US 20030190635A1
`
`(19) United States
`(12) Patent Application Publication (10) Pub. No.: US 2003/0190635 A1
`Oct. 9, 2003
`McSwiggen
`(43) Pub. Date:
`
`(54)
`
`RNA INTERFERENCE MEDIATED
`TREATMENT OF ALZHEIMER’S DISEASE
`USING SHORT INTERFERING RNA
`
`(76)
`
`Inventor:
`
`James A. McSWiggen, Boulder, CO
`(US)
`
`Correspondence Address:
`MCDONNELL BOEHNEN HULBERT &
`BERGHOFF
`300 SOUTH WACKER DRIVE
`SUITE 3200
`
`CHICAGO, IL 60606 (US)
`
`(21)
`
`Appl. N0.:
`
`10/205,309
`
`(22)
`
`Filed:
`
`Jul. 25, 2002
`
`Related US. Application Data
`
`(60)
`
`Provisional application No. 60/358,580, filed on Feb.
`20, 2002. Provisional application No. 60/363,124,
`
`filed on Mar. 11, 2002. Provisional application No.
`60/386,782, filed on Jun. 6, 2002.
`
`Publication Classification
`
`Int. C1.7 ............................ C12Q 1/68; C07H 21/04,
`C07H 21/02, A61K 48/00
`............... 435/6; 435/375, 514/44, 536/232
`
`US. Cl.
`
`ABSTRACT
`
`(51)
`
`(52)
`
`(57)
`
`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 associated With Alzhe-
`imer’s disease. Specifically, the invention relates to small
`interfering RNA (siRNA) molecules capable of mediating
`RNA interference (RNAi) against beta-secretase (BACE),
`PIN-1, presenillin-l
`(PS-1)
`and presenillin-2 (PS-2)
`polypeptide and polynucleotide targets.
`
`Exhibit 1014
`
`Exhibit 1014
`
`i
`
`

`

`Patent Application Publication
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`Oct. 9, 2003 Sheet 1 0f 8
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`US 2003/0190635 A1
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`Figure 1
`
`
`(1) FIRST STRAND
`
`
`(2)
`
`SECOND STRAND
`|IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII—O_R
`
`* DEPROTECTION
`
`IIIlIllllllllllllIIIIIIIIIIIIIIIIIIII—O_R
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`* PURIFICATION
`
`(DETRITYLATION)
`
`
`
`siRNA DUPLEX
`
`= SOLID SUPPORT
`
`= TERMINAL PROTECTING GROUP
`
`FOR EXAMPLE:
`
`DIMETHOXYTRITYL (DMT)
`
` R
`
`I
`WQAN = CLEAVABLE LINKER
`(FOR EXAMPLE: NUCLEOTIDE SUCCINATE OR
`INVERTED DEOXYABASIC SUCCINATE)
`(2)
`WWW : CLEAVABLE LINKER
`
`(FOR EXAMPLE: NUCLEOTIDE SUCCINATE OR
`INVERTED DEOXYABASIC SUCCINATE)
`
`
`
`INVERTED DEOXYABASIC SUCCINATE LINKAGE
`
`ii
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`Patent Application Publication
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`Oct. 9, 2003 Sheet 2 0f 8
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`US 2003/0190635 A1
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`Patent Application Publication
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`US 2003/0190635 A1
`
`Oct. 9, 2003
`
`RNA INTERFERENCE MEDIATED TREATMENT
`OF ALZHEIMER’S DISEASE USING SHORT
`INTERFERING RNA
`
`BACKGROUND OF THE INVENTION
`
`invention concerns methods and
`[0001] The present
`reagents useful in modulating gene expression associated
`with Alzheimer’s disease in a variety of applications, includ-
`ing use in therapeutic, diagnostic,
`target validation, and
`genomic discovery applications. Specifically, the invention
`relates to short interfering nucleic acid molecules capable of
`mediating RNA interference (RNAi) against beta-secretase
`(BACE), pin-1, presenillin 1 (PS-1) and presenillin 2 (PS-2)
`expression.
