(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`
`(19) World Intellectual Property Organization
`International Bureau
`
`1111111111111111111111111111111111111111111111111111111111111111111111111111111111111
`
`(43) International Publication Date
`28 August 2003 (28.08.2003)
`
`PCT
`
`(10) International Publication Number
`WO 03/070918 A2
`
`(51) International Patent Classification7:
`
`C12N
`
`(21) International Application Number: PCT/US03/05346
`
`(22) International Filing Date: 20 February 2003 (20.02.2003)
`
`(25) Filing Language:
`
`(26) Publication Language:
`
`English
`
`English
`
`(30) Priority Data:
`60/358,580
`60/363,124
`60/386,782
`60/406,784
`60/408,378
`60/409,293
`60/440,129
`
`20 February 2002 (20.02.2002) us
`11 March 2002 (11.03.2002) us
`6 June 2002 (06.06.2002) us
`29 August 2002 (29.08.2002) us
`5 September 2002 (05.09.2002) us
`9 September 2002 (09.09.2002) us
`15 January 2003 (15.01.2003) us
`
`(63) Related by continuation (CON) or continuation-in-part
`(CIP) to earlier applications:
`us
`Filed on
`us
`Filed on
`
`us 60/358,580 (CON)
`20 February 2002 (20.02.2002)
`us 60/336,124 (CON)
`11 March 2002 (11.03.2002)
`
`us
`Filed on
`us
`Filed on
`us
`Filed on
`us
`Filed on
`us
`Filed on
`
`us 60/386,782 (CON)
`6 June 2002 (06.06.2002)
`us 60/406,784 (CON)
`29 August 2002 (29.08.2002)
`us 60/408,378 (CON)
`5 September 2002 (05.09.2002)
`us 60/409,293 (CON)
`9 September 2002 (09.09.2002)
`us 60/440,129 (CON)
`15 January 2003 (15.01.2003)
`
`(71) Applicant (jor all designated States except US): RI(cid:173)
`BOZYME
`PHARMACEUTICALS,
`INCORPO(cid:173)
`RATED [US/US]; 2950 Wilderness Place, Boulder, CO
`80301 (US).
`
`(72) Inventors; and
`(75) Inventors/Applicants (jor US only): McSWIGGEN,
`James [US/US]; 4866 Franklin Drive, Boulder, CO
`80301 (US). BEIGELMAN, Leonid [US/US]; 5530 Colt
`Drive, Longmont, CO 80503 (US). MACEJAK, Dennis
`[US/US]; 6595 Union Street, Arvada, CO 80004 (US).
`ZINNEN, Shawn [US/US]; 2378 Birch Street, Denver,
`CO 80207 (US). PAVCO, Pamela [US/US]; 705 Barberry
`Circle, Lafayette, CO 80026 (US). MORRISSEY, David
`
`[Continued on next page]
`------------------------------------------------------------------------------------------
`(54) Title: RNA INTERFERENCE MEDIATED INHIBITION OF GENE EXPRESSION USING CHEMICALLY MODIFIED
`SHORT INTERFERING NUCLEIC ACID
`
`HCV/Replicmz KJ#I-Clone A Cells transfected
`with 0.5pl/well LFA 2K-72 hours
`
`~ l 0.8
`
`-----
`---iiiiiiii
`iiiiiiii ----
`
`1 . . ; [r
`
`,,.
`••
`
`- -
`
`QO
`,..-.!
`0\
`0
`t"---
`0
`-.... (57) Abstract: The present invention concerns methods and reagents useful in modulating gene expression in a variety of appli-
`~ cations, including use in therapeutic, diagnostic, target validation, and genomic discovery applications. Specifically, the invention
`relates to synthetic chemically modified small nucleic acid molecules, such as short interfering nucleic acid (siNA), short interfering
`0 RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), and short hairpin RNA (shRNA) molecules capable of me(cid:173)
`> diating RNA interference (RNAi) against target nucleic acid sequences. The small nucleic acid molecules are useful in the treatment
`~ of any disease or condition that responds to modulation of gene expression or activity in a cell, tissue, or organism.
`
`Alnylam Exh. 1064
`
`i
`
`

