Electronic Acknowledgement Receipt
`
`EFSID:
`
`Application Number:
`
`24436680
`
`14977710
`
`International Application Number:
`
`Confirmation Number:
`
`8597
`
`Title of Invention:
`
`INTERFERING RNA MOLECULES
`
`First Named Inventor/Applicant Name:
`
`KLAUS GIESE
`
`Customer Number:
`
`23557
`
`Filer:
`
`Frank Christopher Eisenschenk/Amanda Lascala
`
`Filer Authorized By:
`
`Frank Christopher Eisenschenk
`
`Attorney Docket Number:
`
`ST.101XTD5
`
`Receipt Date:
`
`22-DEC-2015
`
`Filing Date:
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`Time Stamp:
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`09:42:52
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`Application Type:
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`Utility under 35 USC 111 (a)
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`Authorized User
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`yes
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`$1600
`
`9939
`
`190065
`
`EISENSCHENK, FRANK C.
`
`The Director of the USPTO is hereby authorized to charge indicated fees and credit any overpayment as follows:
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`This Acknowledgement Receipt evidences receipt on the noted date by the USPTO of the indicated documents,
`characterized by the applicant, and including page counts, where applicable. It serves as evidence of receipt similar to a
`Post Card, as described in MPEP 503.
`
`New Agglications Under 35 U.S.C. 111
`If a new application is being filed and the application includes the necessary components for a filing date (see 37 CFR
`1.53(b)-(d) and MPEP 506), a Filing Receipt (37 CFR 1.54) will be issued in due course and the date shown on this
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`If a timely submission to enter the national stage of an international application is compliant with the conditions of 35
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`national stage submission under 35 U.S.C. 371 will be issued in addition to the Filing Receipt, in due course.
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`New International Agglication Filed with the USPTO as a Receiving Office
`If a new international application is being filed and the international application includes the necessary components for
`an international filing date (see PCT Article 11 and MPEP 181 O), a Notification of the International Application Number
`and of the International Filing Date (Form PCT/RO/1 OS) will be issued in due course, subject to prescriptions concerning
`national security, and the date shown on this Acknowledgement Receipt will establish the international filing date of
`the application.
`
`v
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`

`

`1
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`ST.101XTD5
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`INTERFERING RNA MOLECULES
`
`CROSS-REFERENCE TO RELATED APPLICATIONS
`
`This application is a continuation of U.S. Patent Application 14/578,636, filed on
`
`5
`
`December 22, 2014, now U.S. Patent No. 9,222,092, which is a continuation of U.S. Patent
`
`Application 13/692, 178, filed on December 3, 2012, now U.S. Patent No. 8,933,215, which is
`
`a continuation of U.S. Patent Application 12/986,389, filed on January 7, 2011, now U.S.
`
`Patent No. 8,324,370, which is a continuation of U.S. Patent Application 12/200,296, filed on
`
`August 28, 2008, now U.S. Patent 7,893,245, which is a continuation of U.S. Patent
`
`10
`
`Application No. 10/633,630, filed on August 5, 2003, now U.S. Patent No. 7,452,987, which
`
`claims the benefit of U.S. Provisional Application No. 60/402,541, filed August 12, 2002.
`
`Each of these applications are incorporated herein by reference in their entirety, including all
`
`figures, tables and amino acid or nucleic acid sequences.
`
`The Sequence Listing for this application is labeled "Seq-List.txt" which was created
`
`15
`
`on August 28, 2008 and is 84 KB. The entire contents of the sequence listing is incorporated
`
`herein by reference in its entirety.
`
`FIELD OF THE INVENTION
`
`The invention provides novel forms of interfering ribonucleic acid molecules having a
`
`20
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`double-stranded structure. The first strand comprises a first 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. 1\riethods for using these molecules, for example for inhibiting expression
`
`of a target gene, and pharmaceutical compositions, cells and organisms containing these
`
`25
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`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
`
`30
`
`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
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`2
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`ST.101XTD5
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`of orgamsms, including plants (Baulcombe (1999), Curr Opin Plant Biol 2, 109-13),
`
`nematodes (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 nematode C. elegans about one third of the genome has already been subjected
`
`5
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`to functional analysis by RNAi (Kim (2001), Curr Biol 11, R85-7, Maeda, et al. (2001), Curr
`
`Biol 11, 171-6).
`
`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 of 21-nt into mammalian cells interfered with gene
`
`10
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`expression and 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 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 complex, a
`
`15
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`member of the RNAse 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 Biochem Biophys Methods 13, 97-102, Cazenave, et al.
`
`20
`
`(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 both stable
`
`and active in a biochemical environment such as a living cell are greatly to be desired.
`
`25
`
`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.
`
`In accomplishing this object, there has been provided, m accordance with a first
`
`30
`
`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 complementary to a target nucleic acid, and the second strand
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`ST.101XTD5
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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, and whereby the double stranded structure is blunt
`
`ended.
`
`In accordance with a second aspect of the present invention there has been provided a
`
`5
`
`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
`
`10
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`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 nucleotides.
