PCT/EP O 3 I O 8 6 6 6
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`REC'D 2 0 OCT 2003
`
`WIPO
`
`PCT
`
`UNITED STA~ DEPARTMENT OF COMMERCE
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`United States Patent and Trademark Office
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`September 04, 2003
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`THIS IS A REQUEST FOR FILING A PROVISIONAL APPLICATION FOR PATENT UNDER 37 C.F.R. § 1.53(C). ~c:::> i:;;'j
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`INVENTOR(S)/APPLICANT(S)
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`KAUFMANN
`Berlin, Germany
`GIESE
`Berlin, Germany
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`Given Name (firsl and m!ddle (if any))
`Jorg
`Klaus
`
`Additional inventors are being named on page 2 attached hereto.
`TITLE OF THE INVENTION (280 characters max)
`Novel Forms of Interfering RNA Molecules
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`)
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`Paga 1 of 1
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`ii
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`

`

`·,
`
`atugen AG
`A 19005 EP
`
`I
`Novel forms of interfering RNA molecules
`
`The present invention is related to a ribonucleic acid comprising a double-stranded structure
`
`whereby the double-stranded structure comprises a first strand and a second strand, whereby
`
`the first strand comprises a first stretch of contiguous nucleotides and whereby said first
`
`stretch is at least partially complementary to the target nucleic acid, and the second strand
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`comprises a second stretch of contiguous nucleotides whereby said second stretch is at least
`
`partially identical to a target nucleic acid, the use of such ribonucleic acid, a cell and an
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`organism, respectively, comprising such ribonucleic acid, a composition containing such
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`ribonucleic acid, a pharmaceutical composition containing such ribonucleic acid and a method
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`for inhibiting expression of a targeted gene.
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`RNA-mediated interference (RNAi) is a post-transcriptional gene silencing mechanism
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`initiated by double stranded RNA {dsRNA) homologous in sequence to the silenced gene
`
`(Fire {1999), Trends Genet 15, 358-63, Tuschl, et al. {1999), Genes Dev 13, 3191-7, ,
`
`Waterhouse, et al. (2001), Nature 411, 834-42, Elbashir, et al. (2001), Nature 411, 494-8, for
`'
`review see Sharp (2001), Genes Dev 15, 485-90, Barstead (2001), Curr Opin Chem Biol 5,
`
`63-6). RNAi has been used extensively to determine gene function in a number of organisms,
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`including plants (Baulcombe (1999), Curr Opin Plant Biol 2, 109-13), nematodes
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`(Montgomery, et al. (1998), Proc Natl Acad Sci US A 95, 15502-7), Drosophila (Kennerdell,
`
`et al. (1998), Cell 95, 1017-26, Kennerdell. et al. (2000), Nat Biotechnol 18, 896-8). In the
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`nematode C. elegans about one third of the genome has already been subjected to functional
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`analysis by RNAi (Kim (2001), Curr Biol 11, R85-7, Maeda, et al. (2001), Curr Biol 11, 171-
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`6).
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`Until recently RNAi in mammalian cells was not generally applicable, with the exception of
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`early mouse development (Wianny, et al. (2000), Nat Cell Biol 2, 70-5). The discovery that
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`transfection of duplexes of 21-nt into mammalian cells interfered with gene expression and
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`did not induce a sequence independent interferon-driven anti-viral response usually obtained
`
`with long dsRNA led to new potential application in differentiated mammalian cells (Elbashir
`
`Co~y provided by USPTO from lhe PACR Image, Database 0~ 008/29/2003
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`(cid:20)(cid:3)(cid:82)(cid:73)(cid:3)(cid:25)(cid:27)
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`2
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`et al. (2001), Nature 411, 494-8). Interestingly these small interfering RNAs (siRNAs )
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`resemble· the processing products from long dsRNAs suggesting a potential bypassing
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`mechanism in differentiated mammalian cells. The Dicer complex, a member of the RNAse
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`III family, necessary for the initial dsRNA processing has been identified (Bernstein, et al.