`

`WO 03/070918 A2
`
`1111111111111111111111111111111111111111111111111111111111111111111111111111111111111
`
`[US/US]; 4769 Tanglewood Trail, Boulder, CO 80301
`(US). FOSNAUGH, Kathy [US/US]; 2400 West 17th
`Avenue 201A, Longmont, CO 80501 (US). MOKLER,
`Victor [US/US]; 153 Divide View Drive, Golden, CO
`80403 (US). JAMISON, Sharon [US/US]; 4985 Twin
`Lakes Rd #89, Boulder, CO 80301 (US).
`
`(74) Agent: TERPSTRA, Anita, J.; McDonnell Boehnen Hul(cid:173)
`bert & Berghoff, 300 South Wacker Drive, Suite 3200,
`Chicago, IL 60606 (US).
`
`(81) Designated States (national): AE, AG, AL, AM, AT, AU,
`AZ, BA, BB, BG, BR, BY, BZ, CA, CH, CN, CO, CR, CU,
`CZ, DE, DK, DM, DZ, EC, EE, ES, Fl, GB, GD, GE, GH,
`GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC,
`LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW,
`MX, MZ, NO, NZ, OM, PH, PL, PT, RO, RU, SC, SD, SE,
`
`SG, SK, SL, TJ, TM, TN, TR, TT, TZ, UA, UG, US, UZ,
`VC, VN, YU, ZA, ZM, ZW.
`
`(84) Designated States (regional): ARIPO patent (GH, GM,
`KE, LS, MW, MZ, SD, SL, SZ, TZ, UG, ZM, ZW),
`Eurasian patent (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM),
`European patent (AT, BE, BG, CH, CY, CZ, DE, DK, EE,
`ES, Fl, FR, GB, GR, HU, IE, IT, LU, MC, NL, PT, SE, SI,
`SK, TR), OAPI patent (BF, BJ, CF, CG, CI, CM, GA, GN,
`GQ, GW, ML, MR, NE, SN, TD, TG).
`
`Published:
`without international search report and to be republished
`upon receipt of that report
`
`For two-letter codes and other abbreviations, refer to the "Guid(cid:173)
`ance Notes on Codes and Abbreviations" appearing at the begin(cid:173)
`ning of each regular issue of the PCT Gazette.
`
`ii
`
`

`

`wo 03/070918
`
`PCT /US03/05346
`
`RNA INTERFERENCE MEDIATED INHIBITION OF GENE EXPRESSION
`
`USING CHEMICALLY MODIFIED SHORT INTERFERING NUCLEIC ACID
`
`(siN A)
`
`This invention claims the benefit of Beigelman USSN 60/358,580 filed February
`
`5
`
`20, 2002, of Beigelman USSN 60/363,124 filed March 11, 2002, of Beigelman USSN
`
`60/386,782 filed June 6, 2002, ofBeigelman USSN 60/406,784 filed August 29,2002, of
`
`Beigelman USSN 60/408,378 filed September 5, 2002, of Beigelman USSN 60/409,293
`
`filed September 9, 2002, and of Beigelman USSN 60/440,129 filed January 15, 2003.
`
`These applications are hereby incorporated by reference herein in their entireties,
`
`10
`
`including the drawings.
`
`Field Of The Invention
`
`The present invention concerns methods and reagents useful in modulating gene
`
`expression in a variety of applications, including use in the~apeutic, diagnostic, target
`
`validation, and genomiC discovery applications. Specifically, the invention relates to
`
`15
`
`synthetic small nucleic acid molecules, such as short interfering nucleic acid (siNA), short
`
`interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), and
`
`short hairpin RNA (shRNA) molecules capable of mediating RNA interference (RNAi).
`
`Background Of The Invention
`
`The following is a discussion of relevant art pertaining to RNAi. The discussion is
`
`20
`
`provided only for understanding of the invention that follows. The summary is not an
`
`admission that any of the work described below is prior art to the claimed invention.
`
`Applicant demonstrates herein that chemically modified short interfering nucleic acids
`
`possess the same capacity to mediate RNAl as do siRNA molecules and are expected to
`
`possess improved stability and activity in vivo; therefore, this discussion is not meant to
`
`25
`
`be limiting only to siRNA and can be applied to siNA as a whole.
`
`RNA interference refers to the process of sequence-specific post-transcriptional
`
`gene silencing in animals mediated by short interfering RNAs (siRNAs) (Fire et al., 1998,
`
`Nature, 391, 806). The corresponding process in plants is commonly referred to as post(cid:173)
`
`transcriptional gene silencing or RNA silencing and is also referred to as quelling in
`
`1
`
`