`
`[0002] The following is a discussion of relevant art per-
`taining to RNAi. The discussion is provided only for under-
`standing 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.
`
`[0003] RNA interference refers to the process of sequence-
`specific post transcriptional gene silencing in animals medi-
`ated by short interfering RNAs (siRNA) (Fire et al., 1998,
`Nature, 391, 806). The corresponding process in plants is
`commonly referred to as post transcriptional gene silencing
`or RNA silencing and is also referred to as quelling in fungi.
`The process of post transcriptional gene silencing is thought
`to be an evolutionarily conserved cellular defense mecha-
`nism used to prevent the expression of foreign genes which
`is commonly shared by diverse flora and phyla (Fire et al.,
`1999, Trends Genet, 15, 358). Such protection from foreign
`gene expression may have evolved in response to the
`production of double stranded RNAs (dsRNA) derived from
`viral
`infection or the random integration of transposon
`elements into a host 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 medi-
`ated activation of protein kinase PKR and 2',5'-oligoadeny-
`late synthetase resulting in non-specific cleavage of mRNA
`by ribonuclease L.
`
`[0004] 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 (siRNA)
`(Berstein et al., 2001, Nature, 409, 363). Short interfering
`RNAs derived from dicer activity are typically about 21-23
`nucleotides in length and comprise about 19 base pair
`duplexes. Dicer has also been implicated in the excision of
`21 and 22 nucleotide small temporal RNAs (stRNA) 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 containing a siRNA, commonly referred to as an
`RNA-induced silencing complex (RISC), which mediates
`cleavage of single stranded RNA having sequence comple-
`mentary to the antisense strand of the siRNA duplex. Cleav-
`age 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).
`
`[0005] 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, describe RNAi
`mediated by dsRNA in mouse embryos. Hammond et al.,
`2000, Nature, 404, 293, describe RNAi in Drosophila cells
`transfected with dsRNA. Elbashir et al., 2001, Nature, 411,
`494, describe RNAi induced by introduction of duplexes of
`synthetic 21-nucleotide RNAs in cultured mammalian cells
`including human embryonic kidney and 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 activ-
`ity. 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 mis-
`match 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 RNA is defined by the 5'-end of the siRNA guide
`sequence rather than the 3'-end (Elbashir et al., 2001, EMBO
`J., 20, 6877). Other studies have indicated that a 5'-phos-
`phate 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).
`[0006] Studies have shown that replacing the 3'-overhang-
`ing 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 nucle-
`otides on each end of the siRNA with deoxyribonucleotides
`has been reported to be well tolerated whereas complete
`substitution with deoxyribonucleotides results in no RNAi
`activity (Elbashir et al., 2001, EMBO J., 20, 6877). In
`addition, Elbashir et al., supra, also report that substitution
`of siRNA with 2'-O-methyl nucleotides completely abol-
`ishes 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
`
`nitrogen or sulfur heteroatom”, however neither application
`teaches to what extent these modifications are tolerated in
`
`siRNA molecules nor provide any examples of such modi-
`fied siRNA. Kreutzer and Limmer, Canadian Patent Appli-
`cation No. 2,359,180, also describe certain chemical modi-
`fications for use in dsRNA constructs in order to counteract
`
`stranded-RNA-dependent protein
`activation of double
`kinase PKR, specifically 2'-amino or 2'-O-methyl nucle-
`otides, 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 do they provide any examples of such
`modified siRNA.
`
`[0007] 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 transcripts.