`
`(2001), Nature 409, 363-6, Billy, et al. (2001), Proc Natl Acad Sci US A 98, 14428-33). One
`
`of the problems previously encountered when using unmodified ribooligonucleotides was the
`rapid degradation in cells or even in the serum-containing medium (Wickstrom (1986), J
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`Biochem Biophys Methods 13, 97-102, Cazenave, et al. (1987), Nucleic Acids Res 15,
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`10507-21). It will depend on the particular gene function and assay systems used whether the
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`respective knock down induced by transfected siRNA will be maintained long enough to
`achieve a phenotypic change.
`
`The problem underlying the present invention was to provide synthetic interfering RNA
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`molecules which are both stable and active in·a biochemical environment such as a living cell.
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`In a first aspect of the present invention the problem is solved by a ribonucleic acid
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`comprising a double stranded structure whereby the double- stranded structure comprises a
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`first strand and a second strand, whereby the first strand comprises a first stretch of
`contiguous nucleotides and whereby said first stretch is at least partially complementary to a
`
`target nucleic acid, and the second strand comprises a second stretch of contiguous
`
`nucleotides whereby said second stretch is at least partially identical to a target nucleic acid,
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`and whereby the double stranded structure is blunt ended.
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`~ a second aspect the problem underlying the present invention is solved by a ribonucleic
`acid comprising a double stranded structure whereby the double- stranded structure comprises
`
`a first strand and a second strand, whereby the first strand comprises a first stretch of
`
`contiguous nucleotides and whereby said first stretch is at least partially complementary to a
`
`target nucleic acid, and the second strand comprises a second stretch of contiguous
`nucleotides, whereby said second stretch is at least partially identical to a target nucleic acid,
`
`whereby the first stretch and/or the second stretch have a length of 18 or 19 nucleotides.
`
`In an embodiment of the ribonucleic acid according to the first aspect of the invention the first
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`stretch and/or the second stretch have a length of 18 or 19 nucleotides.
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`Copy provid!ld by USPTO from the PACR Image Database on 08/29/20'03
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`(cid:21)(cid:3)(cid:82)(cid:73)(cid:3)(cid:25)(cid:27)
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`3
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`In a further embodiment of the ribonucleic acid according to the first aspect of the invention
`the double stranded structure is blunt ended on both sides of the double strand.
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`In an alternative embodiment of the ribonucleic acid according to the first aspect of the
`
`invention the double stranded sbucture is blunt ended on the double stranded structure which
`
`js defined by the 5 '-end of the first strand and the 3 '-end of the second strand.
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`In a further alternative embodiment of the ribonucleic acid according to the first aspect of the
`
`invention the double stranded structure is blunt ended on the double stranded structure which
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`is defined by the 3'-end of the first strand and the 5'-end of the second strand.
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`In a third aspect the problem underlying the present invention is solved by a ribonucleic acid
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`comprising a double stranded structure whereby the double- stranded structure comprises a
`
`first strand and a second strand, whereby the first strand comprises a first stretch of
`
`contiguous nucleotides and whereby said first stretch is at least partially complementary to a
`
`target nucleic acid, and the second strand comprises a second stretch of contiguous
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`nucleotides and whereby said second stretch is at least partially identical to a target nucleic
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`acid, and whereby at least one of the two strands has an overhang of at least one nucleotide at
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`the 5'-end.
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`In an embodiment of the ribonucleic acid according to the third aspect of the present invention
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`the overhang consists of at least one nucleotide which is selected from the group comprising
`
`ribonucleotides and desoxyribonucleotides.
`
`In a more preferred embodiment of the ribonucleic acid according to the third aspect of the
`present invention the nucleotide has a modification whereby said modification is preferably
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`selected from the group comprising nucleotides being an inverted abasic and nucleotides
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`having an NHi-modification at the 2'-position.
`
`In a preferred embodiment of the ribonucleic acid according to the third aspect of the present
`
`invention at least one of the strands has an overhang of at least one nucleotide at the 3 '-end
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`consisting of ribonucleotide or deoxyribonucleotide.
