111111 1111111111 IIIIII IIIII 11111 1111111111 111111111111111 111111111111111 111111111111111111
`US 20030190635Al
`
`(19) United States
`(12) Patent Application Publication
`McSwiggen
`
`(10) Pub. No.: US 2003/0190635 A l
`Oct. 9, 2003
`(43) Pub. Date:
`
`(54) RNA .I TERFERENCE MEOfATEO
`TREATMENT OF ALZHElMER'S DISEASE
`USCNG SHORT INTERFERlNG RNA
`
`(76)
`
`Inventor: James
`(US)
`
`. McSwiggen, Boulder, CO
`
`Correspondence Address:
`MCDONNELL BOERNE HULBERT &
`BERGHOFF
`300 SOUTH WACKER DRIVE
`SUil'E 3200
`CIDCAGO, IL 60606 (U )
`
`(21) Appl. No.:
`
`10/205,309
`
`(22) Filed:
`
`Jul. 25, 2002
`
`Related U.S. Application Data
`
`(60) Provisional application o. 60/358,5 0, filed on Feb.
`20, 2002. Provisiona l application
`o. 60 63,124,
`
`filed on Mar. 11, 2002. Provisional application o.
`60/386,782, filed on Jun. 6, 2002.
`
`Publication Classification
`
`(51) Jnt. Cl.7
`
`(52) U .
`
`. Cl .
`
`............................ C12Q 1/68; C071 I 21/04;
`C07H 21/02; A61K 48/00
`............... 435/6; 435/375; 514/44; 536/23.2
`
`(57)
`
`ABSTRACT
`
`The present invention concerns methods and reagent useful
`in moduJating gene expression in a variety of applica 1ions,
`includi ng use in therapeutic, diagnostic, 1arge1 validation,
`and genomic di covery applications associa1ed with Alzhe(cid:173)
`imer's disease. pecifically, 1he invent ion re lates lo small
`interfering RNA ( iR A) molecules capable of mediating
`RNA interference (R NAi) against be1a-secretase (BA E),
`Pl -1, pre. enillin-1
`(PS-1) and presenillin-2
`(P -2)
`polypeptide and polynucleotide targets.
`
`i
`
`Alnylam Exh. 10(cid:20)(cid:23)
`
`

`

`Patent Application Publication
`
`Oct. 9, 2003 Sheet 1 of 8
`
`US 2003/0190635 Al
`
`Figure 1
`
`FIRST STRAND
`
`(2)
`
`SECOND STRAND
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`
`~ DEPROTECTION
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`
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`FOR EXAMPLE:
`DIMETHOXYTRITYL (DMT)
`
`(l)
`WWW
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`
`= CLEA V ABLE LINKER
`(FOR EXAMPLE: NUCLEOTIDE SUCCINATE OR
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`= CLEA V ABLE LINKER
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`INVERTED DEOXYABASIC SUCCINATE)
`
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`
`5
`I
`0
`
`' 3
`
`INVERTED DEOXY ABASIC SUCCINATE LINKAGE
`
`ii
`
`

`

`Patent Application Publication
`
`Oct. 9, 2003 Sheet 2 of 8
`
`US 2003/0190635 Al
`
`Figure 2
`
`6646.96
`
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`POSITIONS (NN) CAN COMPRISE UNIVERSAL BASE OR DEOXY (eg. THYMIDINE)
`
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`
`ANTISENSE STRAND (SEQ ID NO 656)
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`
`ANTISENSE STRAND (SEQ ID NO 654)
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`
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`
`ANTISENSE STRAND (SEQ ID NO 652)
`
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`
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`
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`
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`
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`
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`
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`
`ANTISENSE STRAND (SEQ ID NO 662)
`
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`
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`
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`
`ANTISENSE STRAND (SEQ ID NO 660)
`
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`
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`
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`5'-
`
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`
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`
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`
`ALL RfBONUCLEOTIDES EXCEPT POSITIONS (N N)
`
`ANTISENSE STRAND (SEQ ID NO 658)
`
`(N5N) N N N N N N N N N N N N N N5 N5 N5 N5 N5 N
`
`N5N5N5N5 N5 NN NNNNNNNN N5N5N5Ns(N5N)
`
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`
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`
`ALL RJBONUCLEOTIDES EXCEPT POSITIONS (N N)
`
`SEl\SE STRAND (SEQ ID NO 657)
`
`Fif(ure 4
`
`vi
`
`

