Europaisches
`Patentamt
`
`European
`Patent Office
`
`Office europeen
`des brevets
`
`Bescheinigung Certificate
`
`Attestation
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`Die angehefteten Unterla-
`gen stimmen mit der
`urspriinglich eingereichten
`Fassung der auf dem nach-
`sten Blatt bezeichneten
`europaischen Patentanmel-
`dung iiberein.
`
`The attached documents
`are exact copies of the
`European patent application
`described on the following
`page, as originally filed.
`
`Les documents fixes a
`cette attestation sont
`conformes a la version
`initialement deposee de
`la demande de brevet
`europeen specifiee a la
`page suivante.
`
`Patentanmeldung Nr. Patent application No. Demande de brevet n°
`
`02017601.2
`
`Der President des Europaischen Patentamts:
`I m Auftrag
`
`For the President of the European Patent Office
`
`Le President de ('Office europeen des brevets
`p.o.
`
`4 (cid:9)
`
`4.
`
`R C van Dijk
`
`EPA/EPO/OEB Form 1014.1 - 02.2000 (cid:9)
`
`7001014
`
`Alnylam Exh. 1006
`
`i
`
`(cid:9)
`

`

`Europaisches
`Patentamt
`
`European (cid:9)
`Patent Office (cid:9)
`
`Office europeen
`des brevets
`
`Anmeldung Nr:
`Application no.:
`Demande no:
`
`02017601.2
`
`Anmeldetag:
`Date of filing: (cid:9)
`Date de depot:
`
`05.08.02
`
`Anmelder/Applicant(s)/Demandeur(s):
`
`atugen AG
`Robert-Rossle-Strasse 10
`13125 Berlin
`ALLEMAGNE
`
`Bezeichnung der Erfindung/Title of the invention/Titre de l'invention:
`(Falls die Bezeichnung der Erfindung nicht angegeben ist, siehe Beschreibung.
`If no title is shown please refer to the description.
`Si aucun titre n'est indigue se referer a la description.)
`
`Novel forms of interfering RNA molecules
`
`In Anspruch genommene Prioriat(en) / Priority(ies) claimed /Priori-Ws)
`revendiqude(s)
`Staat/Tag/Aktenzeichen/State/Date/File no./Pays/Date/Numero de depot:
`
`Internationale Patentklassifikation/International Patent Classification/
`Classification internationale des brevets:
`
`C12Q/
`
`Am Anmeldetag benannte Vertragstaaten/Contracting states designated at date of
`filing/Etats contractants designees lors du depot:
`
`AT BE BG CH CY CZ DE DK EE ES FI FR GB GR IE IT LI LU MC NL PT SE SK TR
`
`02017601.2 (cid:9)
`EPA/EPO/OEB Form 1014.2 — 01.2000 (cid:9)
`
`7001014
`
`2
`
`ii
`
`(cid:9)
`(cid:9)
`(cid:9)
`(cid:9)
`

`

`atugen AG
`A 19005 EP
`
`EPO Munk*,
`67
`0 5, Aug. 2002
`
`Novel forms of interfering RNA molecules
`
`The present invention is related to a ribonucleic acid comprising a double-stranded structure
`
`whereby the double-stranded structure comprises a first strand and a second strand, whereby
`
`the first strand comprises a first stretch of contiguous nucleotides and whereby said first
`
`stretch is at least partially complementary to the target nucleic acid, and the second strand
`
`comprises a second stretch of contiguous nucleotides whereby said second stretch is at least
`
`partially identical to a target nucleic acid, the use of such ribonucleic acid, a cell and an
`
`organism, respectively, comprising such ribonucleic acid, a composition containing such
`
`ribonucleic acid, a pharmaceutical composition containing such ribonucleic acid and a method
`
`for inhibiting expression of a targeted gene.
`
`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 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
`(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, 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 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
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`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 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 U S 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. (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.
`
`The problem underlying the present invention was to provide synthetic interfering RNA
`molecules which are both stable and active in a biochemical environment such as a living cell.