`The authors describe the introduction of thiophosphate resi-
`dues into these siRNA transcripts by incorporating thiophos-
`phate nucleotide analogs with T7 and T3 RNA polymerase
`and observed that “RNAs with two [phosphorothioate]
`
`

`

`US 2003/0190635 A1
`
`Oct. 9, 2003
`
`modified bases also had substantial decreases in effective-
`
`ness 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. at 1081. The authors also tested certain
`
`modifications at the 2'-position of the nucleotide sugar in the
`long siRNA transcripts and observed that substituting
`deoxynucleotides for ribonucleotides “produced a substan-
`tial 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-bro-
`mouracil, 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-bromou-
`racil were all well tolerated, inosine “produced a substantial
`decrease in interference activity” when incorporated in
`either strand. Incorporation of 5-iodouracil and 3-(aminoal-
`lyl)uracil
`in the antisense strand resulted in substantial
`decrease in RNAi activity as well.
`[0008] Beach et 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. 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, describes the use of
`specific dsRNAs for use in attenuating the expression of
`certain target genes. Zernicka-Goetz et al., International
`PCT Publication No. WO 01/36646, describes certain meth-
`ods for inhibiting the expression of particular genes in
`mammalian cells using certain dsRNA molecules. Fire et al.,
`International PCT Publication No. WO 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 et al., International
`PCT Publication No. WO 01/29058, describes the identifi-
`cation of specific genes involved in dsRNA mediated RNAi.
`Deschamps Depaillette et al., International PCT Publication
`No. WO 99/07409, describes specific compositions consist-
`ing of particular dsRNA molecules combined with certain
`anti-viral agents. Driscoll et al., International PCT Publica-
`tion No. WO 01/49844, describes specific DNA constructs
`for use in facilitating gene silencing in targeted organisms.
`Parrish et al., 2000,M0lecular Cell, 6, 1977-1087, describes
`specific chemically modified siRNA constructs targeting the
`unc-22 gene of C. elegans. Tuschl et al., International PCT
`Publication No. WO 02/44321, describe certain synthetic
`siRNA constructs.
`
`[0009] Alzheimer’s disease (AD) is a progressive, degen-
`erative disease of the brain which affects approximately 4
`million people in the United States alone. An estimated 14
`million Americans will have Alzheimer’s disease by the
`middle of the next century if no cure or definitive prevention
`of the disease is found. Nearly one out of ten people over age
`65 and nearly half of those over 85 have Alzheimer’s
`disease. Alzheimer’s disease is not confined to the elderly, a
`
`small percentage of people in their 30’s and 40’s are afflicted
`with early onset AD. Alzheimer’s disease is the most com-
`mon form of dementia, and amounts to the third most
`expensive disease in the US following heart disease and
`cancer. An estimated 100 billion dollars are spent annually
`on Alzheimer’s disease (National Alzheimer’s Association,
`1999).
`
`[0010] Alzheimer’s disease is characterized by the pro-
`gressive formation of insoluble plaques and vascular depos-
`its in the brain consisting of the 4 kD amyloid B peptide
`(AB). These plaques are characterized by dystrophic neurites
`that show profound synaptic loss, neurofibrillary tangle
`formation, and gliosis. AB arises from the proteolytic cleav-
`age of the large type I transmembrane protein, B-amyloid
`precursor protein (APP) (Kang et al., 1987, Nature, 325,
`733). Processing of APP to generate AB requires two sites of
`cleavage by a B-secretase and a y-secretase. B-secretase
`cleavage of APP results in the cytoplasmic release of a 100
`kD soluble amino-terminal fragment, APPsB, leaving behind
`a 12 kD transmembrane carboxy-terminal fragment, C99.
`Alternately, APP can be cleaved by a ot-secretase to generate
`cytoplasmic APPsot and transmembrane C83 fragments.
