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`REC'D 10 APR 2003
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`April 04, 2003
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`FILING DATE:February 20, 2002
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`M. SIAS
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`“AL lade
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`PROVISIONAL APPLICATIONFOR PATENTCGOVER’SHEer’
`QZ Oeee a ee ee pea
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`This is a requestfor filing a PROVISIONAL APPLICATION FOR PATENTunder37 CFR §1.53(b)(2).
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`INVENTOR(S)VAPPLICANTS(S) ioS
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`RESIDENCE
`(City and either state or foreign country)
`
`Leonid
`Beigelman
`
`
`
`
`Macejak
`Dennis
`
`
`
`Zinnen
`Shawn
`
`Pamela 705 Barberry Cir.
`Pavco
`
`Lafayette, CO 80026
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`
`METHODS AND REAGENTS FOR RNA INTERFERENCE MEDIATEDINHIBITION OF GENE
`EXPRESSION USING CHEMICALLY MODIFIED SYNTHETIC SHORT INTERFERING RNAS
`
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`CORRESPONDENCE ADDRESS
`
`McDonnell Boehnen Bulbert & Berghoff
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`Kathy
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`| INVENTOR(S)/APPLICANTS(S)
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`2400 West 17th Avenue
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`LAST NAME
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`FIRST NAME
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`MIDDLE
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`Fosnaugh
`
`Mokler
`
`Victor
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` Sharon Jamison
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`Page 2 of 2
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`" Express Mail No.
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`Application for Provisional Patent of Beigelmanetal.
`
`Title: METHODS AND REAGENTS FOR RNA INTERFERENCE MEDIATED
`INHIBITION OF GENE EXPRESSION USING CHEMICALLY MODIFIED
`SYNTHETIC SHORT INTERFERING RNAS
`
`x.
`
`Provisional Patent Application (68 pages of specification and 10
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`Docket No. 02-128 (900/024)
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`SUP AGBt ab Ss pap gs
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`DESCRIPTION
`
`METHODS AND REAGENTS FOR RNA INTERFERENCE MEDIATED INHIBITION
`OF GENE EXPRESSION USING CHEMICALLY MODIFIED SYNTHETIC SHORT
`INTERFERING RNAS
`
`Background Of The Invention
`
`in modulating gene
`invention concerns methods and reagents useful
`The present
`expression in a variety of applications, including use in therapeutic, diagnostic, target validation,
`and genomic discovery applications. Specifically, the invention relates to synthetic chemically
`modified short
`interfering nucleic acid molecules capable of mediating RNA interference
`(RNAi).
`,
`
`The following is a discussion of relevant art pertaining to RNAi. The discussion is
`provided only for understanding of the invention that follows. The summary is not an admission
`that any of the work described below is priorart to the claimed invention.
`
`RNAinterference refers to the process of sequence-specific post transcriptional gene
`silencing in animals mediated by short interfering RNAs (siRNA)(Fire et al., 1998, Nature, 391,
`806). The corresponding process in plants is commonly referred to as post transcriptional gene
`silencing or RNAsilencing and is also referred to as quelling in fungi. The process of post
`transcriptional gene silencing is thought to be an evolutionarily conserved cellular defense
`mechanism used to prevent the expression of foreign genes which is commonly shared by
`diverse flora and phyla (Fire et al., 1999, Trends Genet., 15, 358). Such protection from foreign
`gene expression may have evolved in response to the production of double stranded RNAs
`(dsRNA) derived from viral infection or the random integration of transposon elements into a
`host genomevia a cellular response that specifically destroys homologous single stranded RNA
`or viral genomic RNA. The presence of dsRNAin cells triggers the RNAi response though a
`mechanism that has yet to be fully characterized. This mechanism appears to be different from
`the interferon response that results from dsRNA mediated activation of protein kinase PKR and
`2’,5’-oligoadenylate synthetase resulting in non-specific cleavage of mRNA byribonucleaseL.
`
`The presence of long dsRNAsincells stimulates the activity of a ribonuclease III enzyme
`referred to as dicer. Dicer is involved in the processing of the dsRNA into short pieces of
`dsRNA known as short interfering RNAs (siRNA) (Berstein et al., 2001, Nature, 409, 363).
