Electronic Acknowledgement Receipt
`
`EFSID:
`
`Application Number:
`
`3856817
`
`12200296
`
`International Application Number:
`
`Confirmation Number:
`
`7489
`
`Title of Invention:
`
`INTERFERING RNA MOLECULES
`
`First Named Inventor/Applicant Name:
`
`KLAUS GIESE
`
`Customer Number:
`
`23557
`
`Filer:
`
`Frank Christopher Eisenschenk/Sherry Loke
`
`Filer Authorized By:
`
`Frank Christopher Eisenschenk
`
`Attorney Docket Number:
`
`ST-l0lXTDl
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`Receipt Date:
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`28-AUG-2008
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`Filing Date:
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`Time Stamp:
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`16:16:21
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`Application Type:
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`Utility under 35 USC 111 (a)
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`$2100
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`2158
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`190065
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`EISENSCHENK,FRANK C.
`
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`Specification
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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 A~~lications Under 35 U.S.C. 111
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`
`

`

`5
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`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-med1ated interference (R.i"'fAi) 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, I 09-13), nematodes
`
`25
`
`(Montgomery, et al. (1998), Proc Natl Acad Sci US A 95, 15502-7), Drosophila
`
`(Kennerde11, 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, 171-6).
`
`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 of 21-nt into
`
`mammalian cells interfered with gene expression and did not induce a sequence
`
`independent interferon-driven anti-viral response usually obtained with long
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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 RNAse III family, necessary for the initial dsRNA
`
`processing has been identifjed (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 strncture 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 strncture 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
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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 nuc1eic 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.
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`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 1 7 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,
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`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
`
`first, second or third aspect of the present invention.
`
`10
`
`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
`
`15
`
`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 a preferred embodiment of the ribonucleic acid according to the fourth
`
`20
`
`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 present invention the pattern of modified nucleotides of said first
`
`25
`
`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
`
`30
`
`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 ribonucJeic acid according to the fourth aspect of
`
`the present invention the modification is selected from the group comprising
`
`amino, fluoro, methoxy, alkoxy and alkyl.
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`5
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`Atty. Dkt. No. 39078-0005
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`In another embodiment of the ribonucleic acid according to the fourth
`
`aspect of the 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
`
`5
`
`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.
`
`10
`
`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
`
`15
`
`aspect of the present invention at least one of the two strands has an overhang of
`
`at least one nucleotide at the 5 '-end.
`
`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.
`
`20
`
`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
`
`25
`
`from about 1 7 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, 1 7 to 21 bases and 18
`
`30
`
`or 19 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.
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`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 first strand and the target nucleic acid forming said
`
`5
`
`double-stranded structure.
`
`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
`
`comprise at least one group of modified nucleotides and at least one flanking
`
`group of nucleotides, whereby each group of modified nucleotides comprises at
`
`10
`
`least one nucleotide and whereby each flanking group of nucleotides comprising
`
`at least one nucleotide; with each group of modified nucleotides of the first strand
`
`being aligned with a flanking group of nucleotides on the second strand, whereby
`
`the most terminal 5' nucleotide of the first strand is a nucleotide of the group of
`
`modified nucleotides, and the most terminal 3' nucleotide of the second strand is a
`
`15
`
`nucleotide of the flanking group of nucleotides.
`
`In a preferred embodiment of the ribonucleic acid according to the fourth
`
`aspect, wherein each group of modified nucleotides consists of a single nucleotide
`
`and/or each flanking group of nucleotides consists of a single nucleotide.
`
`In a further embodiment of the ribonucleic acid according to the fourth
`
`20
`
`aspect, wherein on the first strand the nucleotide forming the flanking group of
`
`nucleotides is an unmodified nucleotide which is arranged in a 3' direction
`
`relative to the nucleotide forming the group of modified nucleotides, and wherein
`
`on the second strand the nucleotide forming the group of modified nucleotides is a
`
`modified nucleotide which is arranged in 5' direction relative to the nucleotide
`
`25
`
`forming the flanking group of nucleotides.
`
`In another embodiment of the ribonucleic acid according to the fourth
`
`aspect, wherein the first strand comprises eight to twelve, preferably nine to
`
`eleven, groups of modified nucleotides, and wherein the second strand comprises
`
`seven to eleven, preferably eight to ten, groups of modified nucleotides.