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`lower case= 2'-0-Methyl; italic lower case = 2'-deoxy-2'-jluoro
`
`ANTISENSE STRAND (SEQ ID NO 668)
`
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`iB-c Au G G cu G c c Au c u G C G cc T T-iB
`
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`
`ANTISENSE STRAND (SEQ ID NO 666)
`
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`
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`
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`
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`
`ANTlSENSE STRAND (SEQ ID NO 664)
`
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`
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`
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`
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`
`Figure 5
`
`vii
`
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`
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`
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`
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`
`SENSE STRAND (SEQ ID NO 671)
`
`ANTISEKSE STRAND (SEQ ID NO 670)
`
`CsAsUsGsGsC U G CC AU CU G CsGsCsCsTsT
`
`Ts T G u A C C G A C G G u A G AsCsGsCsGsG
`
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`
`Figure 5
`
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`
`I
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`
`TsT Gu Ace G Ac G G uA GA cGcGG
`
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`
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`Figure 6
`
`ix
`
`

`

`US 2003/0190635 Al
`
`Oct. 9, 2003
`
`1
`
`RNA INTERFERENCE MEDIATED TREATMENT
`OF ALZHEIMER'S DISEASE USING SHORT
`INTERFERING RNA
`BACKGROUND OF THE INVENTION
`[0001) Tbe present
`invention concerns metbods and
`reagents uscfuJ in modulating gene expression associated
`wilb Alzheimer's disease in a variety of applications, includ(cid:173)
`ing use in therapeutic, diagnostic, target val idation, aod
`genomic discovery applications. Specifically, the invention
`relates to sbort 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(cid:173)
`taining to RNAi. The discussion is provided only for under(cid:173)
`standing of tbe invention that follows. The summary is not
`an admission that any of the work described below is prior
`an to the claimed invention.
`[0003) RNA interference refers to tbe process of sequence(cid:173)
`specific post transcriptional gene silencing in animals medi(cid:173)
`ated by short interfering RNAs (siRNA) (Fire et al., 1998,
`Na111re, 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 fongi.
`The process of post transcriptional gene silencing is thought
`to be an evolutionarily conserved cellular defense mccba(cid:173)
`nism used to prevent the expression of foreign genes which
`is commonly sbared by diverse flora and phyla (Fire et al.,
`1999, Trends Genet., 15,358). Such protection from foreign
`gene express.ion may have evolved in response to the
`production of double stranded RNAs (dsRNA) derived from
`viral infection or tbe random integration of transposon
`elements into a host genome via a cellular response that
`specifically destroys homologous single stranded RNA or
`viraJ genomic RNA. The presence of dsRNA in cells triggers
`the RNAi response though a mechanism 1ba1 has yet to be
`ful ly characterized. T his mechanism appears to be different
`from the interferon response that results from dsRNA medi(cid:173)
`ated activation of protein kinase PKR and 2',5'-oligoadcny(cid:173)
`late synthetase resul ting in non-specific cleavage of mRNA
`by ribonuclcase L.
`[0004) The presence of long dsRNAs in cells stimulates
`the activity of a ribonuclease 111 enzyme referred 10 as dicer.
`Dicer is involved in the processing of the dsRNA into short
`pieces of dsRNA known as short interfering RNA~ (siRNA)
`(Berstein ct al., 2001, Nature, 409, 363). Short interfering
`RNA5 derived from dicer activity arc typically about 21-23
`nucleotides in length aod comprise about 19 base pair