`Both remaining transmembrane fragments, C99 and C83,
`can be further cleaved by a y-secretase, leading to the release
`and secretion of Alzheimer’s related AB and a non-patho-
`genic peptide, p3, respectively (Vassar et al., 1999, Science,
`286, 735-741). Early onset familial Alzheimer’s disease is
`characterized by mutant APP protein with a Met to Leu
`substitution at position P1, characterized as the “Swedish”
`familial mutation (Mullan et al., 1992, Nature Genet, 1,
`345). This APP mutation is characterized by a dramatic
`enhancement in B-secretase cleavage (Citron et al., 1992,
`Nature, 360, 672).
`
`[0011] The identification of B-secretase, and y-secretase
`constituents involved in the release of B-amyloid protein is
`of primary importance in the development of treatment
`strategies for Alzheimer’s disease. Characterization of
`ot-secretase is also important in this regard since ot-secretase
`cleavage may compete with B-secretase cleavage resulting
`in non-pathogenic vs. pathogenic protein production.
`Involvement of the two metalloproteases, ADAM 10, and
`TACE has been demonstrated in ot-cleavage of AAP (Bux-
`baum et al., 1999,]. Biol. Chem, 273, 27765, and Lammich
`et al., 1999,Pr0c. Natl.Acad. Sci. U.SA., 96, 3922). Studies
`of y-secretase activity have demonstrated presenilin depen-
`dence (De Stooper et al., 1998, Nature, 391, 387, and De
`Stooper et al., 1999, Nature, 398, 518), and as such, pres-
`enilins have been proposed as y-secretase even though
`presenilin does not present proteolytic activity (Wolfe et al.,
`1999, Nature, 398, 513).
`
`supra reported
`[0012] Recently, Vassar et al., 1999,
`B-secretase cleavage of AAP by the transmembrane aspartic
`protease beta site APP cleaving enzyme, BACE. While other
`potential candidates for B-secretase have been proposed (for
`review see Evin et al., 1999, Proc. Natl. Acad. Sci. USA,
`96, 3922), none have demonstrated the full range of char-
`acteristics expected from this enzyme. Vassar et al, supra,
`demonstrate that BACE expression and localization are as
`expected for B-secretase, that BACE overexpression in cells
`results in increased B-secretase cleavage of APP and Swed-
`ish APP,
`that
`isolated BACE demonstrates site specific
`proteolytic activity on APP derived peptide substrates, and
`
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`US 2003/0190635 A1
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`that antisense mediated endogenous BACE inhibition results
`in dramatically reduced B-secretase activity.
`
`[0013] Current treatment strategies for Alzheimer’s dis-
`ease rely on either the prevention or the alleviation of
`symptoms and/or the slowing down of disease progression.
`Two drugs approved in the treatment of Alzheimer’s, done-
`pezil (Aricept®) and tacrine (Cognex®), both cholinomi-
`metics, attempt
`to slow the loss of cognitive ability by
`increasing the amount of acetylcholine available to the
`brain. Antioxidant therapy through the use of antioxidant
`compounds such as alpha-tocopherol (vitamin E), melato-
`nin, and selegeline (Eldepryl®) attempt
`to slow disease
`progression by minimizing free radical damage. Estrogen
`replacement therapy is thought to incur a possible preven-
`tative benefit in the development of Alzheimer’s disease
`based on limited data. The use of anti-inflammatory drugs
`may be associated with a reduced risk of Alzheimer’s as
`well. Calcium channel blockers such as Nimodipine® are
`considered to have a potential benefit in treating Alzheimer’s
`disease due to protection of nerve cells from calcium over-
`load,
`thereby prolonging nerve cell survival. Nootropic
`compounds, such as acetyl-L-carnitine (Alcar®) and insulin,
`have been proposed to have some benefit in treating Alzhe-
`imer’s due to enhancement of cognitive and memory func-
`tion based on cellular metabolism.