`
`In a first aspect of the present invention the problem is solved by a ribonucleic acid
`comprising a double stranded structure whereby the double- stranded structure comprises a
`first strand and a second strand, whereby the first strand comprises a first stretch of
`contiguous nucleotides and whereby said first stretch is at least partially complementary to a
`target nucleic acid, and the second strand comprises a second stretch of contiguous
`nucleotides whereby said second stretch is at least partially identical to a target nucleic acid,
`and whereby the double stranded structure is blunt ended.
`
`In a second aspect the problem underlying the present invention is solved by a ribonucleic
`acid comprising a double stranded structure whereby the double- stranded structure comprises
`a first strand and a second strand, whereby the first strand comprises a first stretch of
`contiguous nucleotides and whereby said first stretch is at least partially complementary to a
`target nucleic acid, and the second strand comprises a second stretch of contiguous
`nucleotides, whereby said second stretch is at least partially identical to a target nucleic acid,
`whereby the first stretch and/or the second stretch have a length of 18 or 19 nucleotides.
`
`In an embodiment of the ribonucleic acid according to the first aspect of the invention the first
`stretch and/or the second stretch have a length of 18 or 19 nucleotides.
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`In a further embodiment of the ribonucleic acid according to the first aspect of the invention
`the double stranded structure is blunt ended on both sides of the double strand.
`
`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 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 a third aspect the problem underlying the present invention is solved by a ribonucleic acid
`comprising a double stranded structure whereby the double- stranded structure comprises a
`first strand and a second strand, whereby the first strand comprises a first stretch of
`contiguous nucleotides and whereby said first stretch is at least partially complementary to a
`target nucleic acid, and the second strand comprises a second stretch of contiguous
`nucleotides 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 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 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 NI-12-modification at the 2"-position.
`
`In a preferred embodiment of the ribonucleic acid according to the third aspect of the present
`invention at least one of the strands has an overhang of at least one nucleotide at the 3 '-end
`consisting of ribonucleotide or deoxyribonucleotide.
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`In another preferred embodiment of the ribonucleic acid according to the third aspect of the
`present invention the first stretch and/or the second stretch have a length of 18 or 19
`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 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.
`
`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 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 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 and
`alkyl.
`
`In a fourth aspect the problem underlying the present invention is solved by a ribonucleic acid
`comprising a double stranded structure, whereby the double- stranded structure comprises a
`first strand and a second strand, whereby the first strand comprises a first stretch of
`contiguous nucleotides and whereby said first stretch is at least partially complementary to a
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`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 sccond 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 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.
`
`In a further embodiment of the ribonucleic acid according to the fourth aspect of the present
`invention said first strand and/or said second strand comprise said plurality of modified
`nucleotides.
`
`In another embodiment of the ribonucleic acid according to the fourth aspect of the present
`invention said first strand comprises said plurality of groups of modified nucleotides.
`
`In yet another embodiment of the ribonucleic acid according to the fourth aspect of the
`present invention said second strand comprises said plurality of groups of modified
`nucleotides.
`
`In an preferred embodiment of the ribonucleic acid according to the fourth aspect of the
`present invention the group of nucleotides and/or the group of flanking nucleotides comprises
`a number of nucleotides whereby the number is selected from the group comprising one
`nucleotide to 10 nucleotides.
`
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`In another embodiment of the ribonucleic acid according to the fourth aspect of the present
`invention the pattern of modified nucleotides of said first strand is the same as the pattern of
`modified nucleotides of said second strand.
`
`In a preferred embodiment of the ribonucleic acid according to the fourth aspect of the present
`invention the pattern of said first strand aligns with the pattern of said second strand.
`
`In an alternative embodiment of the ribonucleic acid according to the fourth aspect of the
`present invention the pattern of said first strand is shifted by one or more nucleotides relative
`to the pattern of the second strand.
`
`In an embodiment of the ribonucleic acid according to the fourth aspect of the present
`invention the modification is selected from the group comprising amino, fluoro, methoxy and
`alkyl.
