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`.9599 10 APR 2893
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`Swat)
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`UNITED STATES DEPARTMENT OF COMNIERCE
`United States Patent and Trademark Office
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`April 04, 2003
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`THIS IS TO CERTIFY THAT ANNEXED HERETO IS A TRUE COPY FROM
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`APPLICATION NUMBER: 60/358,580
`FILING DATE: February 20, 2002
`RELATED PCT APPLICATION NUMBER: PCT/US03/05346
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`P1 987218
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`By Authority of the
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`PROVISIONAL APPLICATION FOR PATENTCOVERSHEET
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`This'Is a request for filing a PROVISIONAL APPLICATION FOR PA TENT under 37 CFR §1.53(b)(2).
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`5530 Colt Dr.
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`LAST NAME
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`e
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`FIRST NAME
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`Beigelman
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`MIDDLE
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`RESIDENCE
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`Macejak
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`Zinnen
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`TITLE OF THE INVENTION 280 character maximum
`
`METHODS AND REAGENTS FOR RNA INTERFERENCE MEDIATED INHIBITION OF GENE
`
`EXPRESSION USING CHEMICALLY MODIFIED SYNTHETIC SHORT INTERFERING RNAS
`
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`Date: February 20. 2002
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`TYPED or PRINTED NAME
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`PROVISIONAL APPLICATION FOR PATENT COVERSHEET
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`INVENTOR(S)IAPPLICANTS(S)
`RESIDENCE
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` 4866 Franklin Dr.
`McSwiggen
`James
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`Boulder, CO 80301
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`Fosnaugh
`Kathy
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`Morrissey
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`David
`
`Mokler
`
`Victor
`
`4769 Tanglewood Traii
`Boulder, CO 80301
`
`2400 West 17th Avenue
`201A
`Longmont, CO 80501
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`Jamison
`Sharon
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`Page 2 of 2
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`CERTIFICATE OF MAILING
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`Assistant Commissioner of Patents, Box Provisional Patent Application, Washington,
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`DC. 20231.
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` BY:
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`. No. 47,132
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`
`
`Application for Provisional Patent of Beigeiman et at.
`
`Title: METHODS AND REAGENTS FOR RNA INTERFERENCE MEDIATED
`INHIBITION OF GENE EXPRESSION USING CHEMICALLY MODIFIED
`SYNTHETIC SHORT INTERFERING RNAS
`
`X_
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`Provisional Patent Application (68 pages of specification and 10
`pages of drawings)
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`Docket No. 02-128 (900/024)
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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
`
`The present
`
`invention concerns methods and reagents useful
`
`in modulating gene
`
`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).
`
`h
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`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
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`that any of the work described below is prior art to the claimed invention.
`
`RNA interference refers to the process of sequence—specific post transcriptional gene
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`silencing in animals mediated by short interfering RNAs (siRNA) (Fire at al., 1998, Nature, 391,
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`806). The corresponding process in plants is commonly referred to as post transcriptional gene
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`silencing or RNA silencing and is also referred to as quelling in fungi. The process of post
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`transcriptional gene silencing is thought to be an evolutionarily conserved cellular defense
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`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
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`(dsRNA) derived from viral infection or the random integration of transposon elements into a
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`host genome via a cellular response that specifically destroys homologous single stranded RNA
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`or viral genomic RNA. The presence of dsRNA in cells triggers the RNAi response though a
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`mechanism that has yet to be fully characterized. This mechanism appears to be different from
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`the interferon response that results from dsRNA mediated activation of protein kinase PKR and
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`2’,5’~oligoadenylate synthetase resulting in non-specific cleavage of mRNA by ribonuclease L.
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`The presence of long dsRNAs in cells 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
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`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
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`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
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`structure that are implicated in translational control (Hutvagner et 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
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`stranded RNA having sequence complimentary to the antisense strand of the siRNA duplex.
`
`Cleavage of the target RNA takes place in the middle of the region complementary to the
`
`antisense strand of the siRNA duplex (Elbashir et al., 2001, Genes Dev., 15, 188).