`
`Copy provided bv USPTO from the PACR Image Oaiaba.se ori 08/2912003
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`(cid:22)(cid:3)(cid:82)(cid:73)(cid:3)(cid:25)(cid:27)
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`...
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`4
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`In another preferred embodiment of the ribonucleic acid according to the third aspect of the
`
`present invention the first stretch and/or the second stretch have a length of 18 or 19
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`nucleotides.
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`In an embodiment of the ribonucleic acid according to any aspect of the present invention the
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`double-stranded structure has a length of 17 to 21 nucleotides, preferably 18 to 19 nucleotides
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`In an embodiment of the ribonucleic acid according to the third aspect of the present invention
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`the overhang at the 5 '-end is on the second strand.
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`In a preferred embodiment of the ribonucleic acid according to the third aspect of the present
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`invention' the first strand comprises also an overhang, preferably at the 5 '-end.
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`In an embodiment of the ribonucleic acid according to the third aspect of the present invention
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`the 3 • -end of the first strand comprises an overhang.
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`In an alternative embodiment of the ribonucleic acid according to the third aspect of the
`
`present invention the overhang at the 5 '-end is on the first strand.
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`In a preferred embodiment thereof the second strand also comprise an overhang, preferably at
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`the 5'-end.
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`In an embodiment of the ribonucleic acid according to the third aspect of the present invention
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`the 3 • -end of the first strand comprises an overhang.
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`In an embodiment of the ribonucleic acid according to any aspect of the present invention at
`least one nucleotide of the ribonucleic acid has a modification at the 2' -position and the
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`modification is preferably selected from the group comprising amino, fluoro, methoxy and
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`alkyl.
`
`In a fourth aspect the problem underlying the present invention is solved by a ribonucleic acid
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`comprising a double stranded structure, whereby the double- stranded structure comprises a
`
`first strand and a second strand, whereby the first strand comprises a first stretch of
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`contiguous nucleotides and whereby said first stretch is at least partially complementary to a
`
`Copy provided by USPTO from the PACR Image Database on 08/29/2003
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`(cid:23)(cid:3)(cid:82)(cid:73)(cid:3)(cid:25)(cid:27)
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`5
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`target nucleic acid, and the second strand comprises a second stretch of contiguous
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`nucleotides and whereby said second stretch is at least partially identical to a target nucleic
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`acid,
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`whereby
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`said first strand and/or said second strand comprises a plurality of groups of modified
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`nucleotides having a modification at the 2'-position whereby within the strand each group of
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`modified nucleotides is flanked on one or both sides by a flanking group of nucleotides
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`whereby the flanking nucleotides forming the flanking group of nucleotides is either an
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`unmodified nucleotide or a nucleotide having a modification different from the modification
`
`of the modified nucleotides.
`
`In an embodiment of the ribonucleic acid according to the fourth aspect of the present
`
`invention the ribonucleic acid is the ribonucleic acid according to the first, second or third
`
`aspect of the present invention.
`
`In a further embodiment of the ribonucleic acid according to the fourth aspect of the present
`
`invention said first strand and/or said second strand comprise said plurality of modified
`
`nucleotides.
`
`In another embodiment of the ribonucleic acid according to the fourth aspect of the present
`
`invention said first strand comprises said plurality of groups of modified nucleotides.
`
`In yet another embodiment of the ribonucleic acid according to the fourth aspect of the
`
`present invention said second strand comprises said plurality of groups of modified
`
`nucleotides.
`
`In an preferred embodiment of the ribonucleic acid according to the fourth aspect of the
`
`present invention the group of nucleotides and/or the group of flanking nucleotides comprises
`
`a number of nucleotides whereby the number is selected from the group comprising one
`
`nucleotide to 10 nucleotides.
`
`Copy provided by USPTO from the PACR lmaqe Database on 08/29/2003
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`(cid:24)(cid:3)(cid:82)(cid:73)(cid:3)(cid:25)(cid:27)
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`6
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`In another embodiment of the ribonucleic acid according to the fourth aspect of the present
`
`invention the pattern of modified nucleotides of said first strand is the same as the pattern of
`modified nucleotides of said second strand.
