(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`
`(19) World Intellectual Property Organization
`International Bureau
`
`(43) International Publication Date
`12 September 2003 (12.09.2003)
`
`
`
`PCT
`
`(10) International Publication Number
`W0 03/074654 A2
`
`(51)
`
`International Patent Classification7:
`
`C 12N
`
`(21)
`
`International Application Number:
`
`PCT/US03/05028
`
`(22)
`
`International Filing Date: 20 February 2003 (20.02.2003)
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`(25)
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`Filing Language:
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`(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)
`
`11 March 2002 (11.03.2002)
`6 June 2002 (06.06.2002)
`29 August 2002 (29.08.2002)
`5 September 2002 (05.09.2002)
`9 September 2002 (09.09.2002)
`15 January 2003 (15.01.2003)
`
`US
`
`US
`US
`US
`US
`US
`US
`
`(63)
`
`Related by continuation (CON) or continuation-in-part
`(CIP) to earlier applications:
`US
`Filed on
`
`60/358,580 (CON)
`20 February 2002 (20.02.2002)
`
`US
`Filed on
`US
`Filed on
`US
`Filed on
`US
`Filed on
`US
`Filed on
`US
`Filed on
`
`60/363, 124 (CON)
`11 March 2002 (11.03.2002)
`60/386,782 (CON)
`6 June 2002 (06.06.2002)
`60/406,784 (CON)
`29 August 2002 (29.08.2002)
`60/408,378 (CON)
`5 September 2002 (05.09.2002)
`60/409,293 (CON)
`9 September 2002 (09.09.2002)
`60/440,129 (CON)
`15 January 2003 (15.01.2003)
`
`(71)
`
`Applicant U’or all designated States except US): Sirna
`Therapeutics,
`Inc [US/US]; 2950 Wilderness Place,
`Boulder, CO 80301 (US).
`
`(72)
`(75)
`
`Inventors; and
`Inventors/Applicants (for US only): MCSWIGGEN,
`James [US/US]; 4866 Franklin Drive, Boulder, CO
`80301 (US). BEIGELMAN, Leonid [US/US]; 5530
`Colt Drive, Longmont, CO 80503 (US). CHOWRIRA,
`Bharat [US/US]; 576 Manorwood Lane, Louisville, CO
`80027 (US). PAVCO, Pamela [US/US]; 705 Barberry
`
`[Continued on next page]
`
`(54) Title: RNA INTERFERENCE MEDIATED INHIBITION OF GENE EXPRESSION USING SHORT INTERFERING NU—
`CLEIC ACID (SINA)
`
`A549 24h PCNA mRNA Expression
`
`
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`
`03/074654A2
`
`(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 small nucleic acid molecules, such as short interfering nucleic acid (siNA), short interfering RNA (siRNA), double—stranded
`O RNA (dsRNA), micro—RNA (miRNA), and short hairpin RNA (shRNA) molecules capable of mediating RNA interference (RNAi)
`against target nucleic acid sequences. The small nucleic acid molecules are useful in the treatment of any disease or condition that
`B
`responds to modulation of gene expression or activity in a cell, tissue, or organism.
`
`MTX1051
`
`1
`
`MTX1051
`
`

`

`W0 03/074654
`
`A2
`
`Circle, Lafayette, CO 80026 (US). FOSNAUGH, Kathy
`[US/US]; 1030 Edinboro Drive, Boulder, Colorado 80305
`(US). JAMISON, Sharon [US/US]; 4985 Twin Lakes
`Rd, #89, Boulder, CO 80301 (US). USMAN, Nassim
`[US/US]; 2129 Night Sky Lane, Lafayette, CO 80026
`(US). THOMPSON, James
`[US/US]; 705 Barberry
`Circle, Lafayette, CO 80026 (US).
`
`(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, FT, 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).
`
`Agent: TERPSTRA, Anita, J.; McDonnell Boehnen Hul—
`bert & Berghoff, 300 South Wacker Drive, Suite 3200,
`Chicago, IL 60606 (US).
`
`Declaration under Rule 4.17:
`
`of inventorship (Rule 4.] 7(iv)) for US only
`
`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, FT, 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.
`
`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-
`ance Notes on Codes andAbbreviations ” appearing at the begin-
`ning ofeach regular issue ofthe PCT Gazette.