`
`Short interfering RNA mediated RNAi has been studied in a variety of systems. Fire et 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 er
`
`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 ill-nucleotide RNAs in cultured mammalian cells including human embryonic kidney
`
`and HeLa cells. Recent work in Drosophila embryonic lysates (Elbashir at 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
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`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
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`RNA is defined by the 5’-end of the siRNA guide sequence rather than the 3’-end (Elbashir at
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`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 et al., 2001, Cell, 107, 309).
`
`_
`
`Beach at al., International PCT Publication No. WO 01/68836, describes specific methods
`
`for attenuating gene expression using endogenously derived dsRNA. Tuschl et al., International
`
`PCT Publication No. WO 01/75164, describes 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. Bioclzem., 2, 239-245, doubts that RNAi can be used to cure
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`genetic diseases or viral infection due “to the danger of activating interferon response”. Li et (11.,
`
`International PCT Publication No. WO 00/44914, describes the use of specific dsRNAs for use
`
`in attenuating the expression of certain target genes. Zernicka—Goetz er 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 at al, International
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`PCT Publication No. W0 99/32619, describes particular methods for introducing certain dsRNA
`
`molecules into cells for use in inhibiting gene expression. Plaetinck et 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 at al., International PCT Publication No. W0 (ll/29058, describes the identification of
`
`specific genes involved in dsRNA mediated RNAi. Deschamps Depaillette et al., International
`
`PCT Publication No. WO 99/07409, describes specific compositions consisting of particular
`
`dsRNA molecules combined with certain anti-viral agents. Driscoll et (.11., International PCT
`
`Publication No. WO 01/49844, describes specific DNA constructs for use in facilitating gene
`
`silencing in targeted organisms. Parrish et 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 viva and/or improved cellular uptake. The chemically modified
`
`siRNA molecules of the instant invention provide useful reagents and methods for a variety of
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`therapeutic, diagnostic, agricultural, target validation, genomic discovery, genetic engineering
`
`and phannacogenomic applications.
`
`The introduction of chemically modified nucleotides into nucleic acid molecules Will
`
`provide a powerful tool in overcoming limitations of in viva 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
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`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 stranded 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 viva. 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
`
`d'eoxyribonucleotides 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 et a], supra, also report
`
`that substitution of siRNA with 2’-O—methyl
`
`nucleotides completely abolishes RNAi activity. Li et al., International PCT Publication No.
`WO 00/44914, and Beach at 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—1nethyl nucleotides, and nucleotides containing a 2’~O or 4’—
`
`C methylene bridge. However, Kreutzer and Limmer similarly fail to show to what extent these
`
`modifications are tolerated in siRNA molecules nor provide any examples of such modified
`siRNA.
`
`Parrish er al., 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
`
`RNAi triggers (data not shown); [phosphorothioate] modification of more than two residues
`greatly destabilized the RNAs in vitro and we were not able to assay interference activities.” Id.
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`at 1081. The authors also tested certain modifications at the 2’-position of the nucleotide sugar
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`in the
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`long siRNA transcripts
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`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.
`
`Id.
`
`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 intemucleotide linkages, 2’-O-methyl
`
`ribonucleotides, 2’—deoxy-2’-fluoro
`
`ribonucleotides, “universal base” nucleotides, 5-C—methy1 nucleotides, and inverted deoxyabasic
`
`residue incorporation. These chemical modifications, when used in various siRNA constructs,
`
`are shown to preserve RNAi activity in cells while at the same time, dramatically increasing the
`
`serum stability of these compounds. Furthermore, contrary to the data published by Parrish et
`al.', supra, applicant demonstrates that multiple (greater than one) phosphorothioate substitutions
`are well 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
`
`intemucleotide linkage having Formula I:
`
`
`
`Z
`
`R1—X—P-—Y——-R2
`
`w
`
`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, 0-
`
`alkyl, S-alkyl, alkaryl, or aralkyl, and wherein W, X, Y and Z are not all 0.
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`The chemically modified intemucleotide 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 intemucleotide linkages having Formulal 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
`
`intemucleotide 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 intemucleotide
`
`linkages having Formula I in the sense strand, antisense strand, or both strands.