`
`30
`
`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, grov\ih 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 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
`
`5
`
`aspects of the present invention the loop structure is comprised of a non-nucleic
`
`acid polymer.
`
`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
`
`10
`
`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
`
`15
`
`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.
`
`20
`
`In accordance with a sixth aspect of the present invention there have been
`
`provided medicaments and pharmaceutical compositions containing a ribonucleic
`
`acid according to any of the aspects of the present invention, and methods of
`
`making such medicaments and compositions.
`
`In a preferred embodiment of the use according to the sixth aspe2t'o:fthe
`
`25
`
`present invention methods are provided 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, colon cancer, pancreatic cancer
`
`and leukaemia, diabetes, obesity, cardiovascular diseases, and metabolic diseases.
`
`In accordance with a seventh aspect of the present invention there has been
`
`30
`
`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
`
`provided an organism, for example a knockdown organism, containing a
`
`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
`
`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
`
`5
`
`provided a pharmaceutical composition containing a ribonucleic acid according to
`
`any of the aspects of the present invention, and a pharmaceutically acceptable
`
`earner.
`
`In accordance with an eleventh aspect of the present invention there has
`
`been provided a method for inhibiting the expression of a target gene in a cell or
`
`10
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`derivative thereof comprising introducing a ribonucleic acid according to any of
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`the aspects of the present invention into a cell in an amount sufficient to inhibit
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`expression of the target gene, wherein the target gene is the target gene of the a
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`ribonucleic acid according to any of the aspects of the present invention.
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`Brief Description of the Drawings
`Fig. 1 sho,vs a schematic illustration defining the terminology as used
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`herein. The upper of the two strands is the first strand and the anti sense strand of
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`the targeted nucleic acid such as mR.i."JA. The second strand is the one which
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`essentially corresponds in its sequence to the targeted nucleic acid and thus forms
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`the sense strand. Both, the first strand and second strand form a double-stranded
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`structure, typically through Watson Crick base pairing.
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`Fig. 2 illustrates some embodiments of the ribonucleic acid molecules of
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`the present invention with patterns of modified and unmodified groups of
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`nucleotides which are also referred to herein as a pattern of modification. The
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`modified groups of nucleotides are also referred to herein as a group of modified
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`nucleotides. The unmodified nucleotides or unmodified groups of nucleotides
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`refened to as flanking group(s) of nucleotides herein, as used herein may also
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`have one or several of the modification(s) as disclosed herein which, however,
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`is/are different from the modification of the nucleotides forming the group(s) of
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`modified nucleotides. In Fig. 2A the modified and unmodified groups of
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`nucleotides, i.e. the groups of modified nucleotides and the flanking groups of
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`nucleotides on both the first stretch and the second stretch are located on
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`conesponding parts of the stretches and are thus aligned to each other (groups of
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`modified nucleotides on the first strand aligned with groups of modified
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`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
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`in Fig. 2B the pattern realised on the first strand is also realised on the second
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`strand, however, with a phase shift such that the modified group of nucleotides of
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`the first stretch is base pairing with an unmodified group of nucleotides of the
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`second stretch and vice versa so that a group of modified nucleotides on the first
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`strand aligns with a flanking group of nucleotides on the second strand. In Fig. 2C
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`a further possibility of arranging the modified and unmodified groups of
`nucleotides is realised. It is also within the present invention that the pattern ofthe
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`first stretch is independent from the pattern of the second stretch and that both
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`patterns partially overlap in terms of relative position to each other in the double(cid:173)
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`stranded structure defined by base pairing. In a further embodiment the extent of
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`this overlapping can vary over the length of the stretch( es) and strand( s ),
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`respectively.
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`Fig. 3 shows the result of a knockdown experiment using RNAi molecules
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`15 with different end protection groups. More particularly Fig. 3A shows that the
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`various forms of end protected RNAi molecules are :functional on the knockdown
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`of PTEN mRNA.
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`Fig. 3B shows the sequence of the different RNAi molecules used in the
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`experiment the result of which is depicted in Fig. 3A. Fig. 3C shows the result of
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`an immunoblot analysis of PTEN protein after treatment with modified RN Ai
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`molecules in comparison to PTEN specific antisense constructs.