`
`In another embodiment of the ribonucleic acid according to the fourth aspect of the present
`invention the double stranded structure is blunt ended.
`
`In an preferred embodiment of the ribonucleic acid according to the fourth aspect of the
`present invention the double stranded structure is blunt ended on both sides.
`
`In another embodiment of the ribonucleic acid according to the fourth aspect of the present
`invention the double stranded structure is blunt ended on the double stranded structure's side
`which is defined by the 5'-end of the first strand and the 3'-end of the second strand.
`
`In still another embodiment of the ribonucleic acid according to the fourth aspect of the
`present invention the double stranded structure is blunt ended on the double stranded
`structure's side which is defined by at the 3'-end of the first strand and the 5' -end of the
`second strand.
`
`In another embodiment of the ribonucleic acid according to the fourth aspect of the present
`invention at least one of the two strands has an overhang of at least one nucleotide at the 5'-
`end.
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`In a preferred embodiment of the ribonucleic acid according to the fourth aspect of the present
`invention the overhang consists of at least one desoxyribonucleotide.
`
`In a further embodiment of the ribonucleic acid according to the fourth aspect of the present
`invention at least one of the strands has an overhang of at least one nucleotide at the 3'-end.
`
`In an embodiment of the ribonucleic acid according to any of the aspects of the present
`invention the length of the double-stranded structure has a length from about 17 to 21 and
`more preferably 18 or 19 bases
`
`In another embodiment of the ribonucleic acid according to any of the aspects of the present
`invention the length of said first strand and/or the length of said second strand is
`independently from each other selected from the group comprising the ranges of from about
`15 to about 23 bases, 17 to 21 bases and 18 or 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.
`
`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 double-stranded structure.
`
`In a preferred embodiment of the ribonucleic acid according to any of the aspects of the
`present invention the target gene is selected from the group comprising structural genes,
`housekeeping genes, transcription factors, motility factors, cell cycle factors, cell cycle
`inhibitors, enzymes, growth factors, cytokines and tumor suppressors.
`
`In a further embodiment of the ribonucleic acid according to any of the aspects of the present
`invention the first strand and the second strand are linked by a loop structure.
`
`In a preferred embodiment of the ribonucleic acid according to any of the aspects of the
`present invention the loop structure is comprised of a non-nucleic acid polymer.
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`In a preferred embodiment thereof the non-nucleic acid polymer is polyethylene glycol.
`
`In an alternative embodiment thereof the loop structure is comprised of a nucleic acid.
`
`In an embodiment of the ribonucleic acid according to any of the aspects of the present
`invention the 5'-terminus of the first strand is linked to the 3 "-terminus of the second strand.
`
`In a further embodiment of the ribonucleic acid according to any of the aspects of the present
`invention the 3'-end of the first strand is linked to the 5'-terminus of the second strand.
`
`In a fifth aspect the problem underlying the present invention is solved by the use of a
`ribonucleic acid according to any of the aspects of the present invention, for target validation.
`
`In a sixth aspect the problem underlying the present invention is solved by the use of a
`ribonucleic acid according to any of the aspects of the present invention, for the manufacture
`of a medicament.
`
`In a preferred embodiment of the use according to the sixth aspect of the present invention the
`medicament is for the treatment of a disease or of a condition which is selected from the
`group comprising glioblastoma, prostate cancer, breast cancer, lung cancer, liver cancer,
`colon cancer, pancreatic cancer and leukaemia, diabetes, obesity, cardiovascular diseases, and
`metabolic diseases.
`
`In a seventh aspect the problem underlying the present invention is solved by a cell,
`preferably a knockdown cell, containing a ribonucleic acid according to any of the aspects of
`the present invention.
`
`In an eighth aspect the problem underlying the present invention is solved by an organism,
`preferably a knockdown organism, containing a ribonucleic acid according to any of the
`aspects of the present invention.
`
`In a ninth aspect the problem underlying the present invention is solved by a composition
`containing a ribonucleic acid according to any of the aspects of the present invention.