`Short interfering RNAs derived from dicer activity are typically about 21-23 nucleotides in
`length and comprise about 19 base pair duplexes. Dicer has also been implicated in the excision
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`of 21 and 22 nucleotide small temporal RNAs (stRNA) from precursor RNA of conserved
`structure that are implicated in translational control (Hutvagneret al., 2001, Science, 293, 834).
`The RNAi response also features an endonuclease complex containing a siRNA, commonly
`referred to as an RNA-induced silencing complex (RISC), which mediates cleavage of single
`stranded RNA having sequence complimentary to the antisense sirand of the siRNA duplex.
`Cleavage of the target RNA takes place in the middle of the region complementary to the
`antisense strand ofthe siRNA duplex (Elbashiret al., 2001, Genes Dev., 15, 188).
`Short interfering RNA mediated RNAi has been studied in a variety of systems. Fireet al.,
`1998, Nature, 391, 806, were the first to observe RNAi in C. Elegans. Wianny and Goetz, 1999,
`Nature Cell Biol., 2, 70, describes RNAi mediated by dsRNA in mouse embryos. Hammond et
`al, 2000, Nature, 404, 293, describe RNAi
`in Drosophila cells transfected with dsRNA.
`' Elbashir et al., 2001, Nature, 411, 494, describe RNAi induced by introduction of duplexes of
`synthetic 21-nucleotide RNAs in cultured mammalian cells including human embryonic kidney
`and HeLacells. Recent work in Drosophila embryonic lysates (Elbashir ef al., 2001, EMBO J,
`20, 6877) has revealed certain requirements for siRNA length, structure, chemical composition,
`and sequence that are essential to mediate efficient RNAi activity. These studies have shown
`that 21 nucleotide siRNA duplexes are most active when containing two nucleotide 3’-
`‘overhangs. Furthermore, complete substitution of one or both siRNA strands with 2’-deoxy (2’-
`H) or 2’-O-methyl nucleotides abolishes RNAi activity, whereas substitution of the 3’-terminal
`siRNA overhang nucleotides with deoxy nucleotides (2’-H) was shown to be tolerated. Single
`mismatch sequences in the center of the siRNA duplex were also shown to abolish RNAi
`activity. In addition, these studies also indicate that the position of the cleavage site in the target
`RNAis defined by the 5’-end of the siRNA guide sequence rather than the 3’-end (Elbashir et
`al, 2001, EMBO J,, 20, 6877). Other studies have indicated that a 5’~phosphate on the target-
`. complementary strand of a siRNA duplex is required for siRNA activity and that ATP is utilized
`to maintain the 5’-phosphate moiety on the siRNA (Nykanen ef al., 2001, Cell, 107, 309).
`
`Beach et al., International PCT Publication No. WO 01/68836, describes specific methods
`for attenuating gene expression using endogenously derived dsRNA. Tuschl et ai, International
`PCT Publication No. WO 01/75164, deseribes a Drosophila in vitro RNAi system and the use of
`specific siRNA molecules for certain functional genomic and certain therapeutic applications;
`although Tuschl, 2001, Chem. Biochem., 2, 239-245, doubts that RNAi can be used to cure
`genetic diseases or viral infection due “to the dangerof activating interferon response”. Li er al.,
`International PCT Publication No. WO 00/44914, describes the use of specific dsRNAsfor use
`in attenuating the expression of certain target genes. Zemicka-Goetz et al., International PCT
`Publication No. WO 01/36646, describes certain methods for inhibiting the expression of
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`particular genes in mammalian cells using certain dsRNA molecules. Fire et ai, International
`PCT Publication No. WO 99/32619, describes particular methods for introducing certain dsRNA
`molecules into cells for use in inhibiting gene expression. Plaetinck e¢ al, International PCT
`Publication No. WO 00/01846, describes certain methods for identifying specific genes
`responsible for conferring a particular phenotype in a cell using specific dsRNA molecules.
`Mello e¢ al., International PCT Publication No. WO 01/29058, describes the identification of
`specific genes involved in dsRNA mediated RNAi. Deschamps Depaillette ef al., International
`PCT Publication No. WO 99/07409, describes specific compositions consisting of particular
`dsRNA molecules combined with certain anti-viral agents. Driscoll et a/., International PCT
`Publication No. WO 01/49844, describes specific DNA constructs for use in facilitating gene
`silencing in targeted organisms. Parrish ef al., 2000, Molecular Cell, 6, 1977-1087, describes
`specific chemically modified siRNA constructs targeting the unc-22 gene of C. elegans.