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`Fig. 4 shows that the 3' overhang ofRNAi molecules is not important for
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`RNA interference. More particularly, Fig. 4A shows a dose response curve of
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`different RNAi molecules and Fig. 4B shows the sequence of the RNAi molecules
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`used in the experiment the result of which is shown in Fig. 4A.
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`Fig. 5 shows that duplex length of the RNAi molecules has to be at least
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`18-19 nucleotides. More particularly, Fig. 5B shows the sequence of the PTEN
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`specific RNAi molecules used in the experiment the result of which is depicted in
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`Fig. SA as dose response curve.
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`Fig. 6 shows that four terminal mismatched nucleotides in RNAi
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`molecules with a length of 19 nucleotides are stiII functional in mediating Aktl
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`knockdown. More particularly, Fig. 6B shows the sequence of the RNAi
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`molecules used in the experiment the result of which is depicted in Fig. 6A.
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`Fig. 7 shows further results on duplex length requirements and tolerance
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`for mutation in siRNAs. More particularly, Fig. 7 A shows the various constructs
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`used (left panel) and the respective impact on inhibition of Aktl mRNA
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`expression in HeLa cells relative to the expression ofpl l0a. used in the indicated
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`amounts of siRNA molecules (right panel). The nucleotide changes in the
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`mismatch siRNA molecules are indicated by arrows; the 3' desoxynucleotides, if
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`any, are indicated in capital letters. Fig. 7B shows the various PTEN specific
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`siRNAs (1eft panel), the inhibition of PTEN mRNA expression in HeLA cells
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`expressed as ratio PTEN/p 11 0a, at variou_s amounts of siR.'l\l"A (middle panel) and
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`Fig. 7C a Western Blot analysis depicting the inhibition of PTEN protein
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`expression using PTEN specific siRNA (30nM) and respective mismatch siRNA
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`after 48 and 96 hours, respectively, with pl 00a. being used as loading control.
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`Fig. 8 shows the result of studies on the stability in serum conferred to
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`RN Ai molecules by 2 '-O-methylation and that end modifications have no
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`beneficial effects on RNAi stability. More particularly, Fig. 8A shows the result
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`of a gel electrophoresis of the various RNAi molecules depicted in Fig. 8B being
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`subject to incubation with fetal calf serum.
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`Fig. 9 shows that an amino end modification results in loss of activity. Fig.
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`9B shows the particular R.i'l\J'Ai molecules used in the experiments the result of
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`20 which is shnwn in Fig. 9A expressed as PTEN/p 11 Oa expression level ratio. Fig.
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`9C shows the design principles which may be deduced from the results depicted
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`in Fig. 9A. As used in Fig. 9C the term functional means functionally active in the
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`particular assay system as described in the example and "not functional" means
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`not functionally active in said system.
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`Fig. 10 shows that 2'-O-Alkyl (methyl) modifications stabilize RNAi
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`molecules but also result in reduction of their activity. More particularly, Fig. lOC
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`shows the sequence of the RNAi molecules used in the experiment the result of
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`which is depicted as a dose response curve in Fig. 1 0A. Fig. 1 OB shows the result
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`of a gel electrophoresis of the various Rl"'\J"Ai molecules depicted in Fig. lOC being
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`30
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`subject to a two hour incubation in fetal calf serum.
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`Fig. 11 shows the result of an experiment on the efficacy of RNAi
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`molecules with blocks of 2' -O-methyl modifications with Fig. 11 A graphically
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`depicting the results of said experiments as a dose response curve and with Fig.
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`11 C showing the sequences of the particular RNAi molecules used in said
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`experiments. Fig. 11 B shows the result of a gel electrophoresis of the various
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`RN Ai molecules depicted in Fig. 11 C being subject to a two hour incubation in
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`fetal calf serum.
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`Fig. 12 shows that alternating 2' -O-methyl modification result in activity
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`5
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`of the modified Ri~Ai molecules compared to unmodified forms. More
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`particularly, Fig. 12B shows the sequence of the RNAi molecules used in this
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`experiment the result of which is depicted in Fig. 12A. Fig. 12C shows the
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`stability of said RNAi molecules following incubation in serum for two hours,
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`whereas Fig. 12D shows an immunoblot for PTEN protein upon application of
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`different RNAi molecules to HeLa cells. As may be taken therefrom RNAi
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`molecules with alternating modification