`duplexes. Dicer has also bee(cid:143) implicated in the excision of
`21 and 22 nucleotide small temporal RNAs (stRNA) from
`precursor RNA of conserved structure 1ba1 are implicated in
`translational control (Hutvagner et al., 2001, Science, 293,
`834). The RNAi response aL50 features an cndonuclease
`complex containing a siRNA, commonly referred to as an
`RNA-induced silencing complex (RISC), which mediates
`cleavage of single stranded RNA baving sequence comple(cid:173)
`meatary 10 the antisensc strand of the siRNA duplex. Cleav(cid:173)
`age of tbe target RNA takes place in the middle of tbe region
`complementary to the antisense strand of the sil?.NA duplex
`(Elbashir et al., 2001, Genes Dev., LS, 188).
`[0005) Short interfering RNA mediated RNAi has been
`studied in a variety of systems. Fire et al., 1998,Nnture, 391,
`
`806, were the first to observe RNAi io C. Elegans. Wiaony
`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
`transfectecl with dsRNA. Elbashir et al., 2001, Nature, 411,
`494, describe RNAi induced by introduction of duplexes of
`synthetic 21-nucleotide RNAs in cu.hured mammalian cells
`including humao embryonic kidney aocl Hel..a cells. Recent
`work in Drosophi la embryonic lysates (Elbashir et al., 200 L,
`EMBO J., 20, 6877) has revealed cert ain requirements for
`siRNA
`length, structure, chemical composition, and
`sequence that arc essential to mediate efficient RNAi activ(cid:173)
`ity. These studies have sbowo that 21 nucleotide siRNA
`duplexes are most active when containing two nucleotide
`3'-overhangs. Furthermore, complete substitution of one or
`both siRNA strands witb 2'-deoxy (2'-H) or 2'-O-methyl
`nucleotides abolishes RN Ai activity, whereas substitution of
`the 3'-terminal siRNA overhang nucleotides with deoxy
`nucleotides (2'-1-1) was shown to be tolerated. Sioglc mis(cid:173)
`match sequences in the center of the siRNAduplex were also
`show(cid:143)
`to abolish RNAi activity. lo addition, these studies
`also indicate that the position of tbe 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). Otber studies have indicated that a 5'-phos(cid:173)
`phate on the target-complementary strand of a siRNAduplex
`is required for siRNA activity and that ATP is utilized to
`maintain the 5'-pbosphate moiety on the siRN A(Nykanen et
`al., 2001, Cell, 107, 309).
`[0006) Studies have shown that replacing the 3'-overhang(cid:173)
`ing segments of a 21-mer siRNA duplex having 2 nucleotide
`3' overhangs with deoxyribonucleoticlcs does not have an
`adverse effect on RNAi activity. Replacing up to 4 nucle(cid:173)
`otides on each end of the siRNA witb 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
`o[ siRNA with 2'-O-mcthyl nucleotides completely abol(cid:173)
`ishes RNAi activity. Li et al., latematioaal PCT Publication
`No. WO 00/44914, aod Beach et al., International PCT
`Publication No. WO 01/68836 both suggest tbat siR.NA
`"may include modifications to either the phosphate-sugar
`back bone or the nucleoside to include al least one of a
`nitrogen or sulfur heteroatom", however neither application
`teaches lo what extent these modificaLioas arc tolerated in
`siRNA molecules nor provide any examples of such modi(cid:173)
`fled siRNA. Kreutzer and Limmer, Ca1Jadiao Patent Appli(cid:173)
`cation No. 2,359,180, al5o describe certain chemical modi(cid:173)
`fications for use i.o dsRNA constructs i11 order to counteract
`activation of double stranded-RNA-dependent protein
`kinase PKR, specifically 2'-amino or 2'-O-methyl nucle(cid:173)
`otides, and nucleotides containing a 2'-0 or 4'-C methylene
`bridge. However, Kreutzer and Limmer similarly fail to
`show 10 wbat extent these modifications are tolerated in
`siRNA molecules oor do tbcy 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(cid:173)
`dues into these siRNA transcripts by incorporating lhiophos(cid:173)
`phate nucleotide analogs with T7 and T3 RN/\ polymerase
`and observed tbat " RNAs witb two [phosphorotbioate]
`
`