`
`SUMMARY OF THE INVENTION
`
`This invention relates to compounds, compositions, and methods useful for modulating
`RNA function and/or gene expression in a cell. Specifically, the instant invention features
`chemically modified synthetic short interfering RNA (siRNA) molecules capable of modulating
`gene expression in cells by RNA inference (RNAi). The use of chemically modified siRNA is
`expected to improve various properties of native siRNA molecules through increased resistance
`to nuclease degradation in vivo and/or improved cellular uptake. The chemically modified
`siRNA molecules of the instant invention provide useful reagents and methods for a variety of
`therapeutic, diagnostic, agricultural, target validation, genomic discovery, genetic engineering
`and pharmacogenomic applications.
`
`The introduction of chemically modified nucleotides into nucleic acid molecules will
`provide a powerful tool in overcoming limitations of in vive stability and bioavailability inherent
`to native RNA molecules The use of chemically modified nucleic acid molecules can enable a
`lower dose of a particular nucleic acid molecule for a given therapeutic effect since chemically
`modified nucleic acid molecules tend to have a longer half-life in serum. Furthermore, certain
`chemical modifications can improve the bioavailability of nucleic acid molecules by targeting
`particular cells or tissues and/or improving cellular uptake of the nucleic acid molecule.
`Therefore, even if the activity of a chemically modified nucleic acid molecule is reduced as
`compared to a native nucleic acid molecule, for example when compared to an all RNA nucleic
`acid molecule, the overall activity of the modified nucleic acid molecule can be greater than the
`native molecule due to improved stability and/or delivery of the molecule. Unlike native
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`unmodified siRNA, chemically modified siRNA can also minimize the possibility of activating
`interferon activity in humans.
`
`The nucleic acid molecules of the invention that act as mediators of the RNA interference
`gene silencing response are chemically modified double stranded RNA molecules. As in their
`native double sttanded RNA counterparts, these siRNA molecules typically consist of duplexes
`containing about 19 base pairs between oligonucleotides comprising about 19 to about 25
`nucleotides. The most active siRNA molecules are thought to have such duplexes with
`overhanging ends of 1-3 nucleotides, for example 21 nucleotide duplexes with 19 base pairs and
`2 nucleotide 3’-overhangs. These overhanging segments
`are
`readily hydrolyzed by
`endonucleases in vivo. Studies have shown that replacing the 3’-overhanging segments of a 21-
`' mer siRNA duplex having 2 nucleotide 3’ overhangs with deoxyribonucleotides does not have an
`adverse effect on RNAi activity. Replacing up to 4 nucleotides on each end of the siRNA with
`deoxyribonucleotides has been reported to be well tolerated whereas complete substitution with
`deoxyribonucleotides results in no RNAi activity (Elbashir et al., 2001, EMBO J., 20, 6877). In
`addition, Elbashir ef ai, supra, also report
`that substitution of siRNA with 2’-O-methyl
`nucleotides completely abolishes RNAiactivity. Li et al, International PCT Publication No.
`WO 00/44914, and Beachet al., International PCT Publication No. WO 01/68836 both suggest
`‘that siRNA “may include modifications to either the phosphate-sugar back bone or the
`nucleoside.
`.
`. to include at least one of a nitrogen or sulfur heteroatom”, however neither
`application teaches to what extent these modifications are tolerated in siRNA molecules nor
`provide any examples of such modified siRNA. Kreutzer and Limmer, Canadian Patent
`Application No. 2,359,180, also describe certain chemical modifications for use in dsRNA
`. constructs in order to counteract activation of double stranded-RNA-dependent protein kinase
`PKR,specifically 2’-amino or 2’-O-methy! nucleotides, and nucleotides containing a 2’-O or 4’-
`C methylene bridge. However, Kreutzer and Limmersimilarly fail to show to what extent these
`modifications are tolerated in siRNA molecules nor provide any examples of such modified
`siRNA.