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`In a tenth aspect the problem underlying the present invention is solved by a pharmaceutical
`composition containing a ribonucleic acid according to any of the aspects of th epresent
`invention, and a pharmaceutically acceptable carrier.
`
`In an eleventh aspect the problem underlying the present invention is solved by method for
`inhibiting the expression of a target gene in a cell or derivative thereof comprising
`introduction of a ribonucleic acid according to any of the aspects of the present invention into
`the cell in an amount sufficient to inhibit expression of the target gene, wherein the target
`gene is the target gene of the a ribonucleic acid according to any of the aspects of the present
`invention.
`
`The present invention is based on the surprising finding that small interfering RNAs can be
`designed such as to be both highly specific and active as well as stable under the reaction
`conditions typically encountered in biological systems such as biochemical assays or cellular
`environments. The various interfering RNAs described in the prior art such as by Tuschl et al.
`(international patent application WO 01/75164) provide for a length of 21 to 23 nucleotides
`and a modification at the 3' end of the double-stranded RNA. It has been surprisingly found
`by the present inventors that the problem of stability of interfering RNA, including small
`interfering RNA (siRNA) which is generally referred to herein in the following as RNAi,
`actually resides in the attack of endonucleases rather than exonucleases as thought earlier.
`Based on this finding several strategies have been perceived by the present inventors which
`are subject to the present application.
`
`The present invention is thus related to new forms of interfering RNA. RNAi consists of a
`ribonucleic acid comprising a double-stranded structure. Said double-stranded structure is
`formed by a first strand and a second strand. Said first strand comprises a stretch of
`contiguous nucleotides, also referred to as first stretch of contiguous nucleotides herein, and
`this first stretch is at least partially complementary to a target nucleic acid. Said second strand
`comprises also a stretch of contiguous nucleotides whereby said second stretch is at least
`partially identical to a target nucleic acid. The very basic structure of this ribonucleic acid is
`schematically shown in Fig. 1. Said first strand and said second strand are preferably
`hybridised to each other and form the double-stranded structure. The hybridisation typically
`occurs by Watson Crick base pairing. The inventive ribonucleic acid, however, is not
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`necessarily limited in its length to said double-stranded structure. There might be further
`nucleotides added to each strand and/or to each end of any of the strands forming the RNAi.
`Depending on the particular sequence of the first stretch and the second stretch, the
`hybridisation or base pairing is not necessarily complete or perfect, which means that the first
`and the second stretch_are not 100 % base paired due to mismatches. There might also be one
`or more mismatches within the duplex. Said mismatches have no effect on the RNAi activity
`if placed outside a stretch of 17 matching nucleotides. If mismatches are placed to yield only
`15 or less contiguous matching nucleotides, the RNAi molecule typically shows a reduced
`activity in down regulating mRNA for a given target compared to a 17 matching nucleotide
`duplex.
`
`The first stretch of contiguous nucleotides is essentially complementary to a target nucleic
`acid, more preferably to a part of the target nucleic acid. Complementary as used herein
`preferably means that the nucleotide sequence of the first strand is hybridising to a nucleic
`acid sequence or a part thereof of a target nucleic acid sequence. Typically, the target nucleic
`acid sequence is, in accordance with the mode of action of interfering ribonucleic acids, a
`single stranded RNA, more preferably an mRNA. Such hybridisation occurs most likely
`through Watson Crick base pairing, however, is not necessarily limited thereto. The extent to
`which said first strand and more particularly the first stretch of contiguous nucleotides of said
`first strand is complementary to a target nucleic acid sequence can be as high as 100% and be
`as little as 80%, preferably 80-100%, more preferably 85-100%, most preferably 90-100%.
`Optimum complementarity seems to be 95-100%. Complementarity in this sense means that
`the aforementioned range of nucleotides, such as, e. g., 80%-100%, depending on the
`particular range, of the nucleotides are perfect by Watson Crick base pairing. It is shown in
`one aspect of the present invention that the complementarity between said first stretch of
`nucleotides and the target RNA has to be 18-19 nucleotides, stretches of as little as 17
`nucleotides even with two sequence specific overhangs are not functional in mediating RNAi.