`
`(74)
`
`(81)
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`2
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`W0 03/074654
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`PCT/US03/05028
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`RNA INTERFERENCE MEDIATED INHIBITION OF GENE EXPRESSION
`
`USING SHORT INTERFERING NUCLEIC ACID (siNA)
`
`This invention claims the benefit of Beigelman USSN 60/358,580 filed February
`
`20, 2002, of Beigelman USSN 60/363,124 filed March 11, 2002, of Beigelman USSN
`
`60/386,782 filed June 6, 2002, of Beigelman 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,l29 filed January 15, 2003.
`
`These applications are hereby incorporated by reference herein in their entireties,
`
`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 therapeutic, diagnostic, target
`
`validation, and genomic discovery applications. Specifically, the invention relates to
`
`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
`
`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 RNAi as do siRNA molecules and are expected to
`
`possess improved stability and activity in Vivo; therefore, this discussion is not meant to
`
`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 (11., 1998,
`
`Nature, 391, 806). The corresponding process in plants is commonly referred to as post—
`
`transcriptional gene silencing or RNA silencing and is also referred to as quelling in
`
`fungi.
`
`The process of post-transcriptional gene silencing is
`
`thought
`
`to be an
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`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
`
`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
`
`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
`
`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
`
`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-
`
`stranded RNA having sequence complementary to the antisense strand of the siMA
`
`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
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`10
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`15
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`20
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`25
`
`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.,
`
`2000, Nature, 404, 293, describe RNAi in Drosophila cells transfected with dsRNA.
`
`30
`
`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 HeLa cells. Recent work in Drosophila embryonic lysates
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`(Elbashir er 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 3'—terminal dinucleotide 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 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
`
`activity. In addition, these studies also indicate that the position of the cleavage site in the
`
`10
`
`target RNA is defined by the 5'-end of the siRNA guide sequence rather than the 3'—end of
`
`the guide sequence (Elbashir er 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).
`
`15
`
`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
`
`to be well tolerated, whereas complete substitution with deoxyribonucleotides results in
`no RNAi activity (Elbashir et al., 2001, EMBO J., 20, 6872). In addition, Elbashir el al.,
`
`20
`
`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 et a1, Intemational PCT Publication No. WO 01/68836 preliminarily suggest that
`
`siRNA may include modifications to either the phosphate~sugar backbone or the
`
`25
`
`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
`
`(21., Canadian Patent Application No. 2,359,180, also describe certain
`
`chemical modifications for use in dsRNA constructs in order to counteract activation of
`
`30
`
`double-stranded RNA—dependent protein kinase PKR, specifically 2'—amino or 2'—O-
`
`methyl nucleotides, and nucleotides containing a 2'-O or 4'-C methylene bridge.
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`However, Kreutzer et al. similarly fails to provide examples or guidance as to what extent
`
`these modifications would be tolerated in siRNA molecules.
`
`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. Further, Parrish et al. reported
`
`that phosphorothioate modification of more than two residues greatly destabilized the
`
`RNAs in vitro such that interference activities could not be assayed.
`
`Id. 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
`
`authors tested certain base modifications, including substituting, in sense and antisense
`
`strands of the siRNA, 4-thiouraci1, 5—br0mouraci1, 5—iodouracil, and 3-(aminoa11yl)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
`
`that incorporation of 5 —iodouraci1 and 3-(aminoallyl)uraci1 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 ez‘ 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, 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, Chem. 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 al.,
`
`International PCT Publication No. WO
`
`00/44914, describe the use of specific dsRNAs for attenuating the expression of certain
`
`target genes. Zernicka-Goetz et al., International PCT Publication No. WO 01/36646,
`
`describe certain methods for inhibiting the expression of particular genes in mammalian
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`cells using certain dsRNA molecules. Fire et al., International PCT Publication No. W0
`
`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
`
`conferring a particular phenotype in a cell using specific dsRNA molecules. Mello et al.,
`
`International PCT Publication No. W0 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 er al,
`
`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,
`
`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
`
`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. W0
`
`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
`
`screenng using transgenic nematodes as Parkinson's Disease models using certain
`
`dsRNAs. Deak er al, International PCT Publication No. W0 01/72774, describe certain
`
`Drosoplzz'la—derived gene products that may be related to RNAi in Drosophz‘la. 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.,
`
`International PCT Publication No. W0 02/44321, describe certain synthetic siRNA
`
`constructs.
