lower alkyl-aryl, lower aryl-alkyl can be substituted by additional heteroatoms and functional groups as defined above and R.sub.4 is, independently lower alkyl, lower aryl, lower alkyl-aryl, lower aryl-alkyl, where lower alkyl, lower aryl, lower alkyl-aryl, lower aryl-alkyl can be substituted by additional heteroatoms and functional groups as defined above. [0022] In a related aspect, the present invention relates to double-stranded RNA with at least one blunt end comprising at least one 3'-end of the formula: wherein R.sub.5 and R.sub.6 are the same or different and are H, alkyl, aryl, alkyl-aryl, aryl-alkyl, where alkyl, aryl, alkyl-aryl, aryl-alkyl can be substituted by additional heteroatoms, preferably a heteroatom selected from the group of N, O or S, or a functional group selected from the group OH, NH.sub.2, SH, carboxylic acid or ester wherein said double-stranded RNA mediates RNA interference. R.sub.5 and R.sub.6 may also form a cyclic structure, e.g. a carbocyclic or heterocyclic ring, the ring structure preferably having from 3 to 7 members. [0023] R.sub.5 and R.sub.6 may further comprise additional heteroatoms, preferably a heteroatom selected from the group of N, O or S. [0024] In preferred embodiment, R.sub.5 and R.sub.6, respectively, together comprise from 1 to 24 C-atoms more preferably from 1 to 12 or from 2 to 10 and most preferably from 1 to 6 or from 2 to 6. In another preferred embodiment, R.sub.5 and R.sub.6 are independently lower alkyl, lower aryl, lower alkyl-aryl, lower aryl-alkyl, where lower alkyl, lower aryl, lower alkyl-aryl, lower aryl-alkyl can be substituted by additional heteroatoms and functional groups as defined above. In another embodiment R.sub.5 and R.sub.6 are not both H. [0025] The RNA molecules of the present invention will have at least at least one strand comprising a 3'-ends of the formula I, II or III. Preferably, both strands of the double-stranded RNA comprise 3'-ends comprising a group of the formula I, II or III. The RNA molecules of the present invention will further have at least one blunt end, preferably two blunt ends. In a particularly preferred embodiment, the RNA molecules of the present invention are blunt ended on both sides and both ends comprise 3'-ends of the formula I, II or III. [0026] The RNA molecules in accordance with the present invention will have at least a partially double-stranded character. In a preferred embodiment, they are fully double-stranded. They may be composed of two separate strands, but may also be composed of one strand forming a hairpin loop. In a particularly preferred embodiment, the RNA molecules of the present invention are composed of two separate strands which are fully double-stranded comprising at least one, preferably two, blunt ends. [0027] The RNA molecules according to the present invention mediate RNA interference ("RNAi"). The term "RNAi" is well known in the art and is commonly understood to mean the inhibition of one or more target genes in a cell by dsRNA with a region which is complementary to the target gene. Various assays are known in the art to test dsRNA for its ability to mediate RNAi (see for instance Elbashir et al., Methods 26 (2002), 199-213). The effect of the dsRNA according to the present invention on gene expression will typically result in expression of the target gene being inhibited by at least 10%, 33%, 50%, 90%, 95% or 99% when compared to a cell not treated with the RNA molecules according to the present invention. [0028] The RNA molecules in accordance with the present invention comprise a double-stranded region which is substantially identical to a region of the mRNA of the target gene. Particularly preferred is a region with 100% identity to the corresponding sequence of the target gene. However, the region may also contain one or two mismatches as compared to the corresponding region of the target gene. The present invention includes RNA molecules which target more than one gene. In a preferred embodiment, the RNA molecules of the present invention specifically target one given gene. In order to only target the desired mRNA, the siRNA reagent should have 100% homology to the target mRNA and at least 2 mismatched nucleotides to all other genes present in the cell or organism. Methods to analyze and identify dsRNAs with sufficient sequence identity in order to effectively inhibit expression of a specific target sequence are known in the art. Sequence identity may be optimized by sequence comparison and alignment algorithms known in the art (see Gribskov and Devereux, Sequence Analysis Primer, Stockton Press, 1991, and references cited therein) and calculating the percent difference between the nucleotide sequences by, for example, the Smith-Waterman algorithm as implemented in the BESTFIT software program using default parameters (e.g., University of Wisconsin Genetic Computing Group). Another factor affecting the efficiency of the RNAi reagent is the target region of the target gene. The region of a target gene effective for inhibition by the RNAi reagent may be determined by experimentation. Most preferred mRNA target region would be the coding region. Also preferred are untranslated regions, particularly the 3'-UTR, splice junctions. For instance, transfection assays as described in Elbashir S. M. et al, 2001 EMBO J., 20, 6877-6888 may be performed for this purpose. A number of other suitable assays and methods exist in the art which are well known to a person skilled in the art. [0029] The length of the complementary region of the RNA molecules in accordance with the present invention is preferably from 10 to 100 nucleotides, more preferably 15 to 50 nucleotides, even more preferably 17 to 30 nucleotides and most preferably 19 to 25 nucleotides. In a particularly preferred embodiment the RNA molecules in accordance with the present invention consist of short dsRNA molecules having a length from 15 to 50 nucleotides, more preferably 17 to 30 nucleotides and most preferably 19 to 25 nucleotides. Continue reading... Full patent description for Interfering rna duplex having blunt-ends and 3'-modifications Brief Patent Description - Full Patent Description - Patent Application Claims Click on the above for other options relating to this Interfering rna duplex having blunt-ends and 3'-modifications patent application. ###  How KEYWORD MONITOR works... a FREE service from FreshPatents1. Sign up (takes 30 seconds). 2. Fill in the keywords to be monitored. 3. Each week you receive an email with patent applications related to your keywords. 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