`
`In a preferred embodiment of the ribonucleic acid according to the fourth aspect of the present
`
`invention the pattern of said first strand aligns with the pattern of said second strand.
`
`In an alternative embodiment of the ribonucleic acid according to the fourth aspect of the
`
`present invention the pattern of said first strand is shifted by one or more nucleotides relative
`
`to the pattern of the second strand.
`
`In an embodiment of the ribonucleic acid according to the fourth aspect of the present
`invention the modification is selected from the group comprising amino, fluoro, methoxy and
`
`alkyl.
`
`In another embodiment of the ribonucleic acid according to the fourth aspect of the present
`invention the double stranded structure is blunt ended.
`
`In an preferred embodiment of the ribonucleic acid according to the fourth aspect of the
`
`present invention the double stranded structure is blunt ended on both sides.
`
`In another embodiment of the ribonucleic acid according to the fourth aspect of the present
`
`invention the double stranded structure is blunt ended on the double stranded structure's side
`
`which is defined by the 5 '-end of the first strand and the 3 '-end of the second strand.
`
`In still another embodiment of the ribonucleic acid according to the fourth aspect of the
`
`present invention the double stranded structure is blunt ended on the double stranded
`
`structure's side which is defined by at the 3'-end of the first strand and the 5'-end of the
`
`second strand.
`
`In another embodiment of the ribonucleic acid according to the fourth aspect of the present
`invention at least one of the two strands has an overhang of at least one nucleotide at the 5'(cid:173)
`
`end.
`
`)
`
`Copv provided by USPTO from lhe PACR Image Database on 08/2912003
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`(cid:25)(cid:3)(cid:82)(cid:73)(cid:3)(cid:25)(cid:27)
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`'·•
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`7
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`In a preferred embodiment of the ribonucleic acid according to the fourth aspect of the present
`
`invention the overhang consists of at least one desoxyribonucleotide.
`
`In a further embodiment of the ribonucleic acid according to the fourth aspect of the present
`
`invention at least one of the strands has an overhang of at least one nucleotide at the 3 '-end.
`
`In an embodiment of the ribonucleic acid according to any of the aspects of the present
`
`invention the length of the double-stranded structure has a length from about 17 to 21 and
`
`more preferably 18 or 19 bases
`
`In another embodiment of the ribonucleic acid according to any of the aspects of the present
`invention the length of said first strand and/or the length of said second strand is
`
`independently from each other selected from the group comprising the ranges of from about
`
`15 to about 23 bases, 17 to 21 bases and 18 or I 9 bases.
`
`In a preferred embodiment of the ribonucleic acid according to any of the aspects of the
`
`present invention the complementarity between said first strand and the target nucleic acid is
`
`perfect.
`
`In an embodiment of the ribonucleic acid according to any of the aspects of the present
`
`invention the duplex formed between the first strand and the target nucleic acid comprises at
`
`least 15 nucleotides wherein there is one mismatch or two mismatches between said 1first
`
`strand and the target nucleic acid fonning said double-stranded structure.
`
`In a preferred embodiment of the ribonucleic acid according to any of the aspects of the
`
`present invention the target gene is selected from the group comprising structural genes,
`
`housekeeping genes, transcription factors, motility factors, cell cycle factors, cell cycle
`
`inhibitors, enzymes, growth factors, cytokines and tumor suppressors.
`
`In a further embodiment of the ribonucleic acid according to any of the aspects of the present
`
`invention the first strand and the second strand are linked by a loop structure.
`
`In a preferred embodiment of the ribonucleic acid according to any of the aspects of the
`
`present invention the loop structure is comprised of a non-nucleic acid polymer.
`
`,,
`._-
`Coov· orovided by USPTO lroin the 'PACR Image Database on 08/29/2003.
`
`..
`
`(cid:26)(cid:3)(cid:82)(cid:73)(cid:3)(cid:25)(cid:27)
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`8
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`In a preferred embodiment thereof the non-nucleic acid polymer is polyethylene glycol.
`
`In an alternative embodiment thereof the loop structure is comprised of a nucleic acid.