`
`In a further embodiment of the ribonucleic acid according to the first aspect of the
`
`15
`
`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 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
`
`20
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`the second aspect of the invention the double stranded structure is 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
`
`25
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`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, and whereby at least one of the two strands has an overhang of at least
`
`30
`
`one 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.
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`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 selected from the group comprising nucleotides being an inverted abasic and
`
`nucleotides having an NHrmodification at the 2' -position.
`
`5
`
`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.
`
`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
`
`10
`
`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 nucleotides, preferably 18 to
`
`19 nucleotides.
`
`In an embodiment of the ribonucleic acid according to the third aspect of the present
`
`15
`
`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, 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.
`
`20
`
`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.
`
`In a preferred embodiment thereof the second strand also comprises an overhang,
`
`preferably at the 5 '-end.
`
`In an embodiment of the ribonucleic acid according to the third aspect of the present
`
`25
`
`invention the 3 '-end of the first strand comprises an overhang.
`
`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 modification is preferably selected from the group comprising amino, fluoro,
`
`methoxy, alkoxy and alkyl.
`
`30
`
`In accordance with a fourth 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 the first strand comprises a
`
`first stretch of contiguous nucleotides and whereby said first stretch is at least partially
`
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`5
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`ST.101XTD5
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`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, 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
`
`5
`
`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 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
`
`10
`
`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.
`
`15
`
`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.
`
`20
`
`In a preferred embodiment of the ribonucleic acid according to the fourth aspect of the
`
`present invention the group of modified 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.
`
`In another embodiment of the ribonucleic acid according to the fourth aspect of the
`
`25
`
`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
`
`30
`
`the present invention the pattern of said first strand is shifted by one or more nucleotides
`
`relative to the pattern of the second strand.
`
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`ST.101XTD5
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`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,
`
`alkoxy and alkyl.
`
`In another embodiment of the ribonucleic acid according to the fourth aspect of the
`
`5
`
`present invention the double stranded structure is blunt ended.
`
`In a 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
`
`10
`
`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 at the 3 '-end of the first strand and the 5 '-end of the second
`
`15
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`strand.
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`In another embodiment of the ribonucleic acid according to the fourth aspect of the
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`present invention 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 a preferred embodiment of the ribonucleic acid according to the fourth aspect of the
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`present invention the overhang consists of at least one desoxyribonucleotide.
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`In a further embodiment of the ribonucleic acid according to the fourth aspect of the
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`present invention at least one of the strands has an overhang of at least one nucleotide at the
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`3'-end.
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`In an embodiment of the ribonucleic acid according to any of the aspects of the
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`present invention the length of the double-stranded structure has a length from about 17 to 21
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`and more preferably 18 or 19 bases.
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`In another embodiment of the ribonucleic acid according to any of the aspects of the
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`present invention the length of said first strand and/or the length of said second strand is
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`independently from each other selected from the group comprising the ranges of from about
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`3 0
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`15 to about 23 bases, 1 7 to 21 bases and 18 or 19 bases.
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`In a preferred embodiment of the ribonucleic acid according to any of the aspects of
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`the present invention the complementarity between said first strand and the target nucleic
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`acid is perfect.
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`In an embodiment of the ribonucleic acid according to any of the aspects of the
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`present invention the duplex formed between the first strand and the target nucleic acid
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`comprises at least 15 nucleotides wherein there is one mismatch or two mismatches between
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`said first strand and the target nucleic acid forming said double-stranded structure.
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`In an embodiment of the ribonucleic acid according to any of the aspects of the
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`present invention, wherein both the first strand and the second strand each comprise at least
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`one group of modified nucleotides and at least one flanking group of nucleotides, whereby
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`each group of modified nucleotides comprises at least one nucleotide and whereby each
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`flanking group of nucleotides comprising at least one nucleotide; with each group of
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`modified nucleotides of the first strand being aligned with a flanking group of nucleotides on
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`the second strand, whereby the most terminal 5' nucleotide of the first strand is a nucleotide
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`of the group of modified nucleotides, and the most terminal 3' nucleotide of the second strand
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`is a nucleotide of the flanking group of nucleotides.
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`In a preferred embodiment of the ribonucleic acid according to the fourth aspect,
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`wherein each group of modified nucleotides consists of a single nucleotide and/or each
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`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 aspect,
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`wherein on the first strand the nucleotide forming the flanking group of nucleotides is an
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`unmodified nucleotide which is arranged in a 3' direction relative to the nucleotide forming
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`the group of modified nucleotides, and wherein on the second strand the nucleotide forming
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`the group of modified nucleotides is a modified nucleotide which is arranged in 5' direction
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`relative to the nucleotide forming the flanking group of nucleotides.
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`In another embodiment of the ribonucleic acid according to the fourth aspect, wherein
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`the first strand comprises eight to twelve, preferably nine to eleven, groups of modified
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`nucleotides, and wherein the second strand comprises seven to eleven, preferably eight to ten,
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`groups of modified nucleotides.