`
`"
`
`-
`
`Parrish et ai, 2000, Molecular Cell, 6, 1977-1087, tested certain chemical modifications
`targeting the unc-22 gene in C. elegans using long (>25 nt) siRNA transcripts. The authors
`describe the introduction of thiophosphate residues into these siRNA transcripts by incorporating
`thiophosphate nucleotide analogs with T7 and T3 RNA polymerase and observed that “RNAs
`with two [phosphorothioate] modified bases also had substantial decreases in effectiveness as
`RNAitriggers (data not shown); [phosphorothioate] modification of more than two residues
`greatly destabilized the RNAsin vitro and we were not able to assay interferenceactivities.” Jd.
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`at 1081. The authors also tested certain modifications at the 2’-position of the nucleotide sugar
`in the
`long siRNA transcripts
`and observed that
`substituting deoxynucleotides
`for
`ribonucleotides “produced a substantial decrease in interference activity”, especially in the case
`of Uridine to Thymidine and/or Cytidine to deoxy-Cytidine substitutions.
`Jd.
`In addition, the
`authors tested certain base modifications, including substituting 4-thiouracil, 5-bromouracil, 5-
`iodouracil, 3-(aminoallyl)uracil for uracil, and inosine for guanosine in sense and antisense
`strands of the siRNA, and found that whereas 4-thiouracil and 5-bromouracil were all well
`tolerated, inosine “produced a substantial decrease in interference activity” when incorporated in
`either strand.
`Incorporation of 5-iodouracil and 3-(aminoallyl)uracil in the antisense strand
`resulted in substantial decrease in RNAi activity as well.
`
`Here, applicant discloses the incorporation of various chemical modifications into siRNA
`constructs. Non-limiting examples of such chemical modifications include without limitation
`phosphorothioate internucleotide linkages, 2’-O-methyl
`ribonucleotides, 2’-deoxy-2’-fluoro
`ribonucleotides, “universal base” nucleotides, 5~C-methyl nucleotides, and inverted deoxyabasic
`residue incorporation. These chemical modifications, when used in various siRNA constructs,
`are shown to preserve RNAiactivity in cells while at the same time, dramatically increasing the
`serum stability of these compounds. Furthermore, contrary to the data published by Parrish ef
`al., supra, applicant demonstrates that multiple (greater than one) phosphorothioate substitutions
`are weil tolerated and confer substantial
`increases in serum stability for modified siRNA
`constructs.
`
`In one embodiment, the invention features a chemically modified short interfering RNA
`(siRNA) molecule capable of mediating RNA interference (RNAi) inside a cell, wherein the
`chemical modification comprises one or more nucleotides comprising a backbone modified
`internucleotide linkage having Formula I:
`
`
`
`R;—-X——_P—Y—R,
`
`Ww
`
`wherein each R1 and R2 is independently any nucleotide, non-nucleotide, or polynucleotide
`which can be naturally occurring or chemically modified, each X and Y is independently O,S,
`N,alkyl, or substituted alkyl, each Z and W is independently O,S, N, alkyl, substituted alkyl, O-
`allyl, S-alkyl, alkaryl, or aralkyl, and wherein W, X, Y and Z are notall O.
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`The chemically modified internucleotide linkages having Formula I, for example wherein
`any Z, W, X, and/or Y independently comprises a sulphur atom, can be present in one or both
`oligonucleotide strands of the siRNA duplex, for example in the sense strand,antisense strand, or
`both strands. -The siRNA molecules of the invention can comprise one or more chemically
`modified internucleotide linkages having Formula J at the 3’-end, 5’-end, or both 3’ and 5’-ends
`of the sense strand, antisense strand, or both strands. For example, an exemplary siRNA
`molecule of the invention can comprise between about
`1 and about 5 chemically modified
`internucleotide linkages having Formula I at the 5’-~end of the sense strand, antisense strand, or
`both strands.
`In another non-limiting example, an exemplary siRNA molecule of the invention
`can comprise one or more pyrimidine nucleotides with chemically modified internucleotide
`linkages having Formula I in the sense strand, antisense strand, or both strands.