`

`US 2003/0190635 Al
`
`Oct. 9, 2003
`
`2
`
`modified bases also had substantial decreases in effective(cid:173)
`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
`deoxynuclcotides for riboouclcotides " produced a substan(cid:173)
`tial decrease in interCerence activity", especially in the case
`of Uridinc to Thymidinc and/or Cytidinc to deoxy-Cytidinc
`subs1i1u1ioos. Id. lo addition, tbc aut hors tested certain base
`modifications, including substituting 4 -thiouracil, 5-bro(cid:173)
`mouracil, 5-iodouracil, 3-(amiooaUyl)uracil for uracil, aod
`inosine for guanosioe in sense and anti.sense strands of the
`siRNA, and found that whereas 4-thiouracil and 5-bromou(cid:173)
`racil were all well 1olerated, inosine " produced a substantial
`decrease in interference activity" when incorporated in
`either strand. Incorporation of 5-iodouracil and 3-(aminoal(cid:173)
`lyl)uracil in the antiscnsc 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 auenuati.og
`gene expression using endogenously derived dsRNA. Tuschl
`et al., International PCT Publication No. WO 01/75164,
`describes a Drosophila in vitro RNA.i system and the use of
`specific siRNA molecules for certain functional genomic
`and certain tbcrapcutic applications; although Tuscbl, 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 i.oterferoa response". Li et al., International
`PCT Publicatjon No. WO 00/44914, describes the use of
`specific dsRNA5 for use in attenuating Lhe expression of
`certain target genes. Zemicka-Goctz et al., International
`PCT Publication No. WO 01/36646, describes certain meth(cid:173)
`ods for inhibiling tbe expression of particular genes in
`mammalian cells using certain dsRNA molecules. Fire e t al.,
`International PCT Publication No. WO 99/32619, describes
`particular methods for introducing certain dsRNA molecules
`ioto cells for use io inhibiting gene expression. Plaetinck ct
`al., International PCT Publication No. WO 00/01846,
`describes certain methods for identifying specific genes
`responsible for conferring a particular pbenotype i.o a cell
`using specific dsRNA molecules. Mello el al., International
`PCT Publication No. WO 01/29058, describes the identiii(cid:173)
`catioa o[ specific genes involved in dsRNA mediated RNfu.
`Deschamps Depaillette et al., JnternaLional PCT Publication
`No. WO 99/07409, describes specific compositions consist(cid:173)
`ing of particular dsRNA molecules combined witb certain
`anti-viral agents. Driscoll et al., International PCT Publica(cid:173)
`tion No. WO 01/49844, describes specific DNA constructs
`for use in facilitating gene silencing in targeted organisms.
`Parrish et al., 2000, Mo!ecu/ar Cell, 6, 1977-1087, describes
`specific chemically modified siRNA construc ts targeting the
`unc-22 gene of C. elegans. Tuscbl ct al., InternatioDal PCT
`Publication No. WO 02/44321, describe certain synthetic
`siRNA constructs.
`[0009] Alzbeimer's disease (AD) is a progressive, degeo(cid:173)
`crative 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 o[ the next century i[ 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 bave Alzheimer's
`disease. Alzheimer's disease is not confined to the elderly, a
`
`small perccotagc of people io their 30' s and 40's arc afflicted
`witb early onset AD. Alzbcirner's disease is the most com(cid:173)