`

`wo 03/070918
`
`PCT/US03/05346
`
`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 and 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
`
`5
`
`in response to the production of double-stranded RNAs ( dsRNAs) derived from viral
`
`infection or from the random integration of transposon elements into a host genome via a
`
`cellular response that specifically destroys homologous single-stranded RNA or viral
`
`genomic RNA. The presence of dsRNA in cells triggers the RNAi response though a
`
`mechanism that has yet to be fully characterized. This mechanism appears to be different
`
`10
`
`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 by ribonuclease L.
`
`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
`
`15
`
`pieces of dsRNA known as short interfering RNAs (siRNAs) (Berstein et al., 2001,
`
`Nature, 409, 363). Short interfering RNAs derived from dicer activity are typically about
`
`21 to about 23 nucleotides in length and comprise about 19 base pair duplexes (Elbashir
`
`et al., 2001, Genes Dev., 15, 188). Dicer has also been implicated in the excision of 21- ·
`
`and 22-nucleotide small temporal RNAs (stRNAs) from precursor RNA of conserved
`
`20
`
`structure that are implicated in translational control (Hutvagner et al., 2001, Science, 293,
`
`834). The RNAi response also features an endonuclease complex, commonly referred to
`
`as an RNA-induced silencing complex (RISC), which mediates cleavage of single(cid:173)
`
`stranded RNA having sequence complementary to the antisense strand of the siRNA
`
`duplex. Cleavage of the target RNA takes place in the middle of the region
`
`25
`
`complementary to the antisense strand of the siRNA duplex (Elbashir et al., 2001, Genes
`Dev., 15, 188).
`
`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, describe RNAi mediated by dsRNA in mouse embryos. Hammond et al.,
`
`30
`
`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
`
`2
`
`

`

`wo 03/070918
`
`PCT/US03/05346
`
`embryonic kidney and HeLa cells. Recent work in Drosophila embryonic lysates
`
`(Elbashir et 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
`
`5
`
`are most active when containing 3'-terminal dinucleotide overhangs. Furthennore,
`
`complete substitution of one or both siRNA strands with 2'-deoxy (2'-H) or 2'-0-methyl
`
`nucleotides abolishes RNAi activity, whereas substitution of the 3'-terminal siRNA
`
`overhang nucleotides with 2'-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
`
`10
`
`activity. In addition, these studies also indicate that the position of the cleavage site in the
`
`target RNA is defined by the 5'-end of the siRNA guide sequence rather than the 3'-end of
`the guide sequence (Elbashir et at 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
`
`15
`
`on the siRNA (Nykanen et al., 2001, Cell, 107, 309).
`
`Studies have shown that replacing the 3'-terminal nucleotide overhanging segments
`
`of a 21-mer
`
`siRNA duplex having
`
`two
`
`-nucleotide 3'-overhangs with
`
`deoxyribonucleotides does not have an adverse effect on RNAi activity. Replacing up to
`
`four nucleotides on each end of the siRNA with deoxyribonucleotides has been reported
`
`20
`
`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 al.,
`
`supra, also report that substitution of siRNA with 2'-0-methyl nucleotides completely
`
`abolishes RNAi activity. Li et al., International PCT Publication No. WO 00/44914, and
`
`Beach et al., International PCT Publication No. WO 01/68836 preliminarily suggest that
`
`25
`
`siRNA may include modifications to either the phosphate-sugar backbone or the
`
`nucleoside to include at least one of a nitrogen or sulfur heteroatom, however, neither
`
`application postulates to what extent such modifications would be tolerated in siRNA
`
`molecules, nor provides any further guidance or examples of such modified siRNA.
`
`Kreutzer et al., Canadian Patent Application No. 2,359,180, also describe certain
`
`30
`
`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'-0-
`
`methyl nucleotides, and nucleotides containing a 2'-0 or 4'-C methylene bridge.
`
`3
`
`