`
`In an embodiment of the ribonucleic acid according to any of the aspects of the present
`
`invention the 5' -terminus of the first strand is linked to the 3 '-terminus of the second strand.
`
`In a further embodiment of the ribonucleic acid according to any of the aspects of the present
`
`invention the 3 '-end of the first strand is linked to the 5 '-terminus of the second strand.
`
`In a fifth aspect the problem underlying the present invention is solved by the use of a
`
`ribonucleic acid according to any of the aspects of the present invention, for target validation.
`
`In a sixth aspect the problem underlying the present invention is solved by the use of a
`
`ribonucleic acid according to any of the aspects of the present invention, for the manufacture
`
`of a medicament.
`
`In a preferred embodiment of the use according to the sixth aspect of the present invention the
`
`medicament is for the treatment of a disease or of a condition which is selected from the
`
`group comprising glioblastoma, prostate cancer, breast cancer, lung cancer, liver cancer,
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`colon cancer, pancreatic cancer and leukaemia, diabetes, obesity, cardiovascular diseases, and
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`metabo lie diseases.
`
`In a seventh aspect the problem underlying the present invention is solved by a cell,
`
`preferably a knockdown cell, containing a ribonucleic acid according to any of the aspects of
`
`the present invention.
`
`In an eighth aspect the problem underlying the present invention is solved by an organism,
`
`preferably a lmockdown organism, containing a ribonucleic acid according to any of the
`
`aspects of the present invention.
`
`In a ninth aspect the problem underlying the present invention is solved by a composition
`
`containing a ribonucleic acid according to any of the aspects of the present invention.
`
`Coov orovided bv USPTO lrom the PACR Image CJatabase on 08/29/2003
`
`(cid:27)(cid:3)(cid:82)(cid:73)(cid:3)(cid:25)(cid:27)
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`9
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`In a tenth aspect the problem underlying the present invention is solved by a pharmaceutical
`
`composition containing a ribonucleic acid according to any of the aspects of th epresent
`invention, and a pharmaceutically acceptable carrier.
`
`In an eleventh aspect the problem underlying the present invention is solved by method for
`inhibiting the expression of a target gene in a cell or derivative thereof comprising
`
`introduction of a ribonucleic acid according to any of the aspects of the present invention into
`
`the cell in an amount sufficient to inhibit expression of the target gene, 1:vherein the target
`
`gene is the target gene of the a ribonucleic acid according to any of the aspects of the present
`
`invention.
`
`The present invention is based on the surprising finding that small interfering RNAs can be
`
`desi~ed such as to be both highly specific and active as well as stable under the reaction
`
`conditions typically encountered in biological systems such as biochemical assays or cellular
`
`environments. The various interfering RN As described in the prior art such as by Tuschl et al.
`
`(international patent application WO 01/75164) provide for a length of 21 to 23 nucleotides
`
`and a modification at the 3' end of the double-stranded RNA. It has been surprisingly found
`
`by the present inventors that the problem of stability of interfering RNA, including small
`
`interfering RNA (siRNA) which is generally referred to herein in the following as RNAi,
`
`actually resides in the attack of endonucleases rather than exonucleases as thought earlier.
`
`Based on this finding several strategies have been perceived by the present inventors which
`
`are subject to the present application.
`
`The present invention is thus related to new forms of interfering RNA. RNAi consists of a
`
`ribonucleic acid comprising a double-stranded structure. Said double-stranded structure is
`
`formed by a first strand and a second strand. Said first strand comprises a stretch of
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`contiguous nucleotides, also referred to as first stretch of contiguous nucleotides herein, and
`
`this first stretch is at least partially complementary to a target nucleic acid. Said second strand
`
`comprises also a stretch of contiguous nucleotides whereby said second stretch is at least
`I
`partially identical to a target nucleic acid. The very basic structure of this ribonucleic acid is
`
`schematically shown in Fig. l. Said first strand and said second strand are preferably
`
`hybridised to each other and form the double-stranded structure. The hybridisation typically
`
`occurs by Watson Crick base pairing. The inventive ribonucleic acid, however, is not
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`·copy provided by US PTO from the PACR Image Database on 0B/29/2003
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`(cid:28)(cid:3)(cid:82)(cid:73)(cid:3)(cid:25)(cid:27)
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`10
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`necessarily limited in its length to said double-stranded structure. There might be further
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`nucleotides added to each strand and/or to each end of any of the strands forming the RNAi.