`mon form of dementia, and amounts to tbe third most
`expensive disease in tbe US followiag bean 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(cid:173)
`gressive formation of insoluble plaques and vascular depos(cid:173)
`its in the brain consisting of the 4 kD amyloid B peptide
`(A~). These plaques are characterized by dyslropbic oeurites
`that show profound synaptic loss, o.eurofibrillary tangle
`formation, and gliosis. AB arises from !be proteolytic cleav(cid:173)
`age of the large type I trans mernbrane protein, B-amyloid
`precursor protein (APP) (Kang ct al., 1987, Nature, 325,
`733). Processing of APP to generate Aj) reqwres two sites of
`cleavage by a B-secretase and a y-secrctase. B-sccretase
`cleavage of APP results in the cytoplasmic release of a 100
`kD soluble amino-terminal fragment, APPsB, leaving behind
`a 12 kD lraosmembrane carboxy-terminal fragment, C99.
`Alternately, APP can be cleaved by a a-secretasc 10 generate
`cytoplasmic APPsa and transmembrane C83 fragments.
`Both remaining lraasmembrane fragments, C99 and C83,
`can be further cleaved by a y-secretase, leading lo the release
`and secretion of Alzheimer's related A~ and a non-patho(cid:173)
`genic peptide, p3, respectively (Vas.5ar ct al., 1999, Science,
`286, 735-741). Early onset familial Alzheimer's disease is
`characterized by mutant APP protein witb a Met to Leu
`substitution at position Pl, characterized as the "Swedish"
`familial mutation (Mullan et al., 1992, Na1ure Gener., l ,
`345). Ibis APP mutation is characterized by a dramatic
`enhancement in B-sccretasc cleavage (Citron et al., 1992,
`Nature, 360, 672).
`
`[OOU] The identification of B-secretase, and y-secretase
`constituents involved in tbe release of B-amyloid protein is
`of primary importance in the development of treatment
`strategies for Alzheimer's ilisease. Characterization of
`a-sccrctasc is also important in this regard since a-secretasc
`c leavage may compete with ~-secrctase cleavage resulting
`in non-pathogenic vs. pathogenic protein production.
`Involvement of 1he two metalloprotea:ses, ADAM 10, and
`TACE bas been demonstrated in a-cleavage of AAP (Bux(cid:173)
`baum e t al., 1999, J. Biol. Chem., 273, 27765, and Lammich
`el al., 1999, Proc. Nari. Acad. Sci. U.SA., 96, 3922). Studies
`of y-secretase activity have demonstrated prcscnilin depen(cid:173)
`dence (De Stooper et al., 1998, Nature, 391, 387, and De
`Stooper ct al., 1999, Nature, 398, 518), and as sucb, prcs(cid:173)
`enilins bave been proposed as y-secretase even tbougb
`presenilin does not present proteolytic activity (Wolfe et al.,
`1999, Nature, 398, 513).
`
`[0012] Recen tly, Vassar et al., 1999, supra reported
`B-secretase cleavage of AAP by the transmembrane aspartic
`protease beta site APP cleaving enzyme, BACE. While other
`potential candidates for ~-secretase have been proposed (for
`review see Evin et al., 1999, Proc. Nall. Acad. Sci. U.S.A.,
`96, 3922), none bavc demonstrated tbc full range of char(cid:173)
`acteristics expected from thls enzyme. Vassar et al, supra,
`demonstrate that BACE expression and localization are as
`expected for B-secretase, tbat BACE overexpression in cells
`results in increa5ed B-secretasc cleavage of APP and Swed(cid:173)
`ish APP, that isolated BACE demonstrates s ite specific
`proteolytic activity on APP derived peptide substrates, aod
`
`