`
`Pachuk er al.,
`
`International PCT Publication No. WO 00/63364, and
`
`Satishchandran er al., International PCT Publication No. WO 01/04313, describe certain
`
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`methods and compositions for inhibiting the fimction of certain polynucleotide sequences
`
`using certain dsRNAs. Echeverri et al., International PCT Publication No. W0 02/38805,
`
`describe certain C. elegans genes identified via RNAi. Kreutzer er al., International PCT
`
`Publications Nos. WO 02/055692, WO 02/055693, and EP 1144623 B1 describes certain
`
`methods for inhibiting gene expression using RNAi. Graham ez‘ al., International PCT
`
`Publications Nos. WO 99/49029 and WO 01/70949, and AU 4037501 describe certain
`vector expressed siRNA molecules. Fire 61‘ al., US 6,506,559, describe certainme'thods
`
`for inhibiting gene expression in vitro using certain long dsRNA (greater than 25
`
`nucleotide) constructs that mediate RNAi.
`
`10
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`20
`
`SUMMARY OF THE INVENTION
`
`This invention relates to compounds, compositions, i 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-
`
`RNA (miRNA), and short hairpin RNA (shRNA) molecules capable of modulating gene
`
`expression in cells by RNA inference (RNAi). The siRNA of the instant invention can be
`
`chemically synthesized, expressed from a vector or enzyrnatically synthesized. 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 molecules of the instant invention provide useful
`
`reagents and methods for a variety of therapeutic, diagnostic, agricultural,
`
`target
`
`validation, genomic discovery, genetic engineering and pharrnacogenomic 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
`
`viva 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
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`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
`
`modified siNA can also minimize the possibility of activating interferon activity in
`
`humans.
`
`The siRNA molecules of the invention can be designed to inhibit gene expression
`
`through RNAi targeting of a variety of RNA molecules.
`
`In one embodiment, the siRNA
`
`molecules of the invention are used to target various RNAs corresponding to a target
`
`10
`
`gene. Non—limiting examples of such RNAs include messenger RNA (mRNA), alternate
`
`RNA splice variants of target gene(s), post-transcriptionally modified RNA of target
`
`gene(s), pre—mRNA of target gene(s). If alternate splicing produces a family of transcipts
`
`that are distinguished by usage of appropriate exons, the instant invention can be used to
`
`inhibit gene expression through the appropriate exons to specifically inhibit or to
`
`15
`
`distinguish among the functions of gene family members. For example, a protein that
`
`contains an alternatively spliced transmembrane domain can be expressed in both
`
`membrane bound and secreted forms. Use of the invention to target the exon containing
`
`the transmembrane domain can be used to determine the functional consequences of
`
`pharmaceutical targeting of membrane bound as opposed to the secreted form of the
`
`20
`
`protein. Non-limiting examples of applications of the invention relating to targeting these
`
`RNA molecules
`
`include
`
`therapeutic pharmaceutical
`
`applications, pharmaceutical
`
`discovery applications, molecular diagnostic and gene function applications, and gene
`
`mapping,
`
`for example using single nucleotide polymorphism mapping with siRNA
`
`molecules of the invention. Such applications can be implemented using known gene
`
`25
`
`sequences or from partial sequences available from an expressed sequence tag (EST).
`
`In another embodiment, the siRNA molecules of the invention are used to target
`
`conserved sequences corresponding to a gene family or gene families. As such, siRNA
`
`can be used to characterize pathways of gene function in a variety of applications. For
`
`example, the present invention can be used to inhibit the activity of target gene(s) in a
`
`30
`
`pathway to determine the function of uncharacterized gene(s) in gene fimction analysis,
`
`mRNA function analysis, or translational analysis.
`
`The invention can be used to
`
`determine potential target gene pathways involved in various diseases and conditions
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`toward pharmaceutical development. The invention can be used to understand pathways
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`of gene expression involved in development, such as prenatal development, postnatal
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`development and/or aging.
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`In one embodiment, the invention features a short interfering nucleic acid (siNA)
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`molecule that down-regulates expression of a gene family by RNA interference. The
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`gene family can comprise more than one splice variant of a target gene, more than one
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`post-transcriptionally modified RNA of a target gene, or more than one RNA trascript
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`having shared homology.