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`Depending on the particular sequence of the first stretch and the second stretch, the
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`hybridisation or base pairing is not necessarily complete or perfect, which means that the first
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`and the second stretch_are not_ 100 % base paired due to mismatches. There might also be one
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`or more mismatches within the duplex. Said mismatches have no effect on the RNAi activity
`
`if placed outside a stretch of 17 matching nucleotides. If mismatches are placed to yield only
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`15 or less contiguous matching nucleotides, the RNAi molecule typically shows a reduced
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`activity in down regulating mRNA for a given target compared to a 17 matching nucleotide
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`duplex.
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`The first stretch of contiguous nucleotides is essentially complementary to a target nucleic
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`acid, more preferably to a part of the target nucleic acid. Complementary as used herein
`
`preferably means that the nucleotide sequence of the first strand is hybridising to a nucleic
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`acid sequence or a part thereof of a target nucleic acid sequence. Typically, the target nucleic
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`acid sequence is, in accordance with the mode of action of interfering ribonucleic acids, a
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`single stranded RNA, more preferably an mRNA. Such hybridisation occurs most likely
`
`through Watson Crick base pairing, however, is not necessarily limited thereto. The extent to
`
`which said first strand and more particularly the first stretch of contiguous nucleotides of said
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`first strand is complementary to a target nucleic acid sequence can be as high as 100% and be
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`as little as 80%, preferably 80-100%, more preferably 85-100%, most preferably 90-100%.
`
`Optimum complementarity seems to be 95-100%. Complementarity in this sense means that
`
`the aforementioned range of nucleotides, such as, e. g., 80%-100%, depending on the
`particular range, of the nucleotides are perfect by Watson Crick base pairing. It is shown in
`
`one aspect of the present invention that the complementarity between said first stretch of
`
`nucleotides and the target RNA has to be 18-19 nucleotides, stretches of as little as 17
`nuc'leotides even with two sequence specific overhangs are not functional in mediating RNAi.
`Accordingly, given a duplex having a length of 19 nucleotides or base pairs a minimum
`complementarity of 17 nucleotides or nucleotide base pairs would be acceptable allowing for
`
`a mismatch of two nucleotides. In case of a duplex consisting of 20 nucleotides or base pairs a
`
`complementarity of 17 nucleotides or nucleotide base pairs would be allowable and
`
`functionally active. The same applies to a duplex of 21 nucleotides or base pairs with a total
`
`of 17 complementary nucleotides or base pairs. Basically, the extent of complementarity
`
`required for a length of a duplex, i. e. of a double stranded structure, can also be based on the
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`Copy provided by USPTO from the PACR lmag~ Database on 08/29/2003
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`11
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`melting temperature of the complex formed by either the double stranded structure as
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`described herein or by the complex of the first stretch of the first strand and the target nucleic
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`acid.
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`It is to be understood that all of the ribonucleic acid of the present invention are suitable to
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`cause or being involved in RNA mediated interference such as, for example, described in
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`international patent applications WO 99/32619, WO 00/44895 and WO 01/75164.
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`The first strategy according to which an interfering ribonucleic acid molecule may be
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`designed according to the present invention is to have an optimum length of 18 or 19
`nucleotides of the stretch which is complementary to the target nucleic acid. It is also within
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`the present invention that said optimum len~ of 18 or 19 nucleotides is the length of the
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`double stranded structure in the RNAi used. This length requirement is clearly different from
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`the technical teaching of the prior art such as, for example, the international patent application
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`WO 01/75164. It is within the present invention that any further design, both according to the
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`present invention and as described in the prior art, can be realised in connection with an
`interfering ribonucleic acid having said length characteristics, i.e. a length of 18 or t 9
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`nucleotides.