`

`US 2003/0190635 Al
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`
`3
`
`that antiscnse mediated endogenous BACE inhibition results
`io dramatically reduced ~-sccrclase activity.
`[0013) Current treatment strategics for Alzheimer's dis(cid:173)
`ease rely oo either the prevention or the alleviation of
`symptoms aod/or the slowing clown of disease progression.
`Two drugs approved in the treatment of Alzheimer's, do(cid:143) e(cid:173)
`pezil (Aricept®) and 1acrine (Cognex®), both choli(cid:143) omi(cid:173)
`metics, at1ernp1 to slow the loss of cognitive ability by
`increasing the amou nt of acetylcholine available 10 the
`brain. Antioxidant therapy through the use of antioxidant
`compounds such as alpha-tocophcrol (vitamin E), melato(cid:173)
`oin, and selegelioc (Eldcpryl®) attempt to slow disease
`progression by minimizing free radical damage. Estrogen
`replacement therapy is thought 10 incur a possible preven(cid:173)
`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 Nimoclipine® arc
`considered to have a potential benefit in treatingAlzbeirner's
`disease clue to protection of nerve cells from calcium over(cid:173)
`load, thereby prolonging nerve cell survival. Nootropic
`compounds, such as acetyl-L-camitine (Alcar®) and insulin,
`have been proposed to have some benefit in treating Alzhe(cid:173)
`imer's due to enhancement of cognitive and memory func(cid:173)
`tion based on cellular metabolism.
`
`[0014) Whereby the above treatment strategics may all
`improve quality of life io Alzbcimer's patients, there exists
`an unmet need in the comprehensive treatment and preven(cid:173)
`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(cid:173)
`tics that can eliminate and/o r reverse the deposition of
`amyloid ~ peptide. The use of compounds to modulate the
`expression of proteases that are instrumental in the release of
`amyloid 13 peptide, namely ~-secretase (BACE), and
`y-secretase (presenilin), is of therapeutic significance.
`[0015] McSwiggen el al., International PCT Publication
`No. WO 01/16312, describes nucleic acid mediated inhibi(cid:173)
`tion of BACE, PS-1, and PS-2 expression.
`
`SUMMARY OF THE LNVENTION
`
`[0016] One embodiment of the invention provides a short
`interfering RNA (siRNA) molecule that clown regulates
`expression of a beta site APP-cleaving enzyme (BACE)
`gene by RNA interference. Tbe siRNA molecule can be
`adapted for use to treat Alzheimer's disease. lne siRNA
`molecule can comprise a sense region and a(cid:143) antisense
`region. The antiscnsc region can comprise sequence comple(cid:173)
`mentary to a(cid:143) RNA sequence encoding BACE and the sense
`region can comprise sequence complementary to the anti(cid:173)
`sense region.
`
`[0017] The siRNA molecule can be as.5embled from two
`nucleic acid fragments wherein one fragment comprises the
`sense region and the second fragment comprises the anti(cid:173)
`sense region of said siRNA molecule. The sense region aod
`aotisense 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. Tbc antiscose 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. l -325, 663,665,667,669,671, or 673.
`The sense region ca(cid:143) comprise a sequence of SEQ ID NO.
`651 and the antisense region can comprise a sequence of
`SEQ ID NO. 652. The sense region ca(cid:143) comprise a sequence
`or SEQ ID NO. 653 and the antisense region can comprise
`a sequence of SEQ ID NO. 654. Tbc seosc 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 cao comprise a sequence of SEQ ID NO. 657
`and tbe antisense region can comprise a sequence of SEQ ID
`NO. 658. Tbc sense region ca(cid:143) 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(cid:173)
`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 s iRNA molecule o( the
`invention can comprise a 3'-tcrmina 1 overhang and the
`aotisense region can comprise a 3'-terminal overhang. The
`3'-termioal overhangs each can comprise about 2 nucle(cid:173)
`otides. The antiscnse region of the 3'-ierminal nucleotide
`overhang can be complementary to RNA encoding BACE.
`
`[0020) The sense region of a siRNA molecule can com(cid:173)
`prise one or more 2'-O-methyl modified pyrimidine nucle(cid:173)
`otides. Tbe 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(cid:173)
`dine nucleotides. The antisense region cao also comprise a
`phosphorothioate interoucleoticle linkage at the 3' end of said
`antisense region. The antisense region can comprise
`between about one and about five phospborothioate inter(cid:173)
`nucleotide linkages a 1 the 5' cad of said anlisease region.
`
`[0022) The 3'-terminal nucleotide overhangs of a siRNA
`molecule can comprise ribonucleoticles or deoxyribonucle(cid:173)
`otidcs that arc chemically modified at a nucleic acid sugar,
`base, or backbone. The 3'-termioal nucleotide overhangs can
`also comprise one or more universal base ribonucleotides.
`Additionally, the 3'-terminal nucleotide. overhangs can com(cid:173)
`prise one or more acyclic nucleotides.
`
`[0023] The 3'-termina l nucleotide overhangs can comprise
`nucleotides comprising inter(cid:143) uclcoticlc linkages having For(cid:173)
`mula I:
`
`z
`II
`R,-X-P-Y-Ro
`I
`-
`w
`
`[0024) wherein eacb Rl a(cid:143) d R2 is independently any
`nucleotide, (cid:143) on-nucleotide, or polynuclcoticle which
`can be naturally occurring or chemically modified,
`each X and Y is inclependeatly O, S, N, alkyl, or
`substituted alkyl, each Zand Wis independently 0,
`S, N, alkyl, substituted a.lkyl, O-alkyl, S-alkyl,
`alkaryl, or aralkyl, and wherein W, X, Y and Z are not
`all 0.
`
`