`In yet another
`non-limiting example, an exemplary siRNA molecule of the invention can comprise one or more
`purine nucleotides with chemically modified internucleotide linkages having Formula I in the
`sense strand, antisense strand, or both strands. In another embodiment, a siRNA molecule of the
`invention having internucleotide linkage(s) of Formula I also comprises a chemically modified
`nucleotide or non-nucleotide having any of Formulae II,ITI, V, or VI.
`
`In one embodiment, the invention features a chemically modified short interfering RNA
`(siRNA) molecule capable of mediating RNA interference (RNAi) inside a cell, wherein the
`chemical modification comprises one or more nucleotides or non-nucleotides having Formula II:
`
`
`
`wherein each R3, R4, RS, R6, R7, R8, R10, R11 and R12 is independently H, OH, alkyl,
`
`substituted alkyl, alkaryl or aralkyl, F, Cl, Br, CN, CF3, OCF3, OCN, O-alkyl, S-alkyl, N-alkyl,
`
`O-alkenyl, S-alkenyl, N-alkenyl, SO-alkyl, alkyl-OSH, alkyl-OH, O-alkyl-OH, O-alkyl-SH, S-
`
`alkyl-OH, S-alkyl-SH, allyl-S-aliyl, alkyl-O-alkyl, ONO2, NO2, N3, NH2, aminoalkyl,
`
`aminoacid, aminoacyl, ONH2, O-aminoalkyl, O-aminoacid, O-aminoacyl, heterocycloalkyl,
`
`heterocycloalkaryl, aminoalkylamino, polyalklylamino,
`
`substituted silyl, or group having
`
`Formula I; R9 is O, S, CH2, S=O, CHF, or CF2, and B is a nucleosidic base such as adenine,
`
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`guanine, uracil, cytosine, thymine, 2-aminoadenosine, 5-methylcytosine, 2,6-diaminopurine, or
`any other non-naturally occurring base that can be employedto formastable duplex with RNA
`or a non-nucleosidic base such as phenyl, naphthyl, 3-nitropyrrole, 5-nitroindole, nebularine,
`pyridone, pyridinone, or any other non-naturally occurring universal base that can be employed.
`
`to form a stable duplex with RNA.
`
`
`
`The chemically modified nucieotide or non-nucleotide of FormulaII can be present in one
`‘or both oligonucleotide strands of the siRNA duplex, for example in the sense strand, antisense
`strand, or both strands. The siRNA molecules of the invention can comprise one or more
`chemically modified nucleotide or non-nucleotide of FormulaII at the 3’-end, 5’-end, or both 3’
`and 5’-ends of the sensestrand, antisense strand, or both strands. For example, an exemplary
`siRNA molecule of the invention can comprise between about
`1 and about 5 chemically
`modified nucleotide or non-nucleotide of Formula Ii at the 5’-end of the sense strand, antisense
`strand, or both strands.
`In anther non-limiting example, an exemplary siRNA molecule of the
`‘invention can comprise between about 1 and about 5 chemically modified nucleotide or non-
`nucleotide of FormulaII at the 3’-end of the sense strand, antisense strand, or both strands.
`
`In one embodiment, the invention features a chemically modified short interfering RNA
`(siRNA) molecule capable of mediating RNA interference (RNAi) inside a cell, wherein the
`chemical modification comprises one or more nucleotides or non-nucleotides having Formula
`I:
`
`
`
`wherein each R3, R4, R5, R6, R7, R8, R10, R11 and R12 is independently H, OH, ally,
`
`substituted alkyl, alkaryl or aralkyl, F, Cl, Br, CN, CF3, OCF3, OCN, O-alkyl, S-alkyl, N-alkyl,
`‘O-alkenyl, S-alkenyl, N-alkenyl, SO-alkyl, alkyl-OSH,alkyl-OH, O-alkyl-OH, O-alkyl-SH, S-
`alkyl-OH, S-alkyl-SH, alkyl-S-alkyl, alkyl-O-alkyl, ONO2, NO2, N3, NH2, aminoalkyl,
`
`aminoacid, aminoacyl, ONH2, O-aminoalkyl, O-aminoacid, O-aminoacyl, heterocycloalkyl,
`
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`heterocycloalkaryl, aminoalkylamino, polyalklylamino, substituted silyl, or group having
`
`Formula I; R9 is O, S, CH2, S=O, CHF, or CF2, and B is a nucleosidic base such as adenine,
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`guanine, uracil, cytosine, thymine, 2-aminoadenosine, 5-methylcytosine, 2,6-diaminopurine, or
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`any other non-naturally occurring base that can be employed to form a stable duplex with RNA
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`or a non-nucleosidic base such as phenyl, naphthyl, 3-nitropyrrole, 5-nitroindole, nebularine,
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`pytidone, pyridinone, or any other non-naturally occurring universal base that can be employed
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`to form a stable duplex with RNA.