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`The second strategy according to which an interfering ribonucleic acid molecule may be
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`designed is to have a free 5' hydroxyl group, also referred to herein as free 5' OH-group at the
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`first strand. A free 5' OH-group means that the most terminal nucleotide forming the first
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`strand is present and is thus not modified, particularly not by an end modification. Typically,
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`the tenninal 5 '-hydroxy group of the second strand, respectively, is also present in an
`unmodified manner. In a more preferred embodiment, also the 3 '-end of the first strand and
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`first stretch, respectively, is unmodified such as to present a free OH-group which is also
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`referred to herein as free 3 'OH-group. Preferably such free OH-group is also present at the 3 ' -
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`end of the second strand and second stretch, respectively. In other embodiments of the
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`ribonucleic acid molecules according to the present invention the 3 '-end of the _first strand and
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`first stretch, respectively, and/or the 3 · -end of the second strand and second stretch,
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`respectively, may have an end modification at the 3' end.
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`As used herein the terms free 5'OH-group and 3'OH-group also indicate that the respective
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`most tenninal nucleotide at the 5 'end and the 3' end of the polynucleotide, respectively,
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`12
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`presents a OH-group. Such OH-group may stem from either the sugar moiety of the
`nucleotide, more preferably from the 5'position in case of the 5'0H-group and from the
`3 'position in case of the 3 'OH-group, or from a phosphate group attached to the sugar moiety
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`of the respective terminal nucleotide. The phosphate group may in principle be attached to
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`any OH-group of the sugar moiety of the nucleotide. Preferably, the phosphate group is
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`attached to the 5'0H-group of the sugar moiety in case of the free 5'0H-group and/or to the
`3'0H-group oflhe sugar moiety in case of the free 3'0H-group.
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`As used herein with any strategy for the design of RNAi or any embodiment of RNAi
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`disclosed herein, the term end modification means a chemical entity added to the most 5' or 3'
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`nucleotide of the first and/or second strand. Examples for such end modifications include, but
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`are not limited to, inverted (deoxy) abasics, amino, fluoro, chloro, bromo, CN, CF, methoxy,
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`imidazole, caboxylate, thioate, C1 to C1o lower alkyl, substituted lower alkyl, alkaryl or
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`aralkyl, OCF3, OCN, 0-, S-, or N-alkyl; 0-, S-, or N-alkenyl; SOCH3; S02CH3; ON02; N02,
`N3; heterozycloalkyl; heterozycloalkaryl; aminoalkylamino; polyalky!amino or substituted
`silyl, as, among others, described in European patents EP O 586 520 Bl or EP O 618 925 Bl.
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`A further end modification is a biotin group. Such biotin group may preferably be attached to
`either the most 5' or the most 3' nucleotide of the first and/or second strand or to both ends. In
`a more preferred embodiment the biotin group is coupled to a polypeptide or a protein. It is
`also within the scope of the present invention that the polypeptide or protein is attached
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`through any of the other aforementioned end modifications. The polypeptide or protein may
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`confer further characteristics to the inventive nucleic acid molecules. Among others the
`polypeptide or protein may act as a ligand to another molecule. If said other molecule is a
`receptor the receptor's function and activity may be activated by the binding ligand. The
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`receptor may show an internalization activity which allows an effective transfection of the
`ligand bound inventive nucleic acid molecules. An example for the ligand to be coupled to the
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`inventive nucleic acid molecule is VEGF and the corresponding receptor is the VEGF
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`receptor.
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`Various possible embodiments of the RNAi of the present invention having different kinds of
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`end modification(s) are presented in the following table I.
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`13
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`Table 1: V~rious embodiments of the interfering ribonucleic acid according to the
`present invention
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`1.) s·-end
`3'-end
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`2.) 5'-end
`3'-end
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`3.) 5'-end
`J'-end
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`4.) 5'-end
`3'-end
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`5.) S'-end
`J'-end
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`6.) 5'-end
`3'-end
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`7.) 5'-end
`3'-end
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`8.) 5'-end
`3'-end
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`1st strand/I st stretch
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`2nd
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`strand/
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`2nd
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`free OH
`free OH
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`stretch
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`free OH
`free OH