`Accordingly, given a duplex having a length of 19 nucleotides or base pairs a minimum
`complementarity of 17 nucleotides or nucleotide base pairs would be acceptable allowing for
`a mismatch of two nucleotides. In case of a duplex consisting of 20 nucleotides or base pairs a
`complementarity of 17 nucleotides or nucleotide base pairs would be allowable and
`functionally active. The same applies to a duplex of 21 nucleotides or base pairs with a total
`of 17 complementary nucleotides or base pairs. Basically, the extent of complementarity
`required for a length of a duplex, i. e. of a double stranded structure, can also be based on the
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`melting temperature of the complex formed by either the double stranded structure as
`described herein or by the complex of the first stretch of the first strand and the target nucleic
`acid.
`
`It is to be understood that all of the ribonucleic acid of the present invention are suitable to
`cause or being involved in RNA mediated interference such as, for example, described in
`international patent applications WO 99/32619, WO 00/44895 and WO 01/75164.
`
`The first strategy according to which an interfering ribonucleic acid molecule may be
`designed according to the present invention is to have an optimum length of 18 or 19
`nucleotides of the stretch which is complementary to the target nucleic acid. It is also within
`the present invention that said optimum length of 18 or 19 nucleotides is the length of the
`double stranded structure in the RNAi used. This length requirement is clearly different from
`the technical teaching of the prior art such as, for example, the international patent application
`WO 01/75164. It is within the present invention that any further design, both according to the
`present invention and as described in the prior art, can be realised in connection with an
`interfering ribonucleic acid having said length characteristics, i.e. a length of 18 or 19
`nucleotides.
`
`The second strategy according to which an interfering ribonucleic acid molecule may be
`designed is to have a free 5' hydroxyl group, also referred to herein as free 5' OH-group at the
`first strand. A free 5' OH-group means that the most terminal nucleotide forming the first
`strand is present and is thus not modified, particularly not by an end modification. Typically,
`the terminal 5 '-hydroxy group of the second strand, respectively, is also present in an
`unmodified manner. In a more preferred embodiment, also the 3 '-end of the first strand and
`first stretch, respectively, is unmodified such as to present a free OH-group which is also
`referred to herein as free 3 'OH-group. Preferably such free OH-group is also present at the 3'-
`end of the second strand and second stretch, respectively. In other embodiments of the
`ribonucleic acid molecules according to the present invention the 3 '-end of the first strand and
`first stretch, respectively, and/or the 3 '-end of the second strand and second stretch,
`respectively, may have an end modification at the 3' end.
`
`As used herein the terms free 5'0H-group and 3'0H-group also indicate that the respective
`most terminal nucleotide at the 5 'end and the 3' end of the polynucleotide, respectively,
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`presents a OH-group. Such OH-group may stem from either the sugar moiety of the
`
`nucleotide, more preferably from the 5'position in case of the 5'0H-group and from the
`
`3'position in case of the 3'0H-group, or from a phosphate group attached to the sugar moiety
`
`of the respective terminal nucleotide. The phosphate group may in principle be attached to
`
`any OH-group of the sugar moiety of the nucleotide. Preferably, the phosphate group is
`
`attached to the 5'0H-group of the sugar moiety in case of the free 5'0H-group and/or to the
`
`3'0H-group of the sugar moiety in case of the free 3'0H-group.
`
`As used herein with any strategy for the design of RNAi or any embodiment of RNAi
`
`disclosed herein, the term end modification means a chemical entity added to the most 5' or 3'
`
`nucleotide of the first and/or second strand. Examples for such end modifications include, but
`
`are not limited to, inverted (deoxy) abasics, amino, fluoro, chloro, bromo, CN, CF, methoxy,
`
`imidazole, caboxylate, thioate, C1 to C10 lower alkyl, substituted lower alkyl, alkaryl or
`
`aralkyl, OCF3, OCN, 0-, S-, or N-alkyl; 0-, S-, or N-alkenyl; SOCH3; SO2CH3; ONO2; NO2,
`
`N3; heterozycloalkyl; heterozycloalkaryl; aminoalkylamino; polyalkylamino or substituted
`
`silyl, as, among others, described in European patents EP 0 586 520 B1 or EP 0 618 925 Bl.