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`In one embodiment, the gene family comprises epidermal
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`growth factor (e. g., EGFR, such as HERl, HERZ, HER3, and/or HER4) genes, vascular
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`endothelial growth factor and vascular endothelial growth factor receptor (e. g., VEGF,
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`VEGFRl, VEGFR2, or VEGFR3) genes, or Viral genes corresponding to different viral
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`strains (e.g., HIV-1 and HIV—2). Such gene families can be established by analysing
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`nucleic acid sequences (e.g., sequences shown by Genbank Accession Nos. in Table V)
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`for homology.
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`In one embodiment,
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`the invention features a double-stranded short
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`interfering
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`nucleic acid (siNA) molecule that down-regulates expression of an endogenous
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`mammalian target gene (e.g., a human gene), wherein the siNA molecule comprises one
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`or more chemical modifications and each strand of the double-stranded siNA is about 21
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`nucleotides long.
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`In one
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`embodiment,
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`a
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`siNA molecule of
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`the
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`invention comprises no
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`ribonucleotides.
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`In another embodiment, a siNA molecule of the invention comprises
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`ribonucleotides.
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`In one embodiment,
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`the invention features a double-stranded short
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`interfering
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`nucleic acid (siNA) molecule that down—regulates expression of an endogenous
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`mammalian target gene (e. g., a human gene), wherein one of the strands of the double—
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`stranded siNA molecule comprises a nucleotide sequence that is complementary to a
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`nucleotide sequence of the endogenous mammalian target gene or a portion thereof, and
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`wherein the second strand of the double-stranded siNA molecule comprises a nucleotide
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`sequence substantially similar to the nucleotide sequence of the endogenous mammalian
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`target gene or a portion thereof.
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`PCT/US03/05028
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`In one embodiment,
`
`the invention features a double—stranded short
`
`interfering
`
`nucleic acid (siNA) molecule that down—regulates expression of an endogenous
`
`mammalian target gene (e.g., a human gene), wherein each strand of the siNA molecule
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`comprises about 19 to about 23 nucleotides, and wherein each strand comprises about 19
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`nucleotides that are complementary to the nucleotides of the other strand.
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`In one embodiment,
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`the invention features a double—stranded short
`
`interfering
`
`nucleic acid (siNA) molecule that down-regulates expression of an endogenous
`
`mammalian target gene (e. g., a human gene), wherein the siNA molecule comprises an
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`antisense region comprising a nucleotide sequence that is complementary to a nucleotide
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`sequence of the endogenous mammalian target gene or a portion thereof, and wherein the
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`siNA further comprises a sense region, wherein the sense region comprises a nucleotide
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`sequence substantially similar to the nucleotide sequence of the endogenous mammalian
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`target gene or a portion thereof.
`
`In one embodiment,
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`the invention features a double—stranded short
`
`interfering
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`15
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`nucleic acid (siNA) molecule that down—regulates expression of an endogenous
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`mammalian target gene (e.g., a human gene), wherein the antisense region and the sense
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`region each comprise about 19 to about 23 nucleotides, and wherein the antisense region
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`comprises about 19 nucleotides that are complementary to nucleotides of the sense
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`region.
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`20
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`In one embodiment,
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`the invention features a double~stranded short
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`interfering
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`nucleic acid (siNA) molecule that down—regulates expression of an endogenous
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`mammalian target gene (e.g., a human gene), wherein the siNA molecule comprises a
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`sense region and an antisense region and wherein the antisense region comprises a
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`nucleotide sequence that is complementary to a nucleotide sequence of RNA encoded by
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`25
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`the endogenous mammalian target gene or a portion thereof and the sense region
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`comprises a nucleotide sequence that is complementary to the antisense region.
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`In one embodiment,
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`the invention features a double—stranded short
`
`interfering
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`nucleic acid (siNA) molecule that down-regulates expression of an endogenous
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`mammalian target gene (e.g., a human gene), wherein the siNA molecule is assembled
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`30
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`from two separate oligonucleotide fragments wherein one fragment comprises the sense
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`region and the second fragment comprises the antisense region of the siNA molecule.
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`The sense region can be connected to the antisense region via a linker molecule, such as a
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`polynucleotide linker or a non—nucleotide linker.
