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`I 3901a-0005
`UTILITY
`First Inventor I Klaus GIESE et al.
`PATENT. APPLICATION
`TRANSMITTAL
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`Attorney Docket No.
`
`INTEFERING RNA MOLECULES
`
`Title
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`\.. (Only for new nonprovisional applications under 37 C.F.R. 1.53(b))
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`130
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`1003
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`750
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`330
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`($)
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`375
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`165
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`260
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`375
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`1252
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`410
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`930
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`2252
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`1,450
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`1255
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`1,970
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`2255
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`205
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`160
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`750
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`160
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`1501
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`470
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`1460
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`1460
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`

`JI/
`
`Atty. 0kt. No. 39078-0005
`
`Inventors:
`
`Klaus Giese
`J org Kaufinann
`Anke Klippel-Giese
`
`5
`
`INTERFERING RNA MOLECULES
`
`Field of the Invention
`The invention provides novel forms of interfering ribonucleic acid
`
`molecules having a double-stranded structure. The first strand comprises a first
`
`10
`
`stretch of contiguous nucleotides that is at least partially complementary to a
`
`target nucleic acid, and the second strand comprises a second stretch of
`
`contiguous nucleotides that is at least partially identical to a target nucleic acid.
`
`Methods for using these molecules, for example for inhibiting expression of a
`
`target gene, and pharmaceutical compositions, cells and organisms containing
`
`15
`
`these molecules also are provided
`
`Background of the Invention
`RNA-mediated interference (RNAi) is a post-transcriptional gene
`
`silencing mechanism initiated by double stranded RNA (dsRNA) homologous in
`
`sequence to the silenced gene (Fire (1999), Trends Genet 15, 358-63, Tuschl, et
`
`20
`
`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, including
`
`plants (Baulcombe (1999), Curr Opin Plant Biol 2, 109-13), nematodes
`
`25
`
`(Montgomery, et al. (1998), Proc Natl Acad Sci U S A 95, 15502-7), Drosophila
`
`(Kennerdell, et al. (1998), Cell 95, 1017-26, Kennerdell, et al. (2000), Nat
`
`Biotechnol 18, 896-8). In the nematode C.elegans about one third of the genome
`
`has already been subjected to functional analysis by RNAi (Kim (2001), Curr Biol
`
`11, R85-7, Maeda, et al. (2001 ), Curr Biol 11, 1 71-6).
`
`30
`
`Until recently RNAi in mammalian cells was not generally applicable,
`
`with the exception of early mouse development (Wianny, et al. (2000), Nat Cell
`
`Biol 2, 70-5). The discovery that transfection of duplexes of21-nt into
`
`mammalian cells interfered with gene expression and did not induce a sequence
`
`independent interferon-driven anti-viral response usually obtained with long
`
`l
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`1 of 79
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`Atty. Dkt. No. 39078-0005
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`dsRNA led to new potential application in differentiated mammalian cells
`
`(Elbashir et al. (2001), Nature 411, 494-8). Interestingly these small interfering
`
`RNAs (siRNAs) resemble the processing products from long dsRNAs suggesting
`
`a potential bypassing mechanism in differentiated mammalian cells. The Dicer
`
`5
`
`complex, a member of the RN Ase 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
`
`10
`
`(1986), J Biochem Biophys Methods 13, 97-102, Cazenave, et al. (1987), Nucleic
`
`Acids Res 15, 10507-21). It will depend on the particular gene function and assay
`
`systems used whether the respective knock down induced by transfected siRNA
`
`will be maintained long enough to achieve a phenotypic change.
`
`It is apparent, therefore, that synthetic interfering RNA molecules that are
`
`15
`
`both stable and active in a biochemical environment such as a living cell are
`
`greatly to be desired.
`
`Summary of the Invention
`It is therefore an object of the present invention to provide compositions
`
`and methods using interfering RNA molecules having enhanced stability.
`
`20
`
`In accomplishing this object, there has been provided, in accordance with
`
`a first aspect of the present invention, 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
`
`25
`
`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, and whereby the double stranded structure is
`
`blunt ended.
`
`In accordance with a second aspect of the present invention there has been
`
`30
`
`provided 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
`
`2
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`2 of 79
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`Atty. Dkt. No. 39078-0005
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`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 stretch and/or the second stretch have a length of 18 or 19
`
`5
`
`nucleotides.
`
`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.