`

`wo 03/070918
`
`PCT/US03/05346
`
`However, Kreutzer et al. similarly fails to provide examples or guidance as to what extent
`
`these modifications would be tolerated in siRNA molecules.
`
`Parrish et al., 2000, Molecular Cell, 6, 1977-1087, tested certain chemical
`
`modifications targeting the unc-22 gene in C. elegans using long (>25 nt) siRNA
`
`5
`
`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. Further, Parrish et al. reported
`
`that phosphorothioate modification of more than two residues greatly destabilized the
`
`10 RNAs in vitro such that interference activities could not be assayed. !d. at 1081. The
`
`authors also tested certain modifications at the 2'-position of the nucleotide sugar in the
`
`long siRNA transcripts and found 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
`
`15
`
`authors tested certain base modifications, including substituting, in sense and antisense
`
`strands of the siRNA, 4-thiouracil, 5-bromouracil, 5-iodouracil, and 3-(aminoallyl)uracil
`
`for uracil, and inosine for guanosine. Whereas 4-thiouracil and 5-bromouracil
`
`substitution appeared to be tolerated, Parrish reported that inosine produced a substantial
`
`decrease in interference activity when incorporated in either strand. Parrish also reported
`
`20
`
`that incorporation of 5-iodouracil and 3-( aminoallyl)uracil in the antisense strand resulted
`
`in a substantial decrease in RNAi activity as well.
`
`The use of longer dsRNA has been described. For example, Beach et al.,
`
`International PCT Publication No. WO 01/68836, describes specific methods for
`
`attenuating gene expression using endogenously-derived dsRNA.
`
`Tuschl et al.,
`
`25
`
`International PCT Publication No. WO 01/75164, describe a Drosophila in vitro RNAi
`
`system and the use of specific siRNA molecules for certain functional genomic and
`
`certain therapeutic applications; although Tuschl, 2001, Chern. Biochem., 2, 239-245,
`
`doubts that RNAi can be used to cure genetic diseases or viral infection due to the danger
`of activating interferon response. Li et a!., International PCT Publication No. WO
`00/44914, describe the use of specific dsRNAs for attenuating the expression of certain
`
`30
`
`target genes. Zernicka-Goetz eta!., International PCT Publication No. WO 01/36646,
`describe certain methods for inhibiting the expression of particular genes in mammalian
`
`4
`
`

`

`wo 03/070918
`
`PCT/US03/05346
`
`cells using certain dsRNA molecules. Fire et al., International PCT Publication No. WO
`
`99/32619, describe 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, describe certain methods for identifying specific genes responsible for
`
`5
`
`conferring a particular phenotype in a cell using specific dsRNA molecules. Mello et al.,
`
`International PCT Publication No. WO 01/29058, describe the identification of specific
`
`genes involved in dsRNA-mediated RNAi. Deschamps Depaillette et al., International
`
`PCT Publication No. WO 99/07409, describe specific compositions consisting of
`
`particular dsRNA molecules combined with certain anti-viral agents. ·waterhouse et al.,
`
`10
`
`International PCT Publication No. 99/53050, describe certain methods for decreasing the
`
`phenotypic expression of a nucleic acid in plant cells using certain dsRNAs. Driscoll et
`
`al., International PCT Publication No. WO 01/49844, describe specific DNA constructs
`
`for use in facilitating gene silencing in targeted organisms.
`
`Others have reported on various RNAi and gene-silencing systems. For example,
`
`15
`
`Parrish et al., 2000, Molecular Cell, 6, 1977-1087, describe specific chemically-modified
`siRNA constructs targeting the unc-22 gene of C. elegans. Grossniklaus, International
`
`PCT Publication No. WO 01/38551, describes certain methods for regulating polycomb
`
`gene expression in plants using certain dsRNAs. Churikov et al., International PCT
`
`Publication No. WO 01/42443, describe certain methods for modifying genetic
`
`20
`
`characteristics of an organism using certain dsRNAs. Cogoni et al., International PCT
`
`Publication No. WO 01/53475, describe certain methods for isolating a Neurospora
`
`silencing gene and uses thereof. Reed et al., International PCT Publication No. WO
`
`01/68836, describe certain methods for gene silencing in plants. Honer et al.,
`
`International PCT Publication No. WO 01/70944, describe certain methods of drug
`
`25
`
`screening using transgenic nematodes as Parkinson's Disease models using certain
`
`dsRNAs. Deak et al., International PCT Publication No. WO 01/72774, describe certain
`
`Drosophila-derived gene products that may be related to RNAi .in Drosophila. Arndt et
`
`al., International PCT Publication No. WO 01/92513 describe certain methods for
`
`mediating gene suppression by using factors that enhance RNAi. Tuschl et al.,
`
`30
`
`International PCT Publication No. WO 02/44321, describe certain synthetic siRNA
`
`constructs. Pachuk et al., International PCT Publication No. WO 00/63364, and
`
`Sa:tishchandran et al., International PCT Publication No. WO 01/04313, describe certain
`
`5
`
`