`
`[0014] Whereby the above treatment strategies may all
`improve quality of life in Alzheimer’s patients, there exists
`an unmet need in the comprehensive treatment and preven-
`tion of this disease. As such,
`there exists the need for
`therapeutics effective in reversing the physiological changes
`associated with Alzheimer’s disease, specifically, therapeu-
`tics that can eliminate and/or reverse the deposition of
`amyloid [3 peptide. The use of compounds to modulate the
`expression of proteases that are instrumental in the release of
`amyloid [3 peptide, namely B-secretase (BACE), and
`y-secretase (presenilin), is of therapeutic significance.
`
`[0015] McSwiggen et al., International PCT Publication
`No. W0 01/16312, describes nucleic acid mediated inhibi-
`tion of BACE, PS-1, and PS-2 expression.
`
`SUMMARY OF THE INVENTION
`
`[0016] One embodiment of the invention provides a short
`interfering RNA (siRNA) molecule that down regulates
`expression of a beta site APP-cleaving enzyme (BACE)
`gene by RNA interference. The siRNA molecule can be
`adapted for use to treat Alzheimer’s disease. The siRNA
`molecule can comprise a sense region and an antisense
`region. The antisense region can comprise sequence comple-
`mentary to an RNA sequence encoding BACE and the sense
`region can comprise sequence complementary to the anti-
`sense region.
`
`[0017] The siRNA molecule can be assembled from two
`nucleic acid fragments wherein one fragment comprises the
`sense region and the second fragment comprises the anti-
`sense region of said siRNA molecule. The sense region and
`antisense region can be covalently connected via a linker
`molecule. The linker molecule can be a polynucleotide
`linker or a non-nucleotide linker.
`
`[0018] The antisense region can comprise a sequence
`complementary to sequence having any of SEQ ID NOs.
`1-325. The antisense region can also comprise sequence
`
`having any of SEQ ID NOs. 326-650, 664, 666, 668, 670,
`672, or 674. The sense region can comprise sequence having
`any of SEQ ID NOs. 1-325, 663, 665, 667, 669, 671, or 673.
`The sense region can comprise a sequence of SEQ ID NO.
`651 and the antisense region can comprise a sequence of
`SEQ ID NO. 652. The sense region can comprise a sequence
`of SEQ ID NO. 653 and the antisense region can comprise
`a sequence of SEQ ID NO. 654. The sense region can
`comprise a sequence of SEQ ID NO. 655 and the antisense
`region can comprise a sequence of SEQ ID NO. 656. The
`sense region can comprise a sequence of SEQ ID NO. 657
`and the antisense region can comprise a sequence of SEQ ID
`NO. 658. The sense region can comprise a sequence of SEQ
`ID NO. 659 and the antisense region can comprise a
`sequence of SEQ ID NO. 660. The sense region can com-
`prise a sequence of SEQ ID NO. 661 and the antisense
`region can comprise a sequence of SEQ ID NO. 662.
`
`[0019] The sense region of a siRNA molecule of the
`invention can comprise a 3'-terminal overhang and the
`antisense region can comprise a 3'-terminal overhang. The
`3'-terminal overhangs each can comprise about 2 nucle-
`otides. The antisense region of the 3'-terminal nucleotide
`overhang can be complementary to RNA encoding BACE.
`
`[0020] The sense region of a siRNA molecule can com-
`prise one or more 2'-O-methyl modified pyrimidine nucle-
`otides. The sense region can comprise a terminal cap moiety
`at the 5'-end, 3'-end, or both 5' and 3' ends of said sense
`region.
`
`[0021] The antisense region of a siRNA molecule can
`comprise one or more 2'-deoxy-2'-fluoro modified pyrimi-
`dine nucleotides. The antisense region can also comprise a
`phosphorothioate internucleotide linkage at the 3' end of said
`antisense region. The
`antisense
`region can comprise
`between about one and about five phosphorothioate inter-
`nucleotide linkages at the 5' end of said antisense region.
`
`[0022] The 3'-terminal nucleotide overhangs of a siRNA
`molecule can comprise ribonucleotides or deoxyribonucle-
`otides that are chemically modified at a nucleic acid sugar,
`base, or backbone. The 3'-terminal nucleotide overhangs can
`also comprise one or more universal base ribonucleotides.