`
`In an alternative embodiment of the ribonucleic acid according to the first
`
`10
`
`aspect of the invention the double stranded structure is blunt ended on the double
`
`stranded structure which is defined by the 5 '-end of the first strand and the 3 '-end
`
`of the second strand.
`
`In a further alternative embodiment of the ribonucleic acid according to
`
`the first and the second aspect of the invention the double stranded structure is
`
`15
`
`blunt ended on the double stranded structure which is defined by the 3 '-end of the
`
`first strand and the 5 '-end of the second strand.
`
`In accordance with a third aspect of the present invention there has been
`
`provided a ribonucleic acid comprising a double stranded structure whereby the
`
`double- stranded structure comprises a first strand and a second strand, whereby
`
`20
`
`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 and
`
`whereby said second stretch is at least partially identical to a target nucleic acid,
`
`and whereby at least one of the two strands has an overhang of at least one
`
`25
`
`nucleotide at the 5 '-end.
`
`In an embodiment of the ribonucleic acid according to the third aspect of
`
`the present invention 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
`
`30
`
`third aspect of the present invention the nucleotide has a modification whereby
`
`said modification is preferably selected from the group comprising nucleotides
`
`being an inverted abasic and nucleotides having an NH2-modification at the 2 ' -
`position.
`
`3
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`3 of 79
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`•
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`Atty. Dkt. No. 39078-0005
`
`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 consisting of ribonucleotide or
`
`deoxyribonucleotide.
`
`5
`
`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 nucleotides.
`
`In an embodiment of the ribonucleic acid according to any aspect of the
`
`present invention the double-stranded structure has a length of 17 to 21
`
`10
`
`nucleotides, preferably 18 to 19 nucleotides
`
`In an embodiment of the ribonucleic acid according to the third aspect of
`
`the present invention the overhang at the 5 '-end is on the second strand.
`
`In a preferred embodiment of the ribonucleic acid according to the third
`
`aspect of the present invention the first strand comprises also an overhang,
`
`15
`
`preferably at the 5 '-end.
`
`In an embodiment of the ribonucleic acid according to the third aspect of
`
`the present invention the 3 '-end of the first strand comprises an overhang.
`
`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.
`
`20
`
`In a preferred embodiment thereof the second strand also comprise an
`
`overhang, preferably at the 5 '-end.
`
`In an embodiment of the ribonucleic acid according to the third aspect of
`
`the present invention the 3 '-end of the first strand comprises an overhang.
`
`In an embodiment of the ribonucleic acid according to any aspect of the
`
`25
`
`present invention at least one nucleotide of the ribonucleic acid has a modification
`
`at the 2' -position and the modification is preferably selected from the group
`
`comprising amino, fluoro, methoxy, alkoxy and alkyl.
`
`In accordance with a fourth aspect of the present invention there has been
`
`provided a ribonucleic acid comprising a double stranded structure, whereby the
`
`30
`
`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 and
`
`whereby said second stretch is at least partially identical to a target nucleic acid,
`
`4
`
`4 of 79
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`Atty. Dkt. No. 39078-0005
`
`whereby said first strand and/or said second strand comprises a plurality of groups
`
`of modified nucleotides having a modification at the 2 '-position whereby within
`
`the strand each group of modified nucleotides is flanked on one or both sides by a
`
`flanking group of nucleotides whereby the flanking nucleotides forming the
`
`5
`
`flanking group of nucleotides is either an 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
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`first, second or third aspect of the present invention.
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`IO
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`In a further embodiment of the ribonucleic acid according to the fourth
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`aspect of the present invention said first strand and/or said second strand comprise
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`said plurality of modified nucleotides.
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`In another embodiment of the ribonucleic acid according to the fourth
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`aspect of the present invention said first strand comprises said plurality of groups
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`of modified nucleotides.
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`In yet another embodiment of the ribonucleic acid according to the fourth
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`aspect of the present invention said second strand comprises said plurality of
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`groups of modified nucleotides.
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`In a preferred embodiment of the ribonucleic acid according to the fourth
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`aspect of the present invention the group of modified nucleotides and/or the group
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`of flanking nucleotides comprises a number of nucleotides whereby the number is
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`selected from the group comprising one nucleotide to 10 nucleotides.
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`In another embodiment of the ribonucleic acid according to the fourth
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`aspect of the present invention the pattern of modified nucleotides of said first
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`strand is the same as the pattern of modified nucleotides of said second strand.
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`In a preferred embodiment of the ribonucleic acid according to the fourth
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`aspect of the present invention the pattern of said first strand aligns with the
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`pattern of said second strand.