`

`wo 03/070918
`
`PCT/US03/05346
`
`methods and compositions for inhibiting the function of certain polynucleotide sequences
`
`using certain dsRNAs. Echeverri et al., International PCT Publication No. WO 02/38805,
`describe certain C. elegans genes identified via RNAi. Kreutzer et al., International PCT
`
`Publications Nos. WO 02/055692, WO 02/055693, and EP 1144623 Bl describes certain
`
`5 methods for inhibiting gene expression using RNAi. Graham eta!., International PCT
`
`Publications Nos. WO 99/49029 and WO 01/70949, and AU 4037501 describe certain
`
`vector expressed siRNA molecules. Fire et al., US 6,506,559, describe certain methods
`
`for inhibiting gene expression in vitro using certain long dsRNA (greater than 25
`
`nucleotide) constructs that mediate RNAi.
`
`10
`
`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 synthetic small nucleic acid molecules, such as short interfering nucleic
`
`acid (siNA), short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-
`
`15 RNA (miRNA), and short hairpin RNA (shRNA) molecules capable of modulating gene
`
`expression in cells by RNA inference (RNAi). The siN A molecules of the invention can
`
`be chemically modified. The use of chemically modified siNA can improve various
`
`properties of native siRNA molecules through increased resistance to nuclease
`
`degradation in vivo and/or improved cellular uptake. The chemically modified siNA
`
`20 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.
`
`In a non-limiting example, the introduction of chemically modified nucleotides into
`
`nucleic acid molecules provides a powerful tool in overcoming potential limitations of in
`
`25
`
`vivo stability and bioavailability inherent to native RNA molecules that are delivered
`
`exogenously. For example, 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
`
`30
`
`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
`
`6
`
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`

`wo 03/070918
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`PCT/US03/05346
`
`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 unmodified siRNA, chemically
`
`5 modified siNA can also minimize the possibility of activating interferon activity in
`
`humans.
`
`In one embodiment, the nucleic acid molecules of the invention that act as
`
`mediators of the RNA interference gene silencing response are chemically modified
`
`double stranded nucleic acid molecules. As in their native double stranded RNA
`
`10
`
`counterparts, these siN A 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'(cid:173)
`
`overhangs. These overhanging segments are readily hydrolyzed by endonucleases in vivo.
`
`15
`
`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,
`
`20
`
`EMBO J., 20, 6877). In addition, Elbashir et al, supra, also report that substitution of
`
`siRNA with 2' -0-methyl nucleotides completely abolishes RNAi activity. Li et al.,
`
`International PCT Publication No. WO 00/44914, and Beach et 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
`
`25
`
`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,
`
`30
`
`specifically 2'-amino or 2'-0-methyl nucleotides, and nucleotides containing a 2'-0 or
`
`4' -C methylene bridge. However, Kreutzer and Limmer similarly fail to show to what
`
`7
`
`