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`
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`The chemically modified nucleotide or non-nucleotide of Formula II can be present in one
`or both oligonucleotide strands of the siRNA duplex, for example in the sense strand, antisense
`strand, or both strands. The siRNA molecules of the invention can comprise one or more
`chemically modified nucleotide or non-nucleotide of FormulaIII at the 3’-end, 5’-end, or both 3”
`and 5’-ends of the sense strand, antisense strand, or both strands. For example, an exemplary
`siRNA molecule of the mvention can comprise between about
`1 and about 5 chemically
`modified nucleotide or non-nucleotide of Formula II] at the 5’-end of the sense strand, antisense
`strand, or both strands.
`In anther non-limiting example, an exemplary siRNA molecule of the
`mvention can comprise between about 1 and about 5 chemically modified nucleotide or non-
`nucleotide ofFormula [II at the 3’-end ofthe sense strand, antisense strand, or both strands.
`
`In another embodiment, a siRNA molecule of the invention comprises a nucleotide having
`Formula II or II, wherein the nucleotide having Formula I or If is in an inverted configuration.
`For example, the nucleotide having Formula II or III is connected to the siRNA construct in a
`°3’,3°, 3°-2’, 2’-3, or 5’,5’ configuration, such as at the 3’-end, 5’-end, or both 3° and 5’ ends of
`one or both siRNAstrands.
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`In one embodiment, the invention features a chemically modified short interfering RNA
`
`(siRNA) molecule capable of mediating RNA interference (RNAi) inside a cell, wherein the
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`chemical modification comprises a 5’-terminal phosphate group having Forula IV:
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`X——P—y—.
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`wherein each X and Y is independently O, S, N, alkyl, substituted allyl, or alkylhalo; cach Z and
`W is independently O, S$, N, alkyl, substituted alkyl, O-alkyl, S-alkyl, alkaryl, aralkyl, or
`alkylhalo; and wherein W, X, Y and Z are not all O,
`
`the invention features a siRNA molecule having a 5’-terminal
`Tn one embodiment,
`phosphate group having Formula IV on the target-complimentary strand wherein the siRNA
`molecule comprises an all RNA siRNA molecule. In another embodiment, the invention features
`a siRNA molecule having a 5’-terminal phosphate group having Formula IV on the target-
`complimentary strand wherein the siRNA molecule also comprises 1-3 nucleotide 3’-overhangs
`having between about 1 and about 4 deoxyribonucleotides on the 3’-end of one or both strands.
`In another embodiment, a 5’-terminal phosphate group having Formula IV is present on the
`target-complimentary strand of a siRNA molecule of the invention, for example a siRNA
`molecule having chemical modifications having Formula I, Formula 1 and/or FormulaII.
`
`Tn one embodiment, the invention features a chemically modified short interfering RNA
`(siRNA) molecule capable of mediating RNA interference (RNAi) inside a cell, wherein the
`chemical modification comprises one or more phosphorothioate internucleotide linkages. For
`_ example,
`in a non-limiting example,
`the invention features a chemically modified short
`interfermg RNA (siRNA) having about 1, 2, 3, 4, 5, 6, 7, or 8 phosphorothioate internucleotide
`linkages in one siRNA strand.
`In yet another embodiment, the invention features a chemically
`modified short interfering RNA (siRNA) individually having about 1, 2, 3, 4, 5, 6, 7, or 8
`phosphorothioate internucleotide linkages in both siRNA strands.
`The phosphorothioate
`internucleotide linkages can be present in one or both oligonucleotide strands of the siRNA
`duplex, for example in the sense strand, antisense strand, or both strands. The siRNA molecules
`of the invention can comprise one or more phosphorothioate internucleotide linkages at the 3’-
`end, 5’-end, or both 3” and 5’-ends of the sense strand, antisense strand, or both strands. For
`example, an exemplary siRNA molecule of the invention can comprise between about 1 and
`about 5 phosphorothioate internucleotide linkages at the 5’-end of the sense strand, antisense
`strand, or both strands.