`
`A further end modification is a biotin group. Such biotin group may preferably be attached to
`
`either the most 5' or the most 3' nucleotide of the first and/or second strand or to both ends. In
`
`a more preferred embodiment the biotin group is coupled to a polypeptide or a protein. It is
`
`also within the scope of the present invention that the polypeptide or protein is attached
`
`through any of the other aforementioned end modifications. The polypeptide or protein may
`
`confer further characteristics to the inventive nucleic acid molecules. Among others the
`
`polypeptide or protein may act as a ligand to another molecule. If said other molecule is a
`
`receptor the receptor's function and activity may be activated by the binding ligand. The
`
`receptor may show an internalization activity which allows an effective transfection of the
`
`ligand bound inventive nucleic acid molecules. An example for the ligand to be coupled to the
`
`inventive nucleic acid molecule is VEGF and the corresponding receptor is the VEGF
`
`receptor.
`
`Various possible embodiments of the RNAi of the present invention having different kinds of
`
`end modification(s) are presented in the following table 1.
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`13
`
`Table 1: Various embodiments of the interfering ribonucleic acid according to the
`present invention
`
`1.) 5'-end
`3'-end
`
`2.) 5'-end
`3'-end
`
`3.) 5'-end
`3 '-end
`
`4.) 5'-end
`3'-end
`
`5.) 5'-end
`3'-end
`
`6.) 5'-end
`3'-end
`
`7.) S'-end
`3'-end
`
`8.) 5'-end
`3'-end
`
`1st strand/1" stretch 2"d strand/ 2nd
`stretch
`
`free OH
`free OH
`
`free OH
`free OH
`
`free OH
`end modification
`
`free OH
`end modification
`
`free OH
`free OH
`
`free OH
`end modification
`
`free OH
`end modification
`
`free OH
`free OH
`
`free OH
`free OH
`
`end modification
`free OH
`
`free OH
`end modification
`
`end modification
`free OH
`
`free OH
`free OH
`
`end modification
`end modification
`
`free OH
`end modification
`
`end modification
`end modification
`
`The various end modifications as disclosed herein are preferably located at the ribose moiety
`
`of a nucleotide of the ribonucleic acid. More particularly, the end modification may be
`
`attached to or replace any of the OH-groups of the ribose moiety, including but not limited to
`
`the 2'0H, 3'0H and 5'0H position, provided that the nucleotide thus modified is a terminal
`
`nucleotide. Inverted abasics are nucleotides, either desoxyribonucleotides or ribonucleotides
`
`which do not have a nucleobase moiety. This kind of compound is, among others, described
`
`in Sternberger et al.(2002), Antisense. Nucl. Ac. Drug Dev. in press.
`
`Any of the aforementioned end modifications may be used in connection with the various
`
`embodiments of RNAi depicted in table 1. In connection therewith it is to be noted that any of
`
`the RNAi forms or embodiments disclosed herein with the sense strand being inactivated,
`
`preferably by having an end modification more preferably at the 5' end, are particularly
`advantageous. This arises from the inactivation of the sense strand which corresponds to the
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`second strand of the ribonucleic acids described herein, which might otherwise interfere with
`an unrelated single-stranded RNA in the cell. Thus the expression and more particularly the
`translation pattern of the transcriptome of a cell is more specifically influenced. This effect is
`also referred to as off-target effect. Referring to table 1 those embodiments depicted as
`embodiments 7 and 8 are particularly advantageous in the above sense as the modification
`results in an inactivation of the — target unspecific — part of the RNAi (which is the second
`strand) thus reducing any unspecific interaction of the second strand with single-stranded
`RNA in a cellular or similar system where the RNAi according to the present invention is
`going to be used to knock down specific ribonucleic acids and proteins, respectively.
`
`A third strategy subject to the present invention is to realise a ribo