`

`wo 03/070918
`
`PCT/US03/05346
`
`extent these modifications are tolerated in siRNA molecules nor provide any examples of
`
`such modified siRNA.
`
`In one embodiment, the invention features chemically modified siNA constructs
`
`having specificity for target nucleic acid molecules in a cell. Non-limiting examples of
`
`5
`
`such chemical modifications include without limitation phosphorothioate intemucleotide
`
`linkages, 2 '-O-m ethyl ribonucleotides, 2' -deoxy-2 '-flu oro ribonucleotides, 2' -deoxy
`
`ribonucleotides, "universal base" nucleotides, 5-C-methyl nucleotides, and inverted
`
`deoxyabasic residue incorporation. These chemical modifications, when used in various
`
`siNA constructs, are shown to preserve RNAi activity in cells while at the same time,
`
`10
`
`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 siNA constructs.
`
`In one embodiment, the chemically-modified siNA molecules of the invention
`
`15
`
`comprise a duplex having two strands, one or both of which can be chemically-modified,
`
`wherein each strand is about 19 to about 29 (e.g., about 19, 20, 21, 22, 23, 24, 25, 26, 27,
`
`28,or 29) nucleotides. In one embodiment, the chemically-modified siNA molecules of
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`the invention comprise a duplex having two strands, one or both of which can be
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`chemically-modified, wherein each strand is about 19 to about 23 (e.g., about 19, 20, 21,
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`22, or 23) nucleotides. In one embodiment, a siNA molecule of the invention comprises
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`modified nucleotides while maintaining the ability to mediate RNAi. The modified
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`nucleotides can be used to improve in vitro or in vivo characteristics such as stability,
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`activity, and/or bioavailability. For example, a siNA molecule of the invention can
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`comprise modified nucleotides as a percentage of the total number of nucleotides present
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`in the siN A molecule. As such, a siNA molecule of the invention can generally comprise
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`modified nucleotides from about 5 to about 100% of the nucleotide positions (e.g., 5%,
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`10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,
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`85%, 90%, 95% or 100% of the nucleotide positions). The actual percentage of modified
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`nucleotides present in a given siN A molecule depends on the total number of nucleotides
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`present in the siNA. If the siNA molecule is single stranded, the percent modification can
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`be based upon the total number of nucleotides present in the single stranded siNA
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`molecules. Likewise, if the siNA molecule is double stranded, the percent modification
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`can be based upon the total number of nucleotides present in the sense strand, antisense
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`strand, or both the sense and antisense strands.
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`In addition, the actual percentage of
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`modified nucleotides present in a given siNA molecule can also depend on the total
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`number of purine and pyrimidine nucleotides present in the siNA, for example, wherein
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`all pyrimidine nucleotides and/or all purine nucleotides present in the siNA molecule are
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`modified.
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`The antisense region of a siNA molecule of the invention can comprise a
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`phosphorothioate intemucleotide linkage at the 3'-end of said antisense region. The
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`antisense region can comprise about one to about five phosphorothioate intemucleotide
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`linkages at the 5'-end of said antisense region. The 3'-tenninal nucleotide overhangs of a
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`siNA molecule of the invention can comprise ribonucleotides or deoxyribonucleotides
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`that are chemically-modified at a nucleic acid sugar, base, or backbone. The 3'-tenninal
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`nucleotide overhangs can comprise one or more universal base ribonucleotides. The 3'(cid:173)
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`tenninalnucleotide overhangs can comprise one or more acyclic nucleotides.
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`In one embodiment, the invention features a double-stranded short interfering
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`nucleic acid (siN A) molecule that down-regulates expression of a target gene, wherein the
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`siNA molecule comprises no ribonucleotides and each strand of the double-stranded siNA
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`comprises about 21 nucleotides.
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`In one embodiment, one of the strands of a double-stranded siN A molecule of the
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`invention comprises a nucleotide sequence that is complementary to a nucleotide
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`sequence or a portion thereof of a target gene, and wherein the second strand of a double(cid:173)
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`stranded siNA molecule comprises a nucleotide sequence substantially similar to the