`In another non-limiting example, an exemplary siRNA molecule of the
`invention can comprise one or more pyrimidine phosphorothioate internucleotide linkages in the
`sense strand, antisense strand, or both strands.
`In yet another non-limiting example, an
`exemplary siRNA molecule of the invention can comprise one or more purine phosphorothioate
`internucleotide linkages in the sense strand, antisense strand, or both strands.
`
`In one embodiment, the invention features a siRNA molecule, wherein the sense strand
`comprises one or more, for example about 1, 2, 3, 4, 5, 6, 7, 8 , 9 , or 10 phosphorothioate
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`internucleotide linkages, and/or one or more 2’-deoxy, 2’-O-methyl, 2’-deoxy-2’-fluoro, and/or
`one or more universal base modified nucleotides, and optionally a terminal cap molecule at the
`3’, 5’, or both 3’ and 5’-endsof the sense strand; and wherein the antisense strand comprises any
`of between 1 and 10, specifically about 1, 2, 3, 4, 5, 6, 7, 8 , 9 , or 10 phosphorothioate
`internucleotide linkages, and/or one or more 2’-deoxy, 2’-O-methyl, 2’-deoxy-2’-fluoro, and/or
`one or more universal base modified nucleotides, and optionally a terminal cap molecule at the
`3°, 5’, or both 3’ and 5’-endsofthe antisense strand.
`In another embodiment, one or more, for
`example about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 pyrimidine nucleotides of the sense and/or antisense
`- siRNA stand are chemically modified with 2’-deoxy, 2’-O-methyl and/or 2’-deoxy-2’-fluoro
`nucleotides, with or without one or more, for example about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10
`phosphorothioate intemucleotide linkages and/or a terminal cap molecule at the 3’, 5’, or both 3’
`and 5’-ends, being present in the sameor different strand.
`
`In another embodiment, the invention features a siRNA molecule, wherein the sense strand
`comprises between 1 and 5, specifically about 1, 2, 3, 4, or 5 phosphorothioate internucleotide
`linkages, and/or one or more 2’-deoxy, 2’-O-methyl, 2’-deoxy-2’-fluoto, and/or one or more
`universal base modified nucleotides, and optionally a terminal cap molecule at the 3’, 5’, or both
`3° and 5’-ends of the sense strand; and wherein the antisense strand comprises any of between 1
`and 5, specifically about 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages, and/or one or
`more 2’-deoxy, 2’-O-methyl, 2’-deoxy-2’-fluoro, and/or one or more universal base modified
`nucleotides, and optionally a terminal cap molecule at the 3’, 5’, or both 3° and 5’-ends of the
`antisense strand. In another embodiment, one or more, for example about1, 2, 3, 4, 5, 6, 7, 8, 9,
`or 10 pyrimidine nucleotides of the sense and/or antisense siRNA stand are chemically modified
`with 2’-deoxy, 2’-O-methyl and/or 2’-deoxy-2’-fluoro nucleotides, with or without between 1
`and 5, for example about 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages and/or a
`terminal cap molecule at the 3’, 5’, or both 3’ and 5’-ends, being present in the sameor different
`strand.
`
`In one embodiment, the invention features a siRNA molecule, wherein the antisense strand
`comprises one or more, for example about 1, 2, 3, 4, 5, 6, 7, 8 , 9 , or 10 phosphorothioate
`internucleotide linkages, and/or between one or more 2’-deoxy, 2’-O-methyl, 2’-deoxy-2’-fluoro,
`and/or one or more universal base modified nucleotides, and optionally a terminal cap molecule
`at the 3°, 5’, or both 3’ and 5’-ends of the: sense strand; and wherein the antisense strand
`comprises any of between 1 and 10, specifically about 1, 2, 3, 4, 5, 6, 7, 8,9, or 10
`phosphorothioate internucleotide linkages, and/or one or more 2’-deoxy, 2’-O-methyl, 2’-deoxy-
`2?-fluoro, and/or one or more universal base modified nucleotides, and optionally a terminal cap
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