`Additionally, the 3'-terminal nucleotide overhangs can com-
`prise one or more acyclic nucleotides.
`
`[0023] The 3'-terminal nucleotide overhangs can comprise
`nucleotides comprising internucleotide linkages having For-
`mula I:
`
`Rl—X—P—Y—Rz
`
`[0024] wherein each R1 and R2 is independently any
`nucleotide, non-nucleotide, or polynucleotide which
`can be naturally occurring or chemically modified,
`each X and Y is independently O, S, N, alkyl, or
`substituted alkyl, each Z and W is independently O,
`S, N, alkyl, substituted alkyl, O-alkyl, S-alkyl,
`alkaryl, or aralkyl, and wherein W, X, Y and Z are not
`all O.
`
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`US 2003/0190635 A1
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`[0025] The 3'-terminal nucleotide overhangs can comprise
`nucleotides or non-nucleotides having Formula II:
`
`R7
`
`R12
`
`R11
`
`B
`
`R9
`
`R4
`
`R6
`
`R8
`
`R10
`
`R5
`
`R3
`
`[0026] wherein each R3, R4, R5, R6, R7, R8, R10,
`R11 and R12 is independently H, OH, alkyl, substi-
`tuted 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,
`NH2, aminoalkyl, aminoacid, aminoacyl, ONH2,
`O-aminoalkyl, O-aminoacid, O-aminoacyl, hetero-
`cycloalkyl, 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 or any other non-naturally
`occurring base that can be complementary or non-
`complementary to BACE RNA or a non-nucleosidic
`base or any other non-naturally occurring universal
`base that can be complementary or non-complemen-
`tary to BACE RNA.
`
`[0027] Another embodiment of the invention provides an
`expression vector comprising a nucleic acid sequence
`encoding at least one siRNA molecule of the invention in a
`manner that allows expression of the nucleic acid molecule.
`The expression vector can be in a mammalian cell, such as
`a human cell. The siRNA molecule can comprise a sense
`region and an antisense region. The antisense region can
`comprise sequence complementary to an RNA sequence
`encoding BACE and the sense region comprises sequence
`complementary to the antisense region. The siRNA mol-
`ecule can comprise two distinct strands having complemen-
`tarity sense and antisense regions or can comprise a single
`strand having complementary sense and antisense regions.
`
`this invention relates to compounds,
`[0028] Therefore,
`compositions, and methods useful for modulating beta-
`secretase (BACE), PIN-1, presenillin 1 (PS-1) and/or pres-
`enillin 2 (PS-2) function and/or gene expression in a cell by
`RNA interference (RNAi) using short
`interfering RNA
`(siRNA). In particular, the instant invention features siRNA
`molecules and methods to modulate the expression of
`BACE, PIN-1, PS-1 and/or PS-2 RNA. The siRNA of the
`invention can be unmodified or chemically modified. The
`siRNA of the instant invention can be chemically synthe-
`sized, expressed from a vector or enzymatically synthesized.
`The instant invention also features various chemically modi-
`fied synthetic short
`interfering RNA (siRNA) molecules
`capable of modulating BACE, PIN-1, PS-1 and/or PS-2 gene
`expression/activity in cells by RNA inference (RNAi). The
`use of chemically modified siRNA is expected to improve
`various properties of native siRNA molecules through
`increased resistance to nuclease degradation in vivo and/or
`improved cellular uptake. The siRNA molecules of the
`instant invention provide useful reagents and methods for a
`
`variety of therapeutic, diagnostic, agricultural, target vali-
`dation, genomic discovery, genetic engineering and phar-
`macogenomic applications.