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`nucleotide sequence or a portion thereof of the target gene.
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`In one embodiment, a siNA molecule of the invention comprises about 19 to about
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`23 nucleotides, and each strand comprises at least about 19 nucleotides that are
`complementary to the nucleotides of the other strand.
`
`In one embodiment, a siNA molecule of the invention comprises an antisense
`region comprising a nucleotide sequence that is complementary to a nucleotide sequence
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`or a portion thereof of a target gene, and the siNA further comprises a sense region,
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`30 wherein the sense region comprises a nucleotide sequence substantially similar to the
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`nucleotide sequence or a portion thereof of the target gene. The antisense region and the
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`sense region each comprise about 19 to about 23 nucleotides, and the antisense region
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`comprises at least about 19 nucleotides that are complementary to nucleotides of the
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`sense region.
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`5
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`In one embodiment, a siN A molecule of the invention comprises a sense region and
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`an antisense region, wherein the antisense region comprises a nucleotide sequence that is
`
`complementary to a nucleotide sequence or a portion thereof of RNA encoded by a target
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`gene and the sense region comprises a nucleotide sequence that is complementary to the
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`antisense region.
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`In one embodiment, a siN A molecule of the invention is assembled from two
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`separate oligonucleotide fragments wherein one fragment comprises the sense region and
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`the second fragment comprises the antisense region of the siNA molecule.
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`In another
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`embodiment, the sense region is connected to the antisense region via a linker molecule,
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`which can be a polynucleotide linker or a non-nucleotide linker.
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`In one embodiment, a siNA molecule of the invention comprises a sense region and
`
`antisense region, wherein pyrimidine nucleotides in the sense region compries 2'-0-
`
`methyl pyrimidine nucleotides and purine nucleotides in the sense region comprise 2'(cid:173)
`
`deoxy purine nucleotides.
`
`In one embodiment, a siNA molecule of the invention comprises a sense region and
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`20
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`antisense region, wherein pyrimidine nucleotides present in the sense region comprise 2'(cid:173)
`
`deoxy-2'-fluoro pyrimidine nucleotides and wherein purine nucleotides present in the
`
`sense region comprise 2'-deoxy purine nucleotides.
`
`In one embodiment, a siN A molecule of the invention comprises a sense region and
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`antisense region, wherein the sense region includes a terminal cap moiety at the 5'-end,
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`the 3'-end, or both of the 5' and 3' ends of the sense region. In another embodiment, the
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`terminal cap moiety is an inverted deoxy abasic moiety.
`
`In one embodiment, a siN A molecule of the invention comprises a sense region and
`
`antisense region, wherein pyrimidine nucleotides of the antisense region comprise 2'-
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`deoxy-2'-fluoro pyrimidine nucleotides and purine nucleotides of the antisense region
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`comprise 2'-0-methyl purine nucleotides.
`
`In one embodiment, a siN A molecule of the invention comprises a sense region and
`
`antisense region, wherein pyrimidine nucleotides present in the antisense region are 2'-
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`5
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`deoxy-2'-fluoro pyrimidine nucleotides and wherein purine nucleotides present in the
`
`antisense region comprise 2'-deoxy- purine nucleotides.
`
`In one embodiment, a siN A molecule of the invention comprises a sense region and
`
`antisense
`
`region, wherein
`
`the antisense
`
`region comprises a phosphorothioate
`
`intemucleotide linkage at the 3' end ofthe antisense region.
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`In one embodiment, a siN A molecule of the invention comprises a sense region and
`
`antisense region, wherein the antisense region comprises a glyceryl modification at the 3'
`
`end ofthe antisense region.
`
`In one embodiment, a siNA molecule of the invention is assembled from two
`
`separate oligonucleotide fragments, wherein each of the two fragments of the siNA
`
`15 molecule comprise 21 nucleotides.
`
`h1 one embodiment, a siNA molecule of the invention is assembled from two
`
`separate oligonucleotide fragments, wherein about 19 nucleotides of each fragment of the
`
`siN A molecule are base-paired to the complementary nucleotides of the other fragment of
`
`the siN A molecule and wherein at least two 3' terminal nucleotides of each fragment of
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`20
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`the siNA molecule are not base-paired to the nucleotides of the other fragment of the
`
`siNA molecule.
`
`In one embodiment, a siNA molecule of the invention is assembled f