`
`In yet another
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`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, III, 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, R5, R6, R7, R8, R10, R11 and R12 is independently H, OH, alkyl,
`
`substituted alkyl, allcaryl 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,
`
`allcyl—S—alkyl, alkyl—O-alkyl, ONOZ, N02, N3, NH2, aminoalkyl,
`
`aminoacid, aminoacyl, ONHZ, 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 CFZ, and B is a nucleosidic base such as adenine,
`
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`guanine, uracil, cytosine, thymine, 2-aminoadenosine, S-methylcytosine, 2,6—diaminopurine, or
`
`any other non-naturally occurring base that can be employed to form a stable 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 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 Formula II 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
`
`1 and about 5 chemically
`siRNA molecule of the invention can comprise between about
`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 Formula II 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
`III:
`
`
`
`wherein each R3, R4, R5, 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, alkyl-S-alkyl, alkyl-O-alkyl, ONOZ, N02, N3, NHZ, aminoalkyl,
`
`aminoacid, aminoacyl, ONHZ, O—aminoalkyl, O~aminoacid, O-aminoacyl, heterocycloalkyl,
`
`
`
`7 of 80
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`7of80
`7 of 80
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`

`

`A’s-L? aim-Ute Fritafiiiiiifil s firtlri‘sdffijlulud
`
`MBHB02-128
`(900/024)
`
`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,
`
`guanine, uracil, cytosine, thymine, 2—aminoadenosine, S—methylcytosine, 2,6-diaminopun'ne, or
`
`any other non-naturally occurring base that can be employed to form a stable duplex with RNA
`
`or a non—nucleosidic base such as phenyl, naphthyl, 3-nitropyrrole, 5-nitroindole, nebulan'ne,
`
`pyridone, pyridinone, or any other non-naturally occurring universal base that can be employed
`
`to form a stable duplex with RNA.
`
`The chemically modified nucleotide or non-nucleotide of Formula III 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 Formula III at the 3’-end, 5’send, 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 nucleotide or non-nucleotide of Formula 111 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 I and about 5 chemically modified nucleotide or non—
`nucleotide of Formula III at the 3’-end of the sense strand, antisense strand, or both strands.
`
`In another embodiment, a siRNA molecule of the invention comprises a nucleotide having
`
`Formula II or III, wherein the nucleotide having Formula II or III 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’-eud, 5’—end, or both 3’ and 5’ ends of
`one or both siRNA 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 a 5’—terminal phosphate group having Forula IV:
`
`Z
`
`x—P—Y—
`
`W
`
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`MBHB02—128
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`
`wherein each X and Y is independently O, S, N, alkyl, substituted alkyl, or alkylhalo; each 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 0.
`
`In one 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 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 11 and/or Formula III.
`
`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 phosphorothioate internucleotide linkages. For
`
`. example,
`
`in a non—limiting example,
`
`the invention features a chemically modified short
`
`interfering RNA (siRNA) having about 1, 2, 3, 4, 5, 6, 7, or 8 phosphorothioate intemucleotide
`
`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 intemucleotide 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
`
`intemucleotide linkages in the sense strand, antisensc strand, or both strands.
`
`In one embodiment, the invention features a siRNA molecule, wherein the sense strand
`
`cdmprises one or more, for example about 1, 2, 3, 4, 5, 6, 7, 8 , 9 , or 10 phosphorothioate
`
`9 of 80
`
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`9 of 80
`
`

`

`Iris {tweets w . starts“ at.“ it .I.I {at
`
`MBHB02-128
`(900/024)
`
`
`
`intemucleotide linkages, and/or one or more 2’-deoxy, 2’-O-methyl, 2’—deoxy—2’-fluoro, and/or
`
`one or more uniyersal 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
`
`intemucleotide 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 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-methy1 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 internucleotide linkages and/or a terminal cap molecule at the 3’, 5’, or both 3’
`
`and 5’—ends, being present in the same or 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’-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 5, specifically about 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages, and/or one or
`
`more 2’-deoxy, 2’-O—methy1, 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 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 between 1
`
`and 5, for example about 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages and/or a
`
`temiinal cap molecule at the 3’, 5’, or both 3’ and 5’-ends, being present in the same or 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,