`[0029]
`In one embodiment, the invention features one or
`more siRNA molecules and methods that independently or
`in combination modulate the expression of gene(s) encoding
`proteins associated with Alzheimer’s disease and other neu-
`rodegenerative disorders or conditions such as dementia,
`and stroke/cardiovascular accident (CVA). Specifically, the
`present invention features siRNA molecules that modulate
`the expression of proteins associated with Alzheimer’s dis-
`ease and related pathologies, for example BACE (such as
`Genbank Accession No. NMi012104), PIN-1 (such as
`Genbank Accession No. NMi006222), PS-1 (such as Gen-
`bank Accession No. L76517) and/or PS-2 (such as Genbank
`Accession No. L43964).
`[0030] The description below of the various aspects and
`embodiments is provided with reference to the exemplary
`BACE protein, including components or subunits thereof.
`However,
`the various aspects and embodiments are also
`directed to other genes which express other BACE related
`proteins or other proteins associated with Alheimer’s dis-
`ease, such as PIN-1, PS-1 and PS-2. Those additional genes
`can be analyzed for target sites using the methods described
`for BACE herein. Thus, the inhibition and the effects of such
`inhibition of the other genes can be performed as described
`herein.
`
`In one embodiment, the invention features a siRNA
`[0031]
`molecule which down regulates expression of a BACE gene,
`for example, wherein the BACE gene comprises BACE
`encoding sequence.
`[0032]
`In one embodiment, the invention features a siRNA
`molecule having RNAi activity against BACE RNA,
`wherein the siRNA molecule comprises a sequence comple-
`mentary to any RNA having BACE encoding sequence, for
`example Genbank Accession No. NMi012104. In another
`embodiment, the invention features a siRNA molecule hav-
`ing RNAi activity against PIN-1 RNA, wherein the siRNA
`molecule comprises a sequence complementary to any RNA
`having PIN-1 encoding sequence, for example Genbank
`Accession No. NMi006222. In another embodiment, the
`invention features a siRNA molecule having RNAi activity
`against PS-1 RNA, wherein the siRNA molecule comprises
`a sequence complementary to any RNA having PS-1 encod-
`ing sequence, for example Genbank Accession No. L76517.
`In another embodiment,
`the invention features a siRNA
`molecule having RNAi activity against PS-2 RNA, wherein
`the siRNA molecule comprises a sequence complementary
`to any RNA having PS-2 encoding sequence, for example
`Genbank Accession No. L43964.
`
`In another embodiment, the invention features a
`[0033]
`siRNA molecule comprising sequences selected from the
`group consisting of SEQ ID NOs: 1-650. In yet another
`embodiment, the invention features a siRNA molecule com-
`prising a sequence, for example the antisense sequence of
`the siRNA construct, complementary to a sequence or por-
`tion of sequence comprising Genbank Accession Nos.
`NMi012104 (BACE), NM006222 (PIN-1), L76517 (PS-1)
`and/or L43964 (PS-2).
`[0034]
`In one embodiment, a siRNA molecule of the
`invention has RNAi activity that modulates expression of
`RNA encoded by a BACE, PIN-1, PS-1, and/or PS-2
`gene(s).
`
`

`

`US 2003/0190635 A1
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`Oct. 9, 2003
`
`In one embodiment, nucleic acid molecules of the
`[0035]
`invention that act as mediators of the RNA interference gene
`silencing response are double stranded RNA molecules. In
`another embodiment, the siRNA molecules of the invention
`consist of duplexes containing about 19 base pairs between
`oligonucleotides comprising about 19 to about 25 nucle-
`otides (e.g., about 19, 20, 21, 22, 23, 24, or 25). In yet
`another embodiment, siRNA molecules of the invention
`comprise duplexes with overhanging ends of 1-3 (e.g., 1, 2,
`or 3) nucleotides, for example 21 nucleotide duplexes with
`19 base pairs and 2 nucleotide 3'-overhangs. These nucle-
`otide overhangs in the antisense strand are optionally
`complementary to the tar