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`In one embodiment,
`
`the invention features a double—stranded short
`
`interfering
`
`nucleic acid (siNA) molecule that down-regulates expression of an endogenous
`
`mammalian target gene (e.g., a human gene), wherein the siNA molecule comprises a
`
`sense region and an antisense region and wherein the antisense region comprises a
`
`nucleotide sequence that is complementary to a nucleotide sequence of RNA encoded by
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`the endogenous mammalian target gene or a portion thereof and the sense region
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`comprises a nucleotide sequence that is complementary to the antisense region, and
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`wherein pyrimidine nucleotides
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`in the sense region are 2'-O-methyl pyrimidine
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`nucleotides, 2'—deoxy nucleotides, and/or 2'—deoxy—2‘-fluoro pyrimidine nucleotides.
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`In one embodiment,
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`the invention features a double-stranded short
`
`interfering
`
`nucleic acid (siNA) molecule that down-regulates expression of an endogenous
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`mammalian target gene (e.g., a human gene), wherein the siNA molecule is assembled
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`from two separate oligonucleotide fragments wherein one fragment comprises the sense
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`region and the second fragment comprises the antisense region of the siNA molecule, and
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`wherein the fragment comprising the sense region includes a terminal cap moiety at the
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`5'-end, the 3'—end, or both of the 5' and 3' ends of the fragment comprising the sense
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`region.
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`In another embodiment, the terminal cap moiety is an inverted deoxy abasic
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`moiety or glyceryl moiety.
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`In another embodiment, each of the two fragments of the
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`siNA molecule comprise 21 nucleotides.
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`In one embodiment,
`
`the invention features a double-stranded short
`
`interfering
`
`nucleic acid (siNA) molecule that down—regulates expression of an endogenous
`
`mammalian target gene (e.g., a human gene), wherein the siNA molecule comprises a
`
`sense region and an antisense region and wherein the antisense region comprises a
`
`nucleotide sequence that is complementary to a nucleotide sequence of RNA encoded by
`
`the endogenous mammalian target gene or a portion thereof and the sense region
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`comprises a nucleotide sequence that is complementary to the antisense region, and
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`10
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`15
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`20
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`wherein the purine nucleotides present in the antisense region comprise 2'-deoxy— purine
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`30
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`nucleotides.
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`In another embodiment, the antisense region comprises a phosphorothioate
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`intemucleotide linkage at the 3' end of the antisense region.
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`In another embodiment, the
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`antisense region comprises a glyceryl modification at the 3' end of the antisense region.
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`In one embodiment,
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`the invention features a doub1e~stranded short interfering
`
`nucleic acid (siNA) molecule that down-regulates expression of an endogenous
`
`mammalian target gene (e.g., a human gene), wherein the siNA molecule is assembled
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`from two separate oligonucleotide fragments wherein one fragment comprises the sense
`
`region and the second fragment comprises the antisense region of the siNA molecule, and
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`wherein about 19 nucleotides of each fragment of the siNA molecule are base-paired to
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`the complementary nucleotides of the other fragment of the siNA molecule and wherein
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`at least two 3’ terminal nucleotides of each fragment of the siNA molecule are not base-
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`paired to the nucleotides of the other fragment of the siNA molecule.
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`In another
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`embodiment, each of the two 3’
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`terminal nucleotides of each fragment of the siNA
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`molecule are 2’-deoxy—pyrimidines, such as 2’-deoxy—thymidine. In another embodiment,
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`all 21 nucleotides of each fiagment of the siNA molecule are base-paired to the
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`complementary nucleotides of the other fragment of the siNA molecule.
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`In another
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`embodiment, about 19 nucleotides of the antisense region are base—paired to the
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`nucleotide sequence or a portion thereof of the RNA encoded by the endogenous
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`mammalian target gene.
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`In another embodiment, 21 nucleotides of the antisense region
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`are base-paired to the nucleotide sequence or a portion thereof of the RNA encoded by
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`20
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`the endogenous mammalian target gene.
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`In another embodiment,
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`the 5’-end of the
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`fragment comprising said antisense region optionally includes a phosphate group.
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`In one embodiment,
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`the invention features a double—stranded short interfering
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`nucleic acid (siNA) molecule that inhibits the expression of an endogenous mammalian
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`target RNA sequence (e. g., wherein said target RNA sequence is encoded by a human
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`25
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`gene), wherein the siNA molecule comprises no ribonucleotides and wherein each strand
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`of the double-stranded siNA molecule comprises about 21 nucleotides.
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`In one embodiment,
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`the invention features a double—stranded short interfering
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`nucleic acid (siNA) molecule that inhibits the expression of an endogenous mammalian
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`target gene (e.g., a human gene such as vascular endothelial growth