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`In a preferred embodiment of the ribonucleic acid according to any of the aspects of
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`the present invention the target gene is selected from the group comprising structural genes,
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`housekeeping genes, transcription factors, motility factors, cell cycle factors, cell cycle
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`inhibitors, enzymes, growth factors, cytokines and tumor suppressors.
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`In a further embodiment of the ribonucleic acid according to any of the aspects of the
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`present invention the first strand and the second strand are linked by a loop structure.
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`In a preferred embodiment of the ribonucleic acid according to any of the aspects of
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`the present invention the loop structure is comprised of a non-nucleic acid polymer.
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`In a preferred embodiment thereof the non-nucleic acid polymer is polyethylene
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`glycol.
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`5
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`In an alternative embodiment thereof the loop structure is comprised of a nucleic acid.
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`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
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`strand.
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`In a further embodiment of the ribonucleic acid according to any of the aspects of the
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`present invention the 3 '-end of the first strand is linked to the 5' -terminus of the second
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`strand.
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`In accordance with a fifth aspect of the present invention there have been provided
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`methods of using a ribonucleic acid according to any of the aspects of the present invention
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`for target validation.
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`In accordance with a sixth aspect of the present invention there have been provided
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`medicaments and pharmaceutical compositions containing a ribonucleic acid according to
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`any of the aspects of the present invention, and methods of making such medicaments and
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`com positions.
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`In a preferred embodiment of the use according to the sixth aspect of the present
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`invention methods are provided for the treatment of a disease or of a condition which is
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`selected from the group comprising glioblastoma, prostate cancer, breast cancer, lung cancer,
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`liver cancer, colon cancer, pancreatic cancer and leukaemia, diabetes, obesity, cardiovascular
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`diseases, and metabolic diseases.
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`In accordance with a seventh aspect of the present invention there has been provided a
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`cell, for example a knockdown cell, containing a ribonucleic acid according to any of the
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`aspects of the present invention.
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`In accordance with an eighth aspect of the present invention there has been provided
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`an organism, for example a knockdown organism, containing a ribonucleic acid according to
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`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 provided a
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`composition containing a ribonucleic acid according to any of the aspects of the present
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`invention.
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`In accordance with a tenth aspect of the present invention there has been provided a
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`pharmaceutical composition containing a ribonucleic acid according to any of the aspects of
`
`the present invention, and a pharmaceutically acceptable carrier.
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`In accordance with an eleventh aspect of the present invention there has been
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`5
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`provided a method for inhibiting the expression of a target gene in a cell or 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 a ribonucleic acid according to any of the aspects of the
`
`present invention.
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`BRIEF DESCRIPTION OF THE DRAWINGS
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`Fig. 1 shows a schematic illustration defining the terminology as used herein. The
`
`upper of the two strands is the first strand and the anti sense strand of the targeted nucleic acid
`
`such as mRNA. The second strand is the one which essentially corresponds in its sequence to
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`15
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`the targeted nucleic acid and thus forms the sense strand. Both, the first strand and second
`
`strand form a double-stranded 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
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`20
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`referred to herein as a group of modified 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 nucleotides, i.e. the
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`25
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`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
`
`strand aligned with flanking group of nucleotides on the second strand), whereas in Fig. 2B
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`30
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`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 of the second stretch and vice versa so that a group of
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`modified nucleotides on the first strand aligns with a flanking group of nucleotides on the
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`second strand. In Fig. 2C a further possibility of arranging the modified and unmodified
`
`groups of nucleotides is realised. It is also within the present invention that the pattern of the
`
`first stretch is independent from the pattern of the second stretch and that both patterns
`
`partially overlap in terms of relative position to each other in the double-stranded structure
`
`5
`
`defined by base pairing. In a further embodiment the extent of this overlapping can vary over
`
`the length of the stretch(es) and strand(s), respectively.
`
`Figs. 3A-3C show the result of a knockdown experiment using RNAi molecules with
`
`different end protection groups. More particularly Fig. 3A shows that the various forms of
`
`end protected RNAi molecules are functional on the knockdown of PTEN mRNA.
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`10
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`Fig. 3B (SEQ ID NOs: 1-8, respectively in order of appearance) shows the sequence
`
`of the different RN Ai molecules used in the experiment the result of which is depicted in Fig.
`
`3A. Fig. 3C shows the result of an immunoblot analysis of PTEN protein after treatment with
`
`modified RNAi molecules in comparison to PTEN specific antisense constructs.
`
`Figs. 4A-4B show that the 3' overhang of RN Ai molecules is not important for RNA
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`15
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`interference. More particularly, Fig. 4A shows a dose response curve of different RNAi
`
`molecules and Fig. 4B (SEQ ID NOs: 9-20, respectively in order of appearance) shows the
`
`sequence of the RN Ai molecules used in the experiment the result of which is shown in Fig.
`
`4A.
`
`Figs. SA-SB show that duplex length of the RNAi molecules has to be at least 18-19
`
`20
`
`nucleotides. More particularly, Fig. SB (SEQ ID NOs: 21-28, respectively in order of
`
`appearance) shows the sequence of the PTEN specific RNAi molecules use