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`In an alternative embodiment of the ribonucleic acid according to the
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`fourth aspect of the present invention the pattern of said first strand is shifted by
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`one or more nucleotides relative to the pattern of the second strand.
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`In an embodiment of the ribonucleic acid according to the fourth aspect of
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`the present invention the modification is selected from the group comprising
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`amino, fluoro, methoxy, alkoxy and alkyl.
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`In another embodiment of the ribonucleic acid according to the fourth
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`aspect of the present invention the double stranded structure is blunt ended.
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`In a preferred embodiment of the ribonucleic acid according to the fourth
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`aspect of the present invention the double stranded structure is blunt ended on
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`5
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`both sides.
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`In another embodiment of the ribonucleic acid according to the fourth
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`aspect of the present invention the double stranded structure is blunt ended on the
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`double stranded structure's side which is defined by the 5'-end of the first strand
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`and the 3 '-end of the second strand.
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`10
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`In still another embodiment of the ribonucleic acid according to the fourth
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`aspect of the present invention the double stranded structure is blunt ended on the
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`double stranded structure's side which is defined by at the 3'-end of the first
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`strand and the 5 '-end of the second strand.
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`In another embodiment of the ribonucleic acid according to the fourth
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`aspect of the present invention at least one of the two strands has an overhang of
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`at least one nucleotide at the 5 '-end.
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`In a preferred embodiment of the ribonucleic acid according to the fourth
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`aspect of the present invention the overhang consists of at least one
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`desoxyribonucleotide.
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`In a further embodiment of the ribonucleic acid according to the fourth
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`aspect of the present invention at least one of the strands has an overhang of at
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`least one nucleotide at the 3 '-end.
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`In an embodiment of the ribonucleic acid according to any of the aspects
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`of the present invention the length of the double-stranded structure has a length
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`from about 17 to 21 and more preferably 18 or 19 bases
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`In another embodiment of the ribonucleic acid according to any of the
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`aspects of the present invention the length of said first strand and/or the length of
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`said second strand is independently from each other selected from the group
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`comprising the ranges of from about 15 to about 23 bases, 17 to 21 bases and 18
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`or 19 bases.
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`In a preferred embodiment of the ribonucleic acid according to any of the
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`aspects of the present invention the complementarity between said first strand and
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`the target nucleic acid is perfect.
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`In an embodiment of the ribonucleic acid according to any of the aspects
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`of the present invention the duplex formed between the first strand and the target
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`nucleic acid comprises at least 15 nucleotides wherein there is one mismatch or
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`two mismatches between said first strand and the target nucleic acid forming said
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`5
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`double-stranded structure.
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`In an embodiment of the ribonucleic acid according to any of the aspects
`
`of the present invention, wherein both the first strand and the second strand each
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`comprise at least one group of modified nucleotides and at least one flanking
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`group of nucleotides, whereby each group of modified nucleotides comprises at
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`10
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`least one nucleotide and whereby each flanking group of nucleotides comprising
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`at least one nucleotide; with each group of modified nucleotides of the first strand
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`being aligned with a flanking group of nucleotides on the second strand, whereby
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`the most terminal 5' nucleotide of the first strand is a nucleotide of the group of
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`modified nucleotides, and the most terminal 3' nucleotide of the second strand is a
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`nucleotide of the flanking group of nucleotides.
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`In a preferred embodiment of the ribonucleic acid according to the fourth
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`aspect, wherein each group of modified nucleotides consists of a single nucleotide
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`and/or each flanking group of nucleotides consists of a single nucleotide.
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`In a further embodiment of the ribonucleic acid according to the fourth
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`aspect, wherein on the first strand the nucleotide forming the flanking group of
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`nucleotides is an unmodified nucleotide which is arranged in a 3' direction
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`relative to the nucleotide forming the group of modified nucleotides, and wherein
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`on the second strand the nucleotide forming the group of modified nucleotides is a
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`modified nucleotide which is arranged in 5' direction relative to the nucleotide
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`forming the flanking group of nucleotides.
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`In another embodiment of the ribonucleic acid according to the fourth
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`aspect, wherein the first strand comprises eight to twelve, preferably nine to
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`eleven, groups of modified nucleotides, and wherein the second strand comprises
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`seven to eleven, preferably eight to ten, groups of modified nucleotides.
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`In a preferred embodiment of the ribonucleic acid according to any of the
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`aspects of the present invention the target gene is selected from the group
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`comprising structural genes, housekeeping genes, transcription factors, motility
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`factors, cell cycle factors, cell cycle inhibitors, enzymes, growth factors, cytokines
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`and tumor suppressors.