`

`US 2003/0190635 Al
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`Oct. 9, 2003
`
`4
`
`[0025) Tbc 3'-terrninal nucleotide overhangs can comprise
`nucleolidcs or non-oucleolides having Fommla II:
`
`R1~R11 B
`Ri,
`R12
`~ R.,
`
`Rs
`
`Rio
`
`Rs R3
`
`[0026] wherein eacb R3, R4, R5, R6, R7, R8, Rl0,
`Rll and Rl2 is independen1ly H, OH, alkyl, substi(cid:173)
`lUlcd alkyl, alkaryl or aralkyl, F, Cl, Br, CN, CF3,
`OCF3, OCN, O-alk')'l, S-alk')'l, N-alkyl, O-alkenyl,
`S-alkeoyl, N-alkenyl, SO-alkyl, alk')'I-OSH, alkyl(cid:173)
`OH, O-alkyl-O11, O-alkyl-SH, S-alkyl-OH, S-a.lkyl(cid:173)
`Sl-1, alkyl-S-alkyl, alkyl-O-alk-yl, ONO2, NO2, N3,
`Nl-12, aminoalkyl, amiooacid, aminoacyl, ONH2,
`O-aminoalkyl, O-aminoacicl, O-aminoacyl, hetero(cid:173)
`cycloalkyl, heterocycloalkaryl, aminoalkylamino,
`polyalklylamino, substituted silyl, or group having
`Formula I; R9 is O, S, CH2, S=O, CJ-IF, or CF2, and
`B is a nuclcosidic base or any other ooo-oaturally
`occurring base tbat can be complementary or non(cid:173)
`complementary to BACE RNA or a oon-oucleosidic
`base or any other ooo-oaturally occurring universal
`base that can be complementary or non-complemen(cid:173)
`tary to BACE RNA
`
`[0027] Anolber embodiment of !be invention provides an
`expression vector comprising a nucleic acid sequence
`encoding at least one siRNA molecule of 1be ioven1ioo in a
`manner 1ha1 allows expression of lhe nucleic acid molecule.
`The expression vector can be in a mammalian cell, such as
`a human cell. The s iRNA molecule can comprise a sense
`region and an antiscose region. The anLiseose region can
`comprise sequence complementary to an RNA sequence
`encoding BACE and tbe sense region comprises sequence
`complementary 10 tbe antiseose region. The siRNA mol(cid:173)
`ecule can comprise 1wo dis1inc1 SI rands baving complemen-
`1ari1y sense and aotisense regions or can comprise a single
`slrand having complemeolary sense and antisense regions.
`[0028] Therefore, this invenlion relates to compounds,
`compositions, and mc1hods useful for modulating beta(cid:173)
`secretase (BACE), PJN- l, presenillio l (PS-1) and/or pres(cid:173)
`eoillio 2 (PS-2) f-unction and/or gene expression in a cell by
`RNA interference (RNAi) using shon interfering RNA
`(siRNA). Io particular, the instanl invention features siRNA
`molecules and methods 10 modulate the expression of
`BACE, PIN-1, PS-1 and/or PS-2 RNA. The siRNA of the
`inven1ion can be unmodified or chemically modified. The
`siRNA of tbe instant invention can be chemicaUy synthe(cid:173)
`sized, expressed from a vector or cnzymaticaUy synthesized.
`The instant invention also features various chemically modi(cid:173)
`fied synthetic short interfering RNA (siRNA) molecules
`capable of modulating BACE, PIN-1, PS-1 and/or PS-2 gene
`expression/activity in ceUs by RNA inference (RNAi). The
`use of chemically modified siRNA is expected 10 improve
`various properties of native siRNA molecules througb
`increased resistance to nuclease degradation in vivo and/or
`improved ceUular uptake. Tbe siRNA molecules of lhe
`instant invention p