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`In a further embodiment of the ribonucleic acid according to any of the
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`aspects of the present invention the first strand and the second strand are linked by
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`a loop structure.
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`In a preferred embodiment of the ribonucleic acid according to any of the
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`5
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`aspects of the present invention the loop structure is comprised of a non-nucleic
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`acid polymer.
`
`In a preferred embodiment thereof the non-nucleic acid polymer is
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`polyethylene glycol.
`
`In an alternative embodiment thereof the loop structure is comprised of a
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`10
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`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
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`15
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`aspects of the present invention the 3 '-end of the first strand is linked to the 5 ' -
`
`terminus of the second strand.
`
`In accordance with a fifth aspect of the present invention there have been
`
`provided methods of using a he ribonucleic acid according to any of the aspects of
`
`the present invention for target validation.
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`20
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`In accordance with a sixth aspect of the present invention there have been
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`provided medicaments and pharmaceutical compositions containing a ribonucleic
`
`acid according to any of the aspects of the present invention, and methods of
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`making such medicaments and compositions.
`
`In a preferred embodiment of the use according to the sixth aspect of the
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`25
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`present invention methods are provided for the treatment of a disease or of a
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`condition which is selected from the group comprising glioblastoma, prostate
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`cancer, breast cancer, lung cancer, liver cancer, colon cancer, pancreatic cancer
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`and leukaemia, diabetes, obesity, cardiovascular diseases, and metabolic diseases.
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`In accordance with a seventh aspect of the present invention there has been
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`30
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`provided a cell, for example a knockdown cell, containing a ribonucleic acid
`
`according to any of the aspects of the present invention.
`
`In accordance with an eighth aspect of the present invention there has been
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`provided an organism, for example a knockdown organism, containing a
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`ribonucleic acid according to any of the aspects of the present invention.
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`In accordance with a ninth aspect of the present invention there has been
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`provided a composition containing a ribonucleic acid according to any of the
`
`aspects of the present invention.
`
`In accordance with a tenth aspect of the present invention there has been
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`5
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`provided a pharmaceutical composition containing a ribonucleic acid according to
`
`any of the aspects of the present invention, and a pharmaceutically acceptable
`
`carrier.
`
`In accordance with an eleventh aspect of the present invention there has
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`been provided a method for inhibiting the expression of a target gene in a cell or
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`10
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`derivative thereof comprising introducing a ribonucleic acid according to any of
`
`the aspects of the present invention into a cell in an amount sufficient to inhibit
`
`expression of the target gene, wherein the target gene is the target gene of the a
`
`ribonucleic acid according to any of the aspects of the present invention.
`
`Brief Description of the Drawings
`Fig. 1 shows a schematic illustration defining the terminology as used
`
`15
`
`herein. The upper of the two strands is the first strand and the antisense strand of
`
`the targeted nucleic acid such as mRNA. The second strand is the one which
`
`essentially corresponds in its sequence to the targeted nucleic acid and thus forms
`
`the sense strand. Both, the first strand and second strand form a double-stranded
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`20
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`structure, typically through Watson Crick base pairing.
`
`Fig. 2 illustrates some embodiments of the ribonucleic acid molecules of
`
`the present invention with patterns of modified and unmodified groups of
`
`nucleotides which are also referred to herein as a pattern of modification. The
`
`modified groups of nucleotides are also referred to herein as a group of modified
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`25
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`nucleotides. The unmodified nucleotides or unmodified groups of nucleotides
`
`referred to as flanking group(s) of nucleotides herein, as used herein may also
`
`have one or several of the modification(s) as disclosed herein which, however,
`
`is/are different from the modification of the nucleotides forming the group(s) of
`
`modified nucleotides. In Fig. 2A the modified and unmodified groups of
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`30
`
`nucleotides, i.e. the groups of modified nucleotides and the flanking groups of
`
`nucleotides on both the first stretch and the second stretch are located on
`
`corresponding parts of the stretches and are thus aligned to each other (groups of
`
`modified nucleotides on the first strand aligned with groups of modified
`
`nucleotides on the second strand and flanking groups of nucleotides on the first
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`strand aligned with flanking group of nucleotides on the second strand), whereas
`
`in Fig. 2B the pattern realised on the first strand is also realised on the second
`
`strand, however, with a phase shift such that the modified group of nucleotides of
`
`the first stretch is base pairing with an